Proteomics

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The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Posted in Amino acids, Ca2+ triggered activation, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, De novo synthesis, Developmental biology, Dialectic, Discovery process, Disease Biology, Embryology, Epigenetics and Cardiovascular Risks, Evolutionary cognition, Explanatory, Gene Regulation and Evolution, Genome Biology, Historical relevance, Innovations in Neurophysiology & Neuropsychology, Metabolism, Metabolomics, Mutant Gene Expression, Na-K-ATPase, Nitric Oxide in Health and Disease, Nutrition, Pentose monophosphate shunt, Proteins, Proteomics, RNA Biology, Signaling, Signaling & Cell Circuits, tagged adaptiaton, Evolution, habitats, metabolites, oligomers, oligonucleotides, phenotypes, predators, protein conformation, protein interactions, protein synthesis, respiration, temperature, trrain, water salinity on February 25, 2015| Leave a Comment »

The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

Writer and Curator: Larry H. Bernstein, MD, FCAP

phylogenetics

http://upload.wikimedia.org/wikipedia/commons/thumb/1/12/CollapsedtreeLabels-simplified.svg/200px-CollapsedtreeLabels-simplified.svg.png

This discussion that completes and is an epicrisis (summary and critical evaluation) of the series of discussions that preceded it.

Innervation of Heart and Heart Rate
Action of hormones on the circulation
Allogeneic Transfusion Reactions
Graft-versus Host reaction
Unique problems of perinatal period
High altitude sickness
Deep water adaptation
Heart-Lung-and Kidney
Acute Lung Injury

The concept inherent in this series is that the genetic code is an imprint that is translated into a message. It is much the same as a blueprint, or a darkroom photographic image that has to be converted to a print. It is biologically an innovation of evolutionary nature because it establishes a simple and reproducible standard for the transcription of the message through the transcription of the message using strings of nucleotides (oligonucleotides) that systematically transfer the message through ribonucleotides that communicate in the cytoplasm with the cytoskeleton based endoplasmic reticulum (ER), composing a primary amino acid sequence. This process is a quite simple and convenient method of biological activity. However, the simplicity ends at this step. The metabolic components of the cell are organelles consisting of lipoprotein membranes and a cytosol which have particularly aligned active proteins, as in the inner membrane of the mitochondrion, or as in the liposome or phagosome, or the structure of the ER, each of which is critical for energy transduction and respiration, in particular, for the mitochondria, cellular remodeling or cell death, with respect to the phagosome, and construction of proteins with respect to the ER, and anaerobic glycolysis and the hexose monophosphate shunt in the cytoplasmic domain. All of this refers to structure and function, not to leave out the membrane assigned transport of inorganic, and organic ions (electrolytes and metabolites).

I have identified a specific role of the ER, the organelles, and cellular transactions within and between cells that is orchestrated. But what I have outlined is a somewhat limited and rigid model that does not reach into the dynamics of cellular transactions. The DNA has expression that may be old, no longer used messages, and this is perhaps only part of a significant portion of “dark matter”. There is also nuclear DNA that is enmeshed with protein, mRNA that is a copy of DNA, and mDNA is copied to ribosomal RNA (rRNA). There is also rDNA. The classic model is DNA to RNA to protein. However, there is also noncoding RNA, which plays an important role in regulation of transcription.

This has been discussed in other articles. But the important point is that proteins have secondary structure through disulfide bonds, which is determined by position of sulfur amino acids, and by van der Waal forces, attraction and repulsion. They have tertiary structure, which is critical for 3-D structure. When like subunits associate, or dissimilar oligomers, then you have heterodimers and oligomers. These constructs that have emerged over time interact with metabolites within the cell, and also have an important interaction with the extracellular environment.

When you take this into consideration then a more complete picture emerges. The primitive cell or the multicellular organism lives in an environment that has the following characteristics – air composition, water and salinity, natural habitat, temperature, exposure to radiation, availability of nutrients, and exposure to chemical toxins or to predators. In addition, there is a time dimension that proceeds from embryonic stage to birth in mammals, a rapid growth phase, a tapering, and a decline. The time span is determined by body size, fluidity of adaptation, and environmental factors. This is covered in great detail in this work. The last two pieces are in the writing stage that completes the series. Much content has already be presented in previous articles.

The function of the heart, kidneys and metabolism of stressful conditions have already been extensively covered in http://pharmaceuticalintelligence.com in the following and more:

The Amazing Structure and Adaptive Functioning of the Kidneys: Nitric Oxide – Part I

http://pharmaceuticalintelligence.com/2012/11/26/the-amazing-structure-and-adaptive-functioning-of-the-kidneys/

Nitric Oxide and iNOS have Key Roles in Kidney Diseases – Part II

http://pharmaceuticalintelligence.com/2012/11/26/nitric-oxide-and-inos-have-key-roles-in-kidney-diseases/

The pathological role of IL-18Rα in renal ischemia/reperfusion injury – Nature.com

http://pharmaceuticalintelligence.com/2014/10/24/the-pathological-role-of-il-18r%CE%B1-in-renal-ischemiareperfusion-injury-nature-com/

Summary, Metabolic Pathways

http://pharmaceuticalintelligence.com/2014/10/23/summary-metabolic-pathways/

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Depth Underwater and Underground

Posted in Amino acids, Best evidence, Biochemical pathways, Biological Networks, Biological Networks, Gene Regulation and Evolution, Biomedical Measurement Science, Cell Biology, Cell Biology, Signaling & Cell Circuits, Curation, Curation methodology, Developmental biology, Diabetes Mellitus, Dialectic, Discovery process, Electrophysiology, Evolutionary cognition, Explanatory, Fatty acids, Gene Regulation, Gene Regulation and Evolution, Genome Biology, Hematopoiesis, Historical relevance, Innovations, Lipid metabolism, Metabolism, Mutant Gene Expression, Myocardial Ischemia, Myocardial Metabolism, Na-K transport, Na-K-ATPase, Neurological Diseases, Nutrition, Proteins, Proteomics, Signaling & Cell Circuits, Theoretic convergence, Translational Research, Translational Science, tagged Biochemistry and Molecular Biology, Bioinformatics, Biology, blind mole rat, Cell Biology, cellular stress, climate, decompression, Hemoglobin, human diving, mtDNA, myoglobin, nitrogen sickness, oxygen delivery, sea lion, seals and otters, whales on February 24, 2015| Leave a Comment »

Depth Underwater and Underground

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

Deep diving for mammals is dangerous for humans and land based animals for too long, and it has dangerous consequences, most notable in nitrogen emboli with very deep underwater diving. Other mammals live in water and have adapted to a water habitat. This is another topic that needs further exploration.

Deep diving has different meanings depending on the context. Even in recreational diving the meaning may vary:

In recreational diving, a depth below about 30 metres (98 ft), where nitrogen narcosis becomes a significant hazard for most divers, may be considered a “deep dive”

In technical diving, a depth below about 60 metres (200 ft) where hypoxic breathing gas becomes necessary to avoid oxygen toxicity may be considered a “deep dive”.

Early experiments carried out by Comex S.A. (Compagnie maritime d’expertises) using hydrox and trimix attained far greater depths than any recreational technical diving. One example being the Comex Janus IV open-sea dive to 501 metres (1,644 ft) in 1977. The open-sea diving depth record was achieved in 1988 by a team of Comex divers who performed pipe line connection exercises at a depth of 534 metres (1,752 ft) in the Mediterranean Sea as part of the Hydra 8 program. These divers needed to breathe special gas mixtures because they were exposed to very high ambient pressure (more than 50 times atmospheric pressure).

Then there is the adaptation to the water habitat as a living environment. The two main types of aquatic ecosystems are marine ecosystems and freshwater ecosystems.

http://en.wikipedia.org/wiki/Deep_diving

Marine ecosystems are part of the earth’s aquatic ecosystem. The habitats that make up this vast system range from the productive nearshore regions to the barren ocean floor. The marine waters may be fully saline, brackish or nearly fresh. The saline waters have a salinity of 35-50 ppt (= parts per thousand). The freshwater has a salinity of less than 0.5 ppt. The brackish water lies in between these 2. Marine habitats are situated from the coasts, over the continental shelf to the open ocean and deep sea. The ecosystems are sometimes linked with each other and are sometimes replacing each other in other geographical regions. The reason why habitats differ from another is because of the physical factors that influence the functioning and diversity of the habitats. These factors are temperature, salinity, tides, currents, wind, wave action, light and substrate.

Marine ecosystems are home to a host of different species ranging from planktonic organisms that form the base of the marine food web to large marine mammals. Many species rely on marine ecosystems for both food and shelter from predators. They are very important to the overall health of both marine and terrestrial environments. Coastal habitats are those above the spring high tide limit or above the mean water level in non-tidal waters. They are close to the sea and include habitats such as coastal dunes and sandy shores, beaches , cliffs and supralittoral habitats. Coastal habitats alone account for approximately 30% of all marine biological productivity.

http://www.marbef.org/wiki/marine_habitats_and_ecosystems

All plant and animal life forms are included from the microscopic picoplankton all the way to the majestic blue whale, the largest creature in the sea—and for that matter in the world. It wasn’t until the writings of Aristotle from 384-322 BC that specific references to marine life were recorded. Aristotle identified a variety of species including crustaceans, echinoderms, mollusks, and fish.
Today’s classification system was developed by Carl Linnaeus external link as an important tool for use in the study of biology and for use in the protection of biodiversity. Without very specific classification information and a naming system to identify species’ relationships, scientists would be limited in attempts to accurately describe the relationships among species. Understanding these relationships helps predict how ecosystems can be altered by human or natural factors.

Preserving biodiversity is facilitated by taxonomy. Species data can be better analyzed to determine the number of different species in a community and to determine how they might be affected by environmental stresses. Family, or phylogenetic, trees for species help predict environmental impacts on individual species and their relatives.

http://marinebio.org/oceans/marine-taxonomy/

For generations, whales and other marine mammals have intrigued humans. 2,400 years ago, Aristotle, a Greek scientist and philosopher, recognized that whales are mammals, not fish, because they nurse their young and breathe air like other mammals. There are numerous myths and legends surrounding marine mammals. The Greeks believed that killing a dolphin was as bad as murdering a human. An Amazon legend said that river dolphins came to shore dressed as men to woo pretty girls during fiestas. During the Middle Ages, there were numerous legends surrounding the narwhals’ amazing tusk, which was thought to have come from the unicorn.

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Marine mammals evolved from their land dwelling ancestors over time by developing adaptations to life in the water. To aid swimming, the body has become streamlined and the number of body projections has been reduced. The ears have shrunk to small holes in size and shape. Mammary glands and sex organs are not part of the external physiology, and posterior (hind) limbs are no longer present.

Mechanisms to prevent heat loss have also been developed. The cylindrical body shape with small appendages reduces the surface area to volume ratio of the body, which reduces heat loss. Marine mammals also have a counter current heat exchange mechanism created by convergent evolution external link where the heat from the arteries is transferred to the veins as they pass each other before getting to extremities, thus reducing heat loss. Some marine mammals also have a thick layer of fur with a water repellent undercoat and/or a thick layer of blubber that can’t be compressed. The blubber provides insulation, a food reserve, and aids with buoyancy. These heat loss adaptations can also lead to overheating for animals that spend time out of the water. To prevent overheating, seals or sea lions will swim close to the surface with their front flippers waving in the air. They also flick sand onto themselves to keep the sun from directly hitting their skin. Blood vessels can also be expanded to act as a sort of radiator.

One of the major behavioral adaptations of marine mammals is their ability to swim and dive. Pinnipeds swim by paddling their flippers while sirenians and cetaceans move their tails or flukes up and down.

Some marine mammals can swim at relatively high speeds. Sea lions swim up to 35 kph and orcas can reach 50 kph. The fastest marine mammal, however, is the common dolphin, which reaches speeds up to 64 kph. While swimming, these animals take very quick breaths. For example, fin whales can empty and refill their huge lungs in less than 2 seconds. Marine mammals’ larynx and esophagus close automatically when they open their mouths to catch prey during dives. Oxygen is stored in hemoglobin in the blood and in myoglobin in the muscles. The lungs are also collapsible so that air is pushed into the windpipe preventing excess nitrogen from being absorbed into the tissues. Decreasing pressure can cause excess nitrogen to expand in the tissues as animals ascend to shallower depths, which can lead to decompression sickness, aka “the bends.” Bradycardia, the reduction of heart rate by 10 to 20%, also takes place to aid with slowing respiration during dives and the blood flow to non-essential body parts. These adaptations allow sea otters to stay submerged for 4 to 5 minutes and dive to depths up to 55 m. Pinnipeds can often stay down for 30 minutes and reach average depths of 150-250 m. One marine mammal with exceptional diving skills is the Weddell seal, which can stay submerged for at least 73 minutes at a time at depths up to 600 m. The length and depth of whale dives depends on the species. Baleen whales feed on plankton near the surface of the water and have no need to dive deeply so they are rarely seen diving deeper than 100 m external link. Toothed whales seek larger prey at deeper depths and some can stay down for hours at depths of up to 2,250 m external link.

http://marinebio.org/oceans/marine-mammals/

Human Experience

Albert Behnke: Nitrogen Narcosis

Casey A. Grover and David H. Grover
The Journal of Emergency Medicine, 2014; 46(2):225–227
http://dx.doi.org/10.1016/j.jemermed.2013.08.080

As early as 1826, divers diving to great depths noted that descent often resulted in a phenomenon of intoxication and euphoria. In 1935, Albert Behnke discovered nitrogen as the cause of this clinical syndrome, a condition now known as nitrogen narcosis. Nitrogen narcosis consists of the development of euphoria, a false sense of security, and impaired judgment upon underwater descent using compressed air below 34 atmospheres (99 to 132 feet). At greater depths, symptoms can progress to loss of consciousness. The syndrome remains relatively unchanged in modern diving when compressed air is used. Behnke’s use of non-nitrogencontaining gas mixtures subsequent to his discovery during the 1939 rescue of the wrecked submarine USS Squalus pioneered the use of non-nitrogencontaining gas mixtures, which are used by modern divers when working at great depth to avoid the effects of nitrogen narcosis.

Behnke’s first duty station as a licensed physician was as assistant medical officer for Submarine Division 20 in San Diego, which was then commanded by one of the Navy’s rising stars, Captain Chester W. Nimitz of World War II fame.
In this setting, Dr. Behnke spent his free time constructively by learning to dive, using the traditional ‘‘hard-hat’’ gear aboard the USS Ortalon, a submarine rescue vessel to which he also rotated. Diving was not a notable specialty of the Navy at the time, and the service was slow in developing the infrastructure for it. Dr. Behnke devoted his efforts to research on the topic of diving medicine, as well as developing a more sound understanding of the biophysics of diving. In 1932, he wrote a letter to the Surgeon General describing some of his observations on arterial gas embolism, which earned him some accolades from the Navy and resulted in his transfer to Harvard’s School of Public Health as a graduate fellow. After 2 years at Harvard, the Navy assigned duty to Dr. Behnke at the Navy’s submarine escape training tower at Pearl Harbor. He worked extensively here on developing techniques for rescuing personnel from disabled submarines on the sea floor. In 1937, he was one of three Navy physicians assigned to the Navy’s Experimental Diving Unit. This team worked on improving the rescue system, plus updating the diving recompression tables originally developed by the British in 1908.

The intoxicating effects of diving were first described by a French physician named Colladon in 1826, who reported that descent in a diving bell resulted in his feeling a ‘‘state of excitement as though I had drunk some alcoholic liquor’’.
The etiology of this phenomenon remained largely unknown until the 1930s, when the British military researcher Damant again highlighted the issue, and reported very unpredictable behavior in his divers during descents as deep as 320 feet during the British Admiralty Deep Sea diving trials. Two initial theories arose as to the etiology for this effect, the first being from psychological causes by Hill and Phillip in 1932, and the second being from oxygen toxicity by Haldane in 1935.

Dr. Behnke and his colleagues at the Harvard School of Public Health had another idea as to the etiology of this phenomenon. In 1935, based on observation of individuals in experiments with a pressure chamber, Dr. Behnke published an article in the American Journal of Physiology in which he posited that nitrogen was the etiology of the intoxicating effects of diving.

Nitrogen narcosis, described as ‘‘rapture of the deep’’ by Jacques Cousteau, still remains a relatively common occurrence in modern diving, despite major advances in diving technology since Behnke’s initial description of the pathophysiologic cause of the condition in 1935. The development of symptoms of this condition varies from diver to diver, but usually begins when a depth of 4 atmospheres (132 feet) is reached in divers using compressed air. More sensitive divers can develop symptoms at only 3 atmospheres (99 feet), and other divers may not be affected up to depths as high as 6 atmospheres (198 feet). Interestingly, tolerance to nitrogen narcosis can be developed by frequent diving and exposure to the effects of compressed air at depth.

Acott C. A brief history of diving and decompression illness. SPUMS J 1999;29:98–109.
2. Bornmann R. Dr. Behnke, founder of UHMS, dies. Pressure 1992; 21:14.
3. Behnke AR, Thomson RM, Motley P. The psychologic effects from breathing air at 4 atmospheric pressures. Am J Physiol 1935; 112:554–8.
4. Behnke AR, Johnson FS, Poppen JR, Motley P. The effect of oxygen on man at pressures from 1 to 4 atmospheres. AmJ Physiol 1934; 110:565–72.

Exhaled nitric oxide concentration and decompression-induced bubble formation: An index of decompression severity in humans?

J.-M. Pontier, Buzzacott, J. Nastorg, A.T. Dinh-Xuan, K. Lambrechts
Nitric Oxide 39 (2014) 29–34
http://dx.doi.org/10.1016/j.niox.2014.04.005

Introduction: Previous studies have highlighted a decreased exhaled nitric oxide concentration (FE NO) in divers after hyperbaric exposure in a dry chamber or following a wet dive. The underlying mechanisms of this decrease remain however unknown. The aim of this study was to quantify the separate effects of submersion, hyperbaric hyperoxia exposure and decompression-induced bubble formation on FE NO after a wet dive.
Methods: Healthy experienced divers (n = 31) were assigned to either

a group making a scuba-air dive (Air dive),
a group with a shallow oxygen dive protocol (Oxygen dive) or

a group making a deep dive breathing a trimix gas mixture (deep-dive).
Bubble signals were graded with the KISS score. Before and after each dive FE NO values were measured using a hand-held electrochemical analyzer.
Results: There was no change in post-dive values of FE NO values (expressed in ppb = parts per billion) in the Air dive group (15.1 ± 3.6 ppb vs. 14.3 ± 4.7 ppb, n = 9, p = 0.32). There was a significant decrease in post-dive values of FE NO in the Oxygen dive group (15.6 ± 6 ppb vs. 11.7 ± 4.7 ppb, n = 9, p = 0.009). There was an even more pronounced decrease in the deep dive group (16.4 ± 6.6 ppb vs. 9.4 ± 3.5 ppb, n = 13, p < 0.001) and a significant correlation between KISS bubble score >0 (n = 13) and percentage decrease in post-dive FE NO values (r = -0.53, p = 0.03). Discussion: Submersion and hyperbaric hyperoxia exposure cannot account entirely for these results suggesting the possibility that, in combination, one effect magnifies the other. A main finding of the present study is a significant relationship between reduction in exhaled NO concentration and dive-induced bubble formation. We postulate that exhaled NO concentration could be a useful index of decompression severity in healthy human divers.

Brain Damage in Commercial Breath-Hold Divers

Kiyotaka Kohshi, H Tamaki, F Lemaıtre, T Okudera, T Ishitake, PJ Denoble
PLoS ONE 9(8): e105006 http://dx.doi.org:/10.1371/journal.pone.0105006

Background: Acute decompression illness (DCI) involving the brain (Cerebral DCI) is one of the most serious forms of diving related injuries which may leave residual brain damage. Cerebral DCI occurs in compressed air and in breath-hold divers, likewise. We conducted this study to investigate whether long-term breath-hold divers who may be exposed to repeated symptomatic and asymptomatic brain injuries, show brain damage on magnetic resonance imaging (MRI).
Subjects and Methods: Our study subjects were 12 commercial breath-hold divers (Ama) with long histories of diving work in a district of Japan. We obtained information on their diving practices and the presence or absence of medical problems, especially DCI events. All participants were examined with MRI to determine the prevalence of brain lesions.
Results: Out of 12 Ama divers (mean age: 54.965.1 years), four had histories of cerebral DCI events, and 11 divers demonstrated ischemic lesions of the brain on MRI studies. The lesions were situated in the cortical and/or subcortical area (9 cases), white matters (4 cases), the basal ganglia (4 cases), and the thalamus (1 case). Subdural fluid collections were seen in 2 cases. Conclusion: These results suggest that commercial breath-hold divers are at a risk of clinical or subclinical brain injury which may affect the long-term neuropsychological health of divers.

Decompression illness

Richard D Vann, Frank K Butler, Simon J Mitchell, Richard E Moon
Lancet 2010; 377: 153–64

Decompression illness is caused by intravascular or extravascular bubbles that are formed as a result of reduction in environmental pressure (decompression). The term covers both arterial gas embolism, in which alveolar gas or venous gas emboli (via cardiac shunts or via pulmonary vessels) are introduced into the arterial circulation, and decompression sickness, which is caused by in-situ bubble formation from dissolved inert gas. Both syndromes can occur in divers, compressed air workers, aviators, and astronauts, but arterial gas embolism also arises from iatrogenic causes unrelated to decompression. Risk of decompression illness is
affected by immersion, exercise, and heat or cold. Manifestations range from itching and minor pain to neurological symptoms, cardiac collapse, and death. First aid treatment is 100% oxygen and definitive treatment is recompression to increased pressure, breathing 100% oxygen. Adjunctive treatment, including fluid administration and prophylaxis against venous thromboembolism in paralyzed patients, is also recommended. Treatment is, in most cases, effective although residual deficits can remain in serious cases, even after several recompressions.

Bubbles can have mechanical, embolic, and biochemical effects with manifestations ranging from trivial to fatal. Clinical manifestations can be caused by direct effects from extravascular (autochthonous) bubbles such as mechanical distortion of tissues causing pain, or vascular obstruction causing stroke-like signs and symptoms. Secondary effects can cause delayed symptom onset up to 24 h after surfacing. Endothelial damage by intravascular bubbles can cause capillary leak, extravasation of plasma, and haemoconcentration. Impaired endothelial function, as measured by decreased effects of vasoactive compounds, has been reported in animals and might occur in man. Hypotension can occur in severe cases. Other effects include platelet activation and deposition, leucocyte-endothelial adhesion, and possibly consequences of vascular occlusion believed to occur in thromboembolic stroke such as ischaemia-reperfusion injury, and apoptosis.

Classification of initial and of all eventual manifestations of decompression illness in 2346 recreational diving accidents reported to the Divers Alert Network from 1998 to 2004 For all instances of pain, 58% consisted of joint pain, 35% muscle pain, and 7% girdle pain. Girdle pain often portends spinal cord involvement. Constitutional symptoms included headache, lightheadedness, inappropriate fatigue, malaise, nausea or vomiting, and anorexia. Muscular discomfort included stiffness, pressure, cramps, and spasm but excluded pain. Pulmonary manifestations included dyspnoea and cough.

Other than depth and time, risk of decompression sickness is affected by other factors that affect inert gas exchange and bubble formation, such as immersion (vs dry hyperbaric chamber exposure), exercise, and temperature. Immersion decreases venous pooling and increases venous return and cardiac output. Warm environments improve peripheral perfusion by promoting vasodilation, whereas cool temperatures decrease perfusion through vasoconstriction. Exercise increases both peripheral perfusion and temperature. The effect of environmental conditions on risk of decompression sickness is dependent on the phase of the pressure exposure. Pressure, exercise, immersion, or a hot environment increase inert gas uptake and risk of decompression sickness. During decom-pression these factors increase inert gas elimination and therefore decrease the risk of decompression sickness. Conversely, uptake is reduced during rest or in a cold environment, hence a diver resting in a cold environment on the bottom has decreased risk of decompression sickness. Rest or low temperatures during decompression increase the risk. If exercise occurs after decompression when super-saturation is present, bubble formation increases and risk of decompression sickness rises.

Exercise at specific times before a dive can decrease the risk of serious decompression sickness in animals and incidence of venous gas emboli in both animals and man. The mechanisms of these effects are unknown but might involve modulation of nitric oxide production and effects on endothelium. Venous gas emboli and risk of decompression sickness increase slightly with age and body-mass index.

Arterial gas embolism should be suspected if a diver has a new onset of altered consciousness, confusion, focal cortical signs, or seizure during ascent or within a few minutes after surfacing from a compressed gas dive.

If the diver spends much time at depth and might have absorbed substantial inert gas before surfacing, arterial gas embolism and serious decompression sickness can coexist, and in such cases, spinal cord manifestations can predominate. Other organ systems, such as the heart, can also be affected, but the clinical diagnosis of gas embolism is not reliable without CNS manifestations. Arterial gas embolism is rare in altitude exposure; if cerebral symptoms occur after altitude exposure, the cause is usually decompression sickness.

Nondermatomal hypoaesthesia and truncal ataxia are common in neurological decompression sickness and can be missed by cursory examination. Pertinent information includes level of consciousness and mental status, cranial nerve function, and motor strength. Coordination can be affected disproportionately, and abnormalities can be detected by assessment of finger-nose movement, and, with eyes open and closed, ability to stand and walk and do heel-toe walking backwards and forwards. Many of these simple tests can be done on the scene by untrained companions.

Panel: Differential diagnosis of decompression illness

Inner-ear barotrauma

Middle-ear or maxillary sinus overinfl ation

Contaminated diving gas and oxygen toxic effects

Musculoskeletal strains or trauma sustained before, during, or after diving

Seafood toxin ingestion (ciguatera, pufferfish, paralytic shellfish poisoning)

Immersion pulmonary edema

Water aspiration

Decompression chamber

Decompression chamber. fluidic or pneumatic ventilator is shown at the left. The infusion pump is contained within a plastic cover, in which 100% nitrogen is used to decrease the fi re risk in the event of an electrical problem. The monitor screen is outside the chamber and can be seen through the viewing port. Photo from Duke University Medical Center, with permission.

Long-term outcomes of 69 divers with spinal cord decompressionsickness, by manifestation
	n	%
No residual symptoms	34	49·3
Any residual symptom	35	50·7
Mild paraesthesias, weakness, or pain	14	20·3
Some impairment of daily activities	21	30·4
Difficulty walking	11	15·9
Impaired micturition	13	18·8
Impaired defecation	15	21·7
Impaired sexual function	15	21·7

Decompression illness occurs in a small population but is an international problem that few physicians are trained to recognise or manage. Although its manifestations are often mild, the potential for permanent injury exists in severe cases, especially if unrecognised or inadequately treated. Emergency medical personnel should be aware of manifestations of decompression illness in the setting of a patient with a history of recent diving or other exposure to substantial pressure change, and should contact an appropriate consultation service for advice.

Diving Medicine: Contemporary Topics and Their Controversies

Michael B. Strauss and Robert C. Borer, Jr
Am J Emerg Med 2001; 19:232-238
http://dx.doi.org:/10.1053/ajem.2001.22654

SCUBA diving is a popular recreational sport. Although serious injuries occur infrequently, when they do knowledge of diving medicine and/or where to obtain appropriate consultation is essential. The emergency physician is likely to be the first physician contact the injured diver has. We discuss 8 subjects
in diving medicine which are contemporary, yet may have controversies associated with them. From this information the physician dealing primarily with the injured diver will have a basis for understanding and managing, as
well as where to find additional help, for his/her patients’ diving injuries.

Over the past 10 years, new knowledge and equipment improvements have made diving safer and more enjoyable. Estimates of actively participating sports divers show a striking increase over this time interval while the number of SCUBA diving deaths annually has remained nearly level at approximately 100. A further indicator of recreational diving safety is that reflected in the nearly constant number of diving injuries (1000 per annum) over the most recent 5 reported years, or approximately 0.53 to 3.4 incidents/10,000 dives.

Divers Alert Network.
The Divers Alert Network (DAN) is a nonprofit organization directed and staffed by experts in the specialty of diving medicine.6 DAN provides immediate consultation for both divers and physicians in the diagnosis and initial management of diving injuries. This 24-hour service is available free world-wide through a dedicated emergency telephone line: 1-919-684-4326. The DAN staff will also identify the nearest appropriate recompression treatment facility and knowledgeable physicians for an expedient referral. General diving medical inquiries can be answered during normal weekday hours either through an information telephone line: 1-919-684-2948 or through an interactive web site http://www.diversalertnetwork.org.

Use of 100% Oxygen for Initial, on the Scene, Management of Diving Accidents
The breathing of pure oxygen is crucial for the initial management of the diving related problems of arterial gas embolism (AGE), decompression sickness (DCS), pulmonary barotrauma (thoracic squeeze), aspiration pneumonitis, and hypoxic encephalopathy associated with near drowning. In 1985, Dick reported that in many cases the neurologic symptoms of AGE and DCS were resolved with the immediate breathing of pure oxygen on the surface. The breathing of pure oxygen reduces bubble size by increasing the differential pressure for the inert gas to diffuse out of the bubble and it also speeds the washout of inert gas from body tissues. The early elimination of the bubble prevents hypoxia and the interaction of the bubble with the blood vessel lining. This interaction leads to secondary problems of capillary leak, bleeding, inflammation, ischemia, and cell death. These secondary problems are the reasons not all DCS symptoms resolve with recompression chamber treatment. The immediate use of pure oxygen for the medical management of these diving problems is analogous to the use of cardiopulmonary resuscitation for the witnessed cardiac arrest; the sooner initiated the better the results.

Diving Education

Medical Fitness for Diving

Asthma has the potential risk for AGE. Neuman reviewed the subject of asthma and diving. He and his coauthors recommend that asthmatics who are asymptomatic, not on medications and have no exercised induced abnormality on pulmonary function studies be allowed to dive.

Conditions leading to loss of consciousness, such as insulin dependent diabetes and epilepsy, can result in drowning. Carefully controlled diving studies in diabetics, who are free from complications, are now defining the safe requirements for diving. Epilepsy remains as a disqualification except in individuals with a history of febrile seizures ending prior to 5 years of age.

Availability of Hyperbaric Oxygen Treatment Facilities

The availability of these chambers makes it possible for divers who become symptomatic after SCUBA diving to readily receive recompression treatment. This is important because the closer the initiation of recompression treatment to the onset of DCS (and AGE) signs and symptoms, the greater the likelihood of full recovery.

Improved Diving Equipment

Mixed and Rebreather Gas Diving
Mixed gas diving involves changing the breathing gas from air which has 20% oxygen to higher oxygen percentages (nitrox). As the amount of oxygen is increased in the gas mixture, the amount of the inert gas (nitrogen) is reduced. With oxygen enriched air there is less tissue deposition of inert gas per unit of time under water for any given depth. However, because of increased oxygen partial pressures, the seizure threshold for oxygen toxicity is lowered. For normal sports diving activities, oxygen toxicity with mixed gas diving is only a theoretical concern.

Decompression Illness is More Than Bubbles

When AGE occurs, DCS symptoms may be concurrent or appear during or after recompression treatment even though the decompression tables were not violated on the dive. When DCS occurs in this situation it appears resistant to recompression treatment (Neuman) perhaps because of the inflammatory reaction generated by the bubble-blood vessel interaction from the AGE. In cases of DCI where components of both DCS and AGE are suspected, the diver should be observed for a minimum of 24 hours after the recompression treatment is completed for the delayed onset of DCS.

No theory of DCS discounts the primary role of bubbles in this condition. However, new information suggests that there are precursors to bubble formation and post-bubbling events that occur as a consequence of the bubbles. As mentioned earlier, venous gas emboli are a common occurrence diving ascent and ordinarily are filtered out harmlessly by the lungs. Precursors to DCS include stasis, dehydration and too rapid of ascents. These conditions allow the ubiquitous VGE to enlarge, coalesce and occlude the venous side of the circulation. Massive venous bubbling to the lungs can cause pulmonary vessel obstruction described as the chokes. If right to left shunts occur in the heart, VGE can become AGE to the brain. If the arterial flow is slow enough and/or the gradients large enough, autochthonus (ie, spontaneous) bubbles can form in the arterial circulation and lead to any of the consequences of AGE. In such situations it could be difficult to determine whether the DCI event was from AGE or DCS even after careful analysis of the dive profile. Hollenbeck’s model for diving paraplegia includes the setting of venous stasis (Batson’s plexus of veins) in the spinal canal, bubble formation, bubble enlargement possibly from off gassing of the spinal cord, blood vessel occlusion, and venous side infarctions of the spinal cord.
Contemporary Management of DCS

Problem	Intervention	Effect
Bubble	Recompression with HBO	Reduce bubble size 1. Washout inert gas. 2. Change bubble composition by diffusion.
Stasis and dehydration	Hydration: oral fluids if alert, IV fluids otherwise.	Improve blood flow.
InflammationCell Ischemia	? Anti-inflammatory medicationsHBO	Reduce interaction between bubble and blood vessel endothelium. Improve oxygen availability to hypoxic tissues, reduce edema and also reduces the interaction between bubble and blood vessel endothelium.

Conclusions

We anticipate that in the future there will be further improvements for the safety and enjoyment of the recreational SCUBA diver. For example, the dive computer of the future will be able to individualize dive profiles for different personal medical parameters such as age, body composition and fitness level. Diver locators could quickly target a missing diver and save time and gas consumption as well as prevent serious diving mishaps. Drugs may be developed that would minimize the effect of bubbles interacting with body tissues and prevent DCS and AGE.

Extracorporeal membrane oxygenation therapy for pulmonary decompression illness

Yutaka Kondo, Masataka Fukami and Ichiro Kukita
Kondo et al. Critical Care 2014; 18:438 http://ccforum.com/content/18/3/438/10.1186/cc13935

Pulmonary decompression illness is rarely observed in clinical settings, and most patients die prior to hospitalization. We administered ECMO therapy to rescue a patient, even though this therapy has rarely been reported with good outcome in patients with decompression illness. In addition, we had to select venovenous ECMO even with the patient showing right ventricular failure. A lot of physicians may select venoarterial ECMO if the patient shows right ventricular failure, but the important physiological mechanism of pulmonary decompression illness is massive air embolism in the pulmonary arteries, and the bubbles diminish within the first 24 hours. The management of decompression illness therefore differs substantially from the usual right-sided heart failure.

Extremes of barometric pressure

Jane E Risdall, David P Gradwell
Anaesthesia and Intensive Care Medicine 16:2
Ascent to elevated altitude, commonly achieved through flight, by climbing or by residence in highland regions, exposes the individual to reduced ambient pressure. Although there are physical manifestations of this exposure as a consequence of Boyle’s law, the primary physiological challenge is of hypobaric hypoxia. The acute physiological and longer-term adaptive responses of the cardiovascular, respiratory, hematological and neurological systems to altitude are described, together with an outline of the presentation and management of acute mountain sickness, high-altitude pulmonary edema and high-altitude cerebral edema. While many millions experience modest exposure to altitude as a result of flight in pressurized aircraft, fewer individuals are exposed to increased ambient pressure. The pressure changes during diving and hyperbaric exposures result in greater changes in gas load and gas toxicity. Physiological effects include the consequences of increased work of breathing and redistribution of circulating volume. Neurological manifestations may be the direct result of pressure or a consequence of gas toxicity at depth. Increased tissue gas loads may result in decompression illness on return to surface or subsequent ascent in flight.

understand the physical effects of changes in ambient pressure and the physiological consequences on the cardiovascular respiratory and neurological systems
gain an awareness that exposure to reduced ambient pressure produces both acute and more chronic effects, with differing signs, symptoms and time to onset at various altitudes
develop an awareness of the toxic effects of ‘inert’ gases at increased ambient pressures and the pathogenesis and management of decompression illness

Decompression illness According to Henry’s law, at a constant temperature the amount of gas which dissolves in a liquid is proportional to the pressure of that gas or its partial pressure, if it is part of a mixture of gases. Breathing gases at increased ambient pressure will increase the amount of each gas dissolved in the fluid phases of body tissues. On ascent this excess gas has to be given up. If the ascent is controlled at a sufficiently slow rate, elimination will be via the respiratory system. If the ascent is too fast, excess gas may come out of solution and form free bubbles in the tissues or circulation. Bubbles may contain any of the gases in the breathing mixture, but it is the presence of inert gas bubbles (nitrogen or helium) that are thought most likely to give rise to problems, since the elimination of excess oxygen is achieved by metabolism as well as ventilation. These bubbles may act as venous emboli or may trigger inflammatory tissue responses giving rise to symptoms of decompression illness (DCI). Signs and symptoms of DCI may appear up to 48 hours after exposure to increased ambient pressure and include joint pains, motor and sensory deficits, dyspnoea, cough and skin rashes.

Neurological effects of deep diving

Marit Grønning, Johan A. Aarli
Journal of the Neurological Sciences 304 (2011) 17–21
http://dx.doi.org:/10.1016/j.jns.2011.01.021

Deep diving is defined as diving to depths more than 50 m of seawater (msw), and is mainly used for occupational and military purposes. A deep dive is characterized by the compression phase, the bottom time and the decompression phase. Neurological and neurophysiologic effects are demonstrated in divers during the compression phase and the bottom time. Immediate and transient neurological effects after deep dives have been shown in some divers. However, the results from the epidemiological studies regarding long term neurological effects from deep diving are conflicting and still not conclusive.

Possible immediate neurological effects of deep diving
Syndrome	Pressure
Hyperoxia/oxygen seizures	>152 kPa (5 msw)
HypoxiaHypercapnia
Nitrogen narcosis	>354 kPa (25 msw)
High pressure nervous syndrome	>1.6 MPa (150 msw)
Neurological decompression sickness

Neurological effects have been demonstrated, both clinically and neurophysiologically in divers during the compression phase and the bottom time. Studies of divers before and after deep dives have shown immediate and transient neurological effects in some divers. However, the results from the epidemiological and clinical studies regarding long term neurological effects from deep diving are conflicting and still not conclusive. Prospective clinical studies with sufficient power and sensitivity are needed to solve this important issue.

Today deep diving to more than 100 msw is routinely performed globally in the oil- and gas industry. In the North Sea remote underwater intervention and maintenance is performed by the use of remotely operated vehicles (ROV), both in conjunction to and as an alternative to manned underwater operations. There will, however, always be a need for human divers in the technically more advanced underwater operations and for contingency repair operations.

P300 latency indexes nitrogen narcosis

Barry Fowler, Janice Pogue and Gerry Porlier
Electroencephalography, and clinical Neurophysiology, 1990, 75:221-229

This experiment investigated the effects of nitrogen narcosis on reaction time (RT) and P300 latency and amplitude, Ten subjects breathed either air or a non-narcotic 20% oxygen-80% helium (heliox) mixture in a hyperbaric chamber at 6.5, 8.3 and 10 atmospheres absolute (ATA), The subjects responded under controlled accuracy conditions to visually presented male or female names in an oddball paradigm. Single-trial analysis revealed a strong relationship between RT and P300 latency, both of which were slowed in a dose-related manner by hyperbaric air but not by heliox. A clear-cut dose-response relationship could not be established for P300 amplitude. These results indicate that P300 latency indexes nitrogen narcosis and are interpreted as support for the slowed processing model of inert gas narcosis.

Adaptation to Deep Water Habitat

Effects of hypoxia on ionic regulation, glycogen utilization and antioxidative ability in the gills and liver of the aquatic air-breathing fish Trichogaster microlepis

Chun-Yen Huang, Hui-Chen Lina, Cheng-Huang Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 25–34
http://dx.doi.org/10.1016/j.cbpa.2014.09.001

We examined the hypothesis that Trichogaster microlepis, a fish with an accessory air-breathing organ, uses a compensatory strategy involving changes in both behavior and protein levels to enhance its gas exchange ability. This compensatory strategy enables the gill ion-regulatory metabolism to maintain homeostasis during exposure to hypoxia. The present study aimed to determinewhether ionic regulation, glycogen utilization and antioxidant activity differ in terms of expression under hypoxic stresses; fish were sampled after being subjected to 3 or 12 h of hypoxia and 12 h of recovery under normoxia. The air-breathing behavior of the fish increased under hypoxia. No morphological modification of the gills was observed. The expression of carbonic anhydrase II did not vary among the treatments. The Na+/K+-ATPase enzyme activity did not decrease, but increases in Na+/K+-ATPase protein expression and ionocyte levels were observed. The glycogen utilization increased under hypoxia as measured by glycogen phosphorylase protein expression and blood glucose level, whereas the glycogen content decreased. The enzyme activity of several components of the antioxidant system in the gills, including catalase, glutathione peroxidase, and superoxidase dismutase, increased in enzyme activity. Based on the above data, we concluded that T. microlepis is a hypoxia-tolerant species that does not exhibit ion-regulatory suppression but uses glycogen to maintain energy utilization in the gills under hypoxic stress. Components of the antioxidant system showed increased expression under the applied experimental treatments.

Divergence date estimation and a comprehensive molecular tree of extant cetaceans

Michael R. McGowen , Michelle Spaulding, John Gatesy
Molecular Phylogenetics and Evolution 53 (2009) 891–906
http://dx.doi.org/10.1016/j.ympev.2009.08.018

Cetaceans are remarkable among mammals for their numerous adaptations to an entirely aquatic existence, yet many aspects of their phylogeny remain unresolved. Here we merged 37 new sequences from the nuclear genes RAG1 and PRM1 with most published molecular data for the group (45 nuclear loci, transposons, mitochondrial genomes), and generated a supermatrix consisting of 42,335 characters. The great majority of these data have never been combined. Model-based analyses of the supermatrix produced a solid, consistent phylogenetic hypothesis for 87 cetacean species. Bayesian analyses corroborated odontocete (toothed whale) monophyly, stabilized basal odontocete relationships, and completely resolved branching events within Mysticeti (baleen whales) as well as the problematic speciose clade Delphinidae (oceanic dolphins). Only limited conflicts relative to maximum likelihood results were recorded, and discrepancies found in parsimony trees were very weakly supported. We utilized the Bayesian supermatrix tree to estimate divergence dates among lineages using relaxed-clock methods. Divergence estimates revealed rapid branching of basal odontocete lineages near the Eocene–Oligocene boundary, the antiquity of river dolphin lineages, a Late Miocene radiation of balaenopteroid mysticetes, and a recent rapid radiation of Delphinidae beginning [1]10 million years ago. Our comprehensive, time calibrated tree provides a powerful evolutionary tool for broad-scale comparative studies of Cetacea.

Mitogenomic analyses provide new insights into cetacean origin and evolution

Ulfur Arnason, Anette Gullberg, Axel Janke
Gene 333 (2004) 27–34
http://dx.doi.org:/10.1016/j.gene.2004.02.010

The evolution of the order Cetacea (whales, dolphins, porpoises) has, for a long time, attracted the attention of evolutionary biologists. Here we examine cetacean phylogenetic relationships on the basis of analyses of complete mitochondrial genomes that represent all extant cetacean families. The results suggest that the ancestors of recent cetaceans had an explosive evolutionary radiation 30–35 million years before present. During this period, extant cetaceans divided into the two primary groups, Mysticeti (baleen whales) and Odontoceti (toothed whales). Soon after this basal split, the Odontoceti diverged into the four extant lineages, sperm whales, beaked whales, Indian river dolphins and delphinoids (iniid river dolphins, narwhals/belugas, porpoises and true dolphins). The current data set has allowed test of two recent morphological hypotheses on cetacean origin. One of these hypotheses posits that Artiodactyla and Cetacea originated from the extinct group Mesonychia, and the other that Mesonychia/Cetacea constitutes a sister group to Artiodactyla. The current results are inconsistent with both these hypotheses. The findings suggest that the claimed morphological similarities between Mesonychia and Cetacea are the result of evolutionary convergence rather than common ancestry.

The order Cetacea traditionally includes three suborders: the extinct Archaeoceti and the recent Odontoceti and Mysticeti. It is commonly believed that the evolution of ancestral cetaceans from terrestrial to marine (aquatic) life was accompanied by a fast and radical morphological adaptation. Such a scenario may explain why it was, for a long time, difficult to morphologically establish the position of Cetacea in the mammalian tree and even to settle whether Cetacea constituted a monophyletic group.

Biochemical analyses in the 1950s and 1960s had shown a closer relationship between cetaceans and artiodactyls (even-toed hoofed mammals) than between cetaceans and any other eutherian order and karyological studies in the late 1960s and early 1970s unequivocally supported cetacean monophyly (Arnason, 1969, 1974). The nature of the relationship between cetaceans and artiodactyls was resolved in phylogenetic studies of mitochondrial (mt) cytochrome b (cytb) genes (Irwin and Arnason, 1994; Arnason and Gullberg, 1996) that placed Cetacea within the order Artiodactyla itself as the sister group of the Hippopotamidae (see also Sarich, 1993). The Hippopotamidae/ Cetacea relationship was subsequently supported in studies of nuclear data (Gatesy et al., 1996; Gatesy, 1997) and statistically established in analysis of complete mt genomes (Ursing and Arnason, 1998). The relationship has also been confirmed in analyses of combined nuclear and mt sequences (Gatesy et al., 1999; Cassens et al., 2000) and in studies of short interspersed repetitive elements (SINEs). Artiodactyla and Cetacea are now commonly referred to as Cetartiodactyla.

Previous analyses of the complete cytb gene of more than 30 cetacean species (Arnason and Gullberg, 1996) identified five primary lineages of recent cetaceans, viz., Mysticeti and the four odontocete lineages Physeteridae (sperm whales), Platanistidae (Indian river dolphins), Ziphiidae (beaked whales) and Delphinoidea (iniid river dolphins, porpoises, narwhals and dolphins). However, these studies left unresolved the relationships of the five lineages as well as those between the three delphinoid families Monodontidae (narwhals, belugas), Phocoenidae (porpoises) and Delphinidae (dolphins). Similarly, the relationships between the four mysticete families Balaenidae (right whales), Neobalaenidae (pygmy right whales), Eschrichtiidae (gray whales) and Balaenopteridae (rorquals) were not conclusively resolved in analyses of cytb genes.

Fig. (not shown). Cetartiodactyl relationships and the estimated times of their divergences. The tree was established on the basis of maximum likelihood analysis of the concatenated amino acid (aa) sequences of 12 mt protein-coding genes. Length of alignment 3610 aa. Support values for branches A–H are shown in the insert.
Cetruminantia (branch A) receives moderate support and Cetancodonta (B) strong support. Cetacea (C) splits into monophyletic Mysticeti (baleen whales) and monophyletic Odontoceti (toothed whales). Odontoceti has four basal lineages, Physeteridae (sperm whales: represented by the sperm and pygmy sperm whales), Ziphiidae (beaked whales: bottlenose and Baird’s beaked whales), Platanistidae (Indian river dolphins: Indian river dolphin) and Delphinoidea. Delphinoidea encompasses the families Iniidae (iniid river dolphins: Amazon river dolphin, La Plata dolphin), Monodontidae (narwhals/belugas: narwhal), Phocoenidae (porpoises: harbour porpoise) and Delphinidae (dolphins: white-beaked dolphin). The common odontocete branch and the branches separating the four cetacean lineages are short. These relationships are therefore somewhat unstable (cf. Section 3.1 and Table 1). Iniid river dolphins (F) are solidly nested within the Delphinoidea (E). Thus, traditional river dolphins (Platanistidae + Iniidae) do not form a monophyletic unit. Molecular estimates of divergence times (Sanderson 2002) were based on two calibration points, A/C-60 and O/M-35 (cf. Section 3.2). Due to the short lengths of internal branches, some estimates for these divergences overlap. NJ: neighbor joining; MP: maximum parsimony; LBP: local bootstrap probability; QP: quartet puzzling. The bar shows the number of aa substitutions per site.

The limited molecular resolution among basal cetacean lineages has been known for some time. Studies of hemoglobin and myoglobin (Goodman, 1989; Czelusniak et al., 1990) have either joined Physeteridae and Mysticeti to the exclusion of Delphinoidea (myoglobin data) or Mysticeti and Delphinoidea to the exclusion of Physeteridae (hemoglobin data). Thus, neither of the data sets identified monophyletic Odontoceti by joining the two odontocete lineages (Physeteridae and Delphinoidea) to the exclusion of Mysticeti. A similar instability was recognized and cautioned against in analyses of some mt data, notably, sequences of rRNA genes (Arnason et al., 1993b). The suggestion (Milinkovitch et al., 1993) of a sister group relationship between Physeteridae and the mysticete family Balaenopteridae (rorquals) was based on a myoglobin data set (which joins Physeteridae and Mysticeti to the exclusion of Delphinoidea) that was complemented with partial data of the mt 16S rRNA gene.

The cetancodont divergence times calculated using A/C-60 and O/M-35 as references have been included in Fig. 1. As a result of the short branches separating several cetacean lineages, the estimates of these divergences overlap. The same observation has been made in calculations based on SINE flanking sequences (Nikaido et al., 2001). There is a general consistency between the current and the flanking sequence datings, except for those involving the Balaenopteridae, which are somewhat younger in our analysis than in the SINEs study. The currently estimated age of the divergence between Hippopotamus and Cetacea (c53.5 MYBP) is consistent with the age (>50 MY) of the oldest archaeocete fossils identified so far (Bajpai and Gingerich, 1998). This suggests that the ages allocated to the two references, A/C-60 (the divergence between ruminant artiodactyls and cetancodonts) and O/M-35 (the divergence between odontocetes and mysticetes) are reasonably accurate.

The dating of the divergence between the blue and fin whales is of interest regarding hybridization between closely related mammalian species. Previous molecular analyses (Arnason et al., 1991b; Spilliaert et al., 1991) demonstrated the occurrence of hybridization between these two species. These studies, which were based on three hybrids (one female and two males), showed that either species could be the mother or father in these hybridizations. The two male hybrids had rudimentary testes, whereas the female hybrid was in her second pregnancy. This suggests that the blue and fin whales may be close to the limit for permissible species hybridization among mammals.

The current data set has allowed examination of the coherence between the molecular results and two prevalent morphological hypotheses related to cetacean evolution. The first hypothesis, which in essence originates from Van Valen (1966, 1968), postulates that monophyletic Artiodactyla and monophyletic Cetacea evolved separately from the extinct Palaeocene group Mesonychia. This hypothesis was recently reinforced in a morphological study (Thewissen et al., 2001) that included mesonychians, two archaeocete taxa (Ambuloocetus and Pakicetus) and some extant and fossil artiodactyls. The study of Thewissen et al. (2001) showed a sister group relationship between monophyletic Artiodactyla and monophyletic Cetacea, with Mesonychia as the basal sister group of Artiodactyla/Cetacea, a conclusion consistent with the palaeontological age of Mesonychia relative to that of Artiodactyla and Cetacea. The second hypothesis favours a sister group relationship between Mesonychia and Cetacea with the Mesonychia/Cetacea clade as the sister group of monophyletic Artiodactyla (O’Leary and Geisler, 1999; see also Gatesy and O’Leary, 2001).

Although the position of Mesonychia differs in the two morphological hypotheses, both correspond to a sister group relationship between Cetacea and monophyletic Artiodactyla among extant cetartiodactyls. Thus, both hypotheses can be tested against the current data set. The result of such a test has been included in Table 1, topology (m)(not shown). As evident, both these morphological hypotheses are incongruent with the mitogenomic findings.

Morphological studies have not provided an answer to the question whether mysticetes and odontocetes had separate origins among the archaeocetes (Fordyce and de Muizon, 2001). However, the long common cetacean branch and the short branches separating the five extant cetacean lineages strongly suggest an origin of modern cetaceans from the same archaeocete group (probably the Dorudontidae).

The limbs of Ambulocetus constitute somewhat of an evolutionary enigma. As evident in Thewissen et al.’s (1994) paper, Ambulocetus has very large hind limbs compared to its forelimbs, a difference that is less pronounced in later silhouette drawings of the animal. It is nevertheless evident that evolution from the powerful hindlimbs of Ambulocetus to their rudimentation in archaeocetes constitutes a remarkable morphological reversal if Ambulocetus is connected to the cetacean branch after the separation of the hippopotamid and cetacean lineages.

For natural reasons, systematic schemes have traditionally been based on external morphological characteristics. The rates of morphological and molecular evolution are rarely (if ever) strictly correlated, however, and this may give rise to inconsistency between traditional systematics and molecular findings. The emerging consensus that the order Cetacea resides within another traditional order, Artiodactyla, makes apparent the incongruity in cetartiodactyl nomenclature (Graur and Higgins, 1994). In this instance, a possible solution for maintaining reasonable consistency between nomenclature and phylogeny would be to recognize Cetartiodactyla as an order with three suborders: Suina, Tylopoda and Cetruminantia. According to such a scheme, Cetacea would (together with the Hippopotamidae) constitute a parvorder within the infraorder Cetancodonta.

Cytochrome b and Bayesian inference of whale phylogeny

Laura May-Collado, Ingi Agnarsson
Molecular Phylogenetics and Evolution 38 (2006) 344–354
http://dx.doi.org//10.1016/j.ympev.2005.09.019

In the mid 1990s cytochrome b and other mitochondrial DNA data reinvigorated cetacean phylogenetics by proposing many novel

and provocative hypotheses of cetacean relationships. These results sparked a revision and reanalysis of morphological datasets, and the collection of new nuclear DNA data from numerous loci. Some of the most controversial mitochondrial hypotheses have now become benchmark clades, corroborated with nuclear DNA and morphological data; others have been resolved in favor of more traditional views. That major conflicts in cetacean phylogeny are disappearing is encouraging. However, most recent papers aim specifically to resolve higher-level conflicts by adding characters, at the cost of densely sampling taxa to resolve lower-level relationships. No molecular study to date has included more than 33 cetaceans. More detailed molecular phylogenies will provide better tools for evolutionary studies. Until more genes are available for a high number of taxa, can we rely on readily available single gene mitochondrial data? Here, we estimate the phylogeny of 66 cetacean taxa and 24 outgroups based on Cytb sequences. We judge the reliability of our phylogeny based on the recovery of several deep-level benchmark clades. A Bayesian phylogenetic analysis recovered all benchmark clades and for the Wrst time supported Odontoceti monophyly based exclusively on analysis of a single mitochondrial gene. The results recover the monophyly, with the exception of only one taxa within Cetacea, and the most recently proposed super- and subfamilies. In contrast, parsimony never recovered all benchmark clades and was sensitive to a priori weighting decisions. These results provide the most detailed phylogeny of Cetacea to date and highlight the utility of both Bayesian methodology in general, and of Cytb in cetacean phylogenetics. They furthermore suggest that dense taxon sampling, like dense character sampling, can overcome problems in phylogenetic reconstruction.

Some long standing debates are all but resolved: our understanding of deeper level cetacean phylogeny has grown strong. However, the strong focus of most recent studies, aiming specifically to resolve these higher level conflicts by adding mostly characters rather than taxa, has left our understanding of lower level relationships among whale species lagging behind. Mitogenomic data, for example, is available only for 16 cetacean species, and no molecular study to date has included more than 33 cetaceans. It seems timely to focus on more detailed (genus, and species level) molecular phylogenies. These will provide better tools for detailed evolutionary studies, and are necessary to test existing morphological phylogenetic hypotheses, and current cetacean classification.

We judge the reliability of our phylogeny based on the recovery of the previously mentioned benchmark clades, in addition to the less controversial clades Perissodactyla, Euungulata (sensu Waddell et al., 2001; Perissodactyla+ Cetartiodactyla), Cetacea, and Mysticeti. Because Cytb is thought to be most reliable at lower taxonomic levels (due to high substitution rates), recovering ‘known’ deeper clades gives credibility to these new findings which have not been addressed by studies using few taxa. We compare the performance of Bayesian analyses versus parsimony under four different models, and briefly examine the sensitivity of the results to taxon sampling. We use our results to discuss agreement and remaining conflict in cetacean phylogenetics, and provide comments on current classification.

The Bayesian analysis recovered all seven benchmark clades. Support for five of the benchmark clades is high (100 posterior probabilities) but rather low for Cetancodonta (79) and marginal for the monophyly of Odontoceti. The analysis also recovered all but one family level, and most sub- and superfamily level cetacean taxa. The results broadly corroborate current cetacean classiffcation, while also pointing to some lower-level groups that may need redefinition.

Many recent cetacean phylogenetic studies include relatively few taxa, in part due to a focus on generating more characters to resolve higher level phylogenetics. While addressing crucial questions and providing the backbone for lower level phylogenies, such studies have limited utility for classification, and for comparative evolutionary studies. In some cases sparse taxon sampling may also confound the results. Of course, taxon sampling is usually simply constrained by the availability of character data, but for some reason many studies have opted to include only one, or a few outgroup taxa, even if many are available.

We find that as long as outgroup taxon sampling was extensive, Bayesian analyses of Cytb recovered all the a priori identified benchmark clades. When only a few outgroups were chosen, however, the Bayesian analysis negated Odontoceti monophyly, as have many previous parsimony analyses of mitochondrial DNA. Furthermore, in almost every detailed comparison possible our results mirror the findings O’Leary et al. (2004), the most ‘character-complete’ (but including relatively few cetacean taxa) analysis to date (37,000 characters from morphology, SINE, and 51 gene fragments). This result gives credibility to our findings, including previously untested lower level clades.

Monophyly and placement of Mysticeti (baleen whales).
Monophyly of Odontoceti (toothed whales)
Delphinoids
River Dolphins
Beaked and sperm whales

A major goal of phylogenetics is a phylogeny of life (i.e., many taxa), based on multiple lines of evidence (many characters of many types). However, when phylogenies based on relatively few characters can be judged reliable based on external evidence (taxonomic congruence with other phylogenies using many characters, but few taxa), they seem like very promising and useful ‘first guess’ hypotheses. The evolution of sexual dimorphism, echolocation, social behavior, and whistles and other communicative signals, and major ecological shifts (e.g., transition to fresh water) are among the numerous interesting questions in cetacean biology that this phylogeny can help answer.

Deep-diving sea lions exhibit extreme bradycardia in long duration dives

Birgitte I. McDonald1, and Paul J. Ponganis
The Journal of Experimental Biology (2014) 217, 1525-1534 http://dx.doi.org:/10.1242/jeb.098558

Heart rate and peripheral blood flow distribution are the primary determinants of the rate and pattern of oxygen store utilization and ultimately breath-hold duration in marine endotherms. Despite this, little is known about how otariids (sea lions and fur seals) regulate heart rate (fH) while diving. We investigated dive fH in five adult female California sea lions (Zalophus californianus) during foraging trips by instrumenting them with digital electrocardiogram (ECG) loggers and time depth recorders. In all dives, dive fH (number of beats/duration; 50±9 beats min⁻¹) decreased compared with surface rates (113±5 beats min⁻¹), with all dives exhibiting an instantaneous fH below resting (<54 beats min⁻¹) at some point during the dive. Both dive fH and minimum instantaneous fH significantly decreased with increasing dive duration. Typical instantaneous fH profiles of deep dives (>100 m) consisted of:

(1) an initial rapid decline in fH resulting in the lowest instantaneous fH of the dive at the end of descent, often below 10 beats min⁻¹ in dives longer than 6 min in duration;
(2) a slight increase in fH to ~10–40 beats min⁻¹ during the bottom portion of the dive; and
(3) a gradual increase in fH during ascent with a rapid increase prior to surfacing.

Thus, fH regulation in deep-diving sea lions is not simply a progressive bradycardia. Extreme bradycardia and the presumed associated reductions in pulmonary and peripheral blood flow during late descent of deep dives should

(a) contribute to preservation of the lung oxygen store,
(b) increase dependence of muscle on the myoglobin-bound oxygen store,
(c) conserve the blood oxygen store and
(d) help limit the absorption of nitrogen at depth.

This fH profile during deep dives of sea lions may be characteristic of deep-diving marine endotherms that dive on inspiration as similar fH profiles have been recently documented in the emperor penguin, another deep diver that dives on inspiration.

The resting ƒH measured in this study (54±6 beats min⁻¹) was lower than predicted for an animal of similar size (~80 beats min⁻¹ for an 80 kg mammal). In part, this may be due to the fact that the sea lions were probably sleeping. The resting ƒH in our study was also lower than previous measurements in captive juvenile California sea lions (87±17 beats min⁻¹, average mass 30 kg) and wild Antarctic fur seals (78±5 beats min⁻¹, body mass 30–50 kg). However, we found a significant negative relationship between mass and resting ƒH even with our small sample size of five sea lions (resting ƒH = –0.58 Mb +100.26, r²=0.81, F_1,3=12.37, P=0.039). For a 30 kg sea lion, this equation predicts a resting ƒH of 83 beats min⁻¹, which is similar to what was measured previously in juvenile sea lions, suggesting this equation may be useful in estimating resting ƒH in sea lions.

The sea lions exhibited a distinct sinus arrhythmia fluctuating between a minimum of 42±9 and a maximum of 87±12 beats min⁻¹, comparable to the sinus arrhythmias described in other diving birds and mammals, including sea lions. The minimum instantaneous ƒH during the sinus arrhythmia was similar to the mean minimum ƒH in dives less than 3 min (37±7 beats min⁻¹), indicating that in dives less than 3 min (estimated cADL), ƒH only decreased to levels observed during exhalation at rest. This is consistent with observations in emperor penguins and elephant seals, where it was proposed that in dives shorter than the aerobic dive limit (ADL) the reduction in ƒH is regulated by a mechanism of cardiorespiratory control similar to that governing the respiratory sinus arrhythmia, with a further reduction only occurring in dives longer than the ADL.

Fig. 3. (not shown) Instantaneous fH and dive depth profiles of a California sea lion (CSL12_2). Data are from (A) a short, shallow dive (1.3 min, 45 m), (B) a mid-duration dive (4.8 min, 239 m) and (C) a long-duration dive (8.5 min, 305 m). Minimum instantaneous fH reached 37 beats min⁻¹ in the short dive
(A) 19 beats min⁻¹ in the mid-duration dive
(B) and 7 beats min⁻¹ in the long duration dive
(C) Prominent features typical of mid- and long-duration dives include

a surface interval tachycardia (pre- and post-dive);
a steady rapid decrease in fH during initial descent;
a gradual decline in fH towards the end of descent with the lowest fH of the dive at the end of descent;
a slight increase and sometimes variable fH during the bottom portion of the dive; and
a slow increase in fH during ascent,
often ending in a rapid increase just before surfacing.

We obtained the first diving ƒH data from wild sea lions on natural foraging trips, demonstrating how they regulate ƒH over a range of dive durations. Sea lions always decreased dive ƒH from surface ƒH values; however, individual sea lions exhibited different dive ƒH, accounting for a significant amount of the variation in the relationship between dive duration and ƒH (intra-individual correlation: 75–81%)). The individual differences in dive ƒH exhibited in this study suggest that different dive capacities of individual sea lions may partially account for the range of dive strategies exhibited in a previous study (Villegas-Amtmann et al., 2011). Despite the individual differences in ƒH, the pattern of the dive ƒH response was similar in all the sea lions. As predicted, sea lions only consistently displayed a true bradycardia on mid- to long- duration dives (>4 min) (Fig. 5A). Additionally, as seen in freely diving phocids, dive ƒH and minimum ƒH were negatively related to dive duration, with the longest duration dives having the lowest dive ƒH and displaying the most intense bradycardia, often below 10 beats min⁻¹ (Fig. 5A,B).

Profiles of mean fH at 10 s intervals of dives

Fig 4. Profiles of mean fH at 10 s intervals of dives for (A) six duration categories and (B) five depth categories. Standard error bars are shown. Data were pooled from 461 dives performed by five sea lions. The number of dives in each category and the number of sea lions performing the dives in each category are provided in the keys.

The mild bradycardia and the dive ƒH profiles observed in the shorter duration dives (<3 min) were similar to those observed in trained juvenile California sea lions and adult Stellar sea lions, but much more intense than ƒH observed in freely diving Antarctic fur seals. Surprisingly, although dive ƒH of trained Steller sea lions was similar, Steller sea lions regularly exhibited lower minimum ƒH, with minimum ƒH almost always less than 20 beats min⁻¹ in dives less than 2 min in duration. In the wild, California sea lions rarely exhibited a minimum ƒH less than 20 beats min⁻¹ in similar duration dives (Fig. 5B), suggesting greater blood oxygen transport during these natural short-duration dives.

Fig. 5. (not shown) fH decreases with increasing dive duration. Dive duration versus (A) dive fH (total number of beats/dive duration), (B) minimum instantaneous fH and (C) bottom fH (total beats at bottom of dive/bottom time) for California sea lions (461 dives from five sea lions).

Although California sea lions are not usually considered exceptional divers, they exhibited extreme bradycardia, comparable to that of the best diving phocids, during their deep dives. In dives greater than 6 min in duration, minimum ƒH was usually less than 10 beats min⁻¹ and sometimes as low as 6 beats mins⁻¹ (Fig. 5B), which is similar to extreme divers such as emperor penguins (3 beats min−1), elephant seals (3 beats min⁻¹), grey seals (2 beats min−1) and Weddell seals (<10 beats min⁻¹), and even as low as what was observed in forced submersion studies. Thus, similar to phocids, the extreme bradycardia exhibited during forced submersions is also a routine component of the sea lion’s physiological repertoire, allowing them to perform long-duration dives.

While the degree of bradycardia observed in long dives of California sea lions was similar to the extreme bradycardia observed in phocids, the ƒH profiles were quite different. In general, phocid ƒH decreases abruptly upon submergence. The intensity of the initial phocid bradycardia either remains relatively stable or intensifies as the dive progresses, and does not start to increase until the seal begins its ascent. In contrast, the ƒH profiles of sea lions were more complex, showing a more gradual decrease during descent, with the minimum ƒH of the dive usually towards the end of descent (Figs 3, 6). There was often a slight increase in ƒH during the bottom portion of the dive, and as soon as the sea lions started to ascend, the ƒH slowly started to increase, often becoming irregular during the middle of ascent, before increasing rapidly as the sea lion approached the surface.

Fig. 6. (not shown) Instantaneous fH and dive depth profiles of the longest dive (10.0 min, 385 m) from a California sea lion (CSL12_1). During this dive, instantaneous fH reached 7 beats min⁻¹ and was less than 20 beats min⁻¹ for over 5.5 min. Post-dive fH was high in the first 0.5–1 min after surfacing, but then declined to ~100 beats min⁻¹ towards the end of the surface interval.

Implications for pulmonary gas exchange

The moderate dive ƒH in short, shallow dives compared with the much slower ƒH of deep long-duration dives suggests more pulmonary blood flow and greater potential for reliance on lung O2. Most of these dives were to depths of less than 100 m (well below the estimated depth of lung collapse near 200 m), so maintenance of a moderate ƒH during these dives may allow sea lions to maximise use of the potentially significant lung O2 stores (~16% of total body O2 stores) throughout the dive. This is supported by venous blood O2 profiles, where, occasionally, there was no decrease in venous blood O2 between the beginning and end of the dive; this can only occur if pulmonary gas exchange continues throughout the dive. Greater utilization of the lung O2 store in sea lions is consistent with higher dive ƒH in other species that both dive on inspiration and typically perform shallow dives (dolphins, porpoises, some penguin species), and in deeper diving species when they perform shallow dives (emperor penguins).

In deeper dives of sea lions, although ƒH was lower and bradycardia more extreme, the diving ƒH profiles suggest that pulmonary gas exchange is also important. In long-duration dives, even though ƒH started to decrease upon or shortly after submergence, the decrease was not as abrupt as in phocids. Additionally, in long deep dives, despite having overall low dive ƒH, there were more heart beats before resting ƒH was reached compared with short, shallow dives. In dives less than 3 min in duration, there were ~10–15 beats until instantaneous ƒH reached resting values. In longer duration dives (>3 min), there were usually ~30–40 beats before instantaneous ƒH reached resting values. We suggest the greater number of heart beats early in these deeper dives enables more gas exchange and blood O2 uptake at shallow depths, thus allowing utilisation of the postulated larger respiratory O2 stores in deeper dives The less abrupt decline in ƒH we observed in sea lions is similar to the more gradual declines documented in emperor penguins and porpoises, where it has also been proposed that the gradual decrease in ƒH allows them to maximise pulmonary gas exchange at shallower depths. However, as sea lions swam deeper, ƒH decreased further (Figs 3, 6), and by 200 m depth (the approximate depth of lung collapse, instantaneous ƒH was 14 beats min⁻¹. Such an extreme decline in ƒH in conjunction with increased pulmonary shunting due to lung compression at greater depths will result in minimization of both O2 and N2 uptake by blood, even before the depth of full lung collapse (100% pulmonary shunt) is reached.

Implications for blood flow

ƒH is often used as a proxy to estimate blood flow and perfusion during diving because of the relative ease of its measurement. This is based on the assumption that stroke volume does not change during diving in sea lions, and, hence, changes in ƒH directly reflect changes in cardiac output. As breath-hold divers maintain arterial pressure while diving, changes in cardiac output should be associated with changes in peripheral vascular resistance and changes in blood flow to tissues. In Weddell seals, a decrease in cardiac output of ~85% during forced submersions resulted in an 80–100% decrease in tissue perfusion in all tissues excluding the brain, adrenal glands and lung. Sea lions exhibited extremely low instantaneous ƒH values that often remained low for significant portions of the dive (Figs 4, 6), suggesting severe decreases in tissue perfusion in dives greater than 5 min in duration. In almost all dives greater than 6 min in duration, instantaneous ƒH reached 10 beats min⁻¹, and stayed below 20 beats min⁻¹ for more than a minute. At a ƒH of 20 beats min⁻¹, cardiac output will be ~36% of resting cardiac output and only about 18% of average surface cardiac output. At these levels of cardiac suppression, most of this flow should be directed towards the brain and heart.

Conclusions

We successfully obtained diving ƒH profiles from a deep-diving otariid during natural foraging trips. We found that

(1) ƒH decreases during all dives, but true and more intense bradycardia only occurred in longer duration dives and
(2) in the longest duration dives, ƒH and presumed cardiac output were as low as 20% of resting values.

We conclude that, although initial high ƒH promotes gas exchange early in deep dives, the extremely low ƒH in late descent of deep dives (a) preserves lung O2, (b) conserves blood O2, (c) increases the dependence of muscle on myoglobin-bound O2 and (d) limits N2 absorption at depth. This ƒH profile, especially during the late descent/early bottom phase of deep dives is similar to that of deep-diving emperor penguins, and may be characteristic of deep diving endotherms that dive on inspiration.

Dive duration was the fixed effect in all models, and to account for the lack of independence caused by having many dives from the same individual, individual (sea lion ID) was included as a random effect. Covariance and random effect structures of the full models were evaluated using Akaike’s information criterion (AIC) and examination of residual plots. AICs from all the tested models are presented with the best model in bold.

Additionally, dives were classified as short-duration (less than 3 min, minimum cADL), mid-duration (3–5 min, range of cADLs) or long-duration (>5 min) dives. Differences in pre-dive ƒH, dive ƒH, minimum ƒH, post-dive ƒH, and heart beats to resting between the categories were investigated using mixed effects ANOVA, followed by post hoc Tukey tests. In all models, dive duration category was the fixed effect and individual (sea lion ID) was included as a random effect. Model fit was accessed by examination of the residuals. All means are expressed ±s.d. and results of the Tukey tests were considered significant at P<0.05. Statistical analysis was performed in R.

Investigating Annual Diving Behaviour by Hooded Seals (Cystophora cristata) within the Northwest Atlantic Ocean

Julie M. Andersen, Mette Skern-Mauritzen, Lars Boehme
PLoS ONE 8(11): e80438. http://dx.doi.org:/10.1371/journal.pone.0080438

With the exception of relatively brief periods when they reproduce and molt, hooded seals, Cystophora cristata, spend most of the year in the open ocean where they undergo feeding migrations to either recover or prepare for the next fasting period. Valuable insights into habitat use and diving behavior during these periods have been obtained by attaching Satellite Relay Data Loggers (SRDLs) to 51 Northwest (NW) Atlantic hooded seals (33 females and 18 males) during icebound fasting periods (200422008). Using General Additive Models (GAMs) we describe habitat use in terms of First Passage Time (FPT) and analyze how bathymetry, seasonality and FPT influence the hooded seals’ diving behavior described by maximum dive depth, dive duration and surface duration. Adult NW Atlantic hooded seals exhibit a change in diving activity in areas where they spend .20 h by increasing maximum dive depth, dive duration and surface duration, indicating a restricted search behavior. We found that male and female hooded seals are spatially segregated and that diving behavior varies between sexes in relation to habitat properties and seasonality. Migration periods are described by increased dive duration for both sexes with a peak in May, October and January. Males demonstrated an increase in dive depth and dive duration towards May (post-breeding/pre-molt) and August–October (post-molt/pre-breeding) but did not show any pronounced increase in surface duration. Females dived deepest and had the highest surface duration between December and January (post-molt/pre-breeding). Our results suggest that the smaller females may have a greater need to recover from dives than that of the larger males. Horizontal segregation could have evolved as a result of a resource partitioning strategy to avoid sexual competition or that the energy requirements of males and females are different due to different energy expenditure during fasting periods.

Novel locomotor muscle design in extreme deep-diving whales

P. Velten, R. M. Dillaman, S. T. Kinsey, W. A. McLellan and D. A. Pabst
The Journal of Experimental Biology 216, 1862-1871
http://dx.doi.org:/10.1242/jeb.081323

Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they

(1) shared muscle design features with other deep divers and
(2) performed dives within their calculated ADLs.

Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinterʼs fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris

Fig. Serial cross-sections of the m. longissimus dorsi of Mesoplodon densirostris (A–D) and Globicephala macrorhynchus (E–H). Scale bars, 50μm. Muscle sections stained for the alkaline (A,E) and acidic (B,F) preincubations of myosin ATPase were used to distinguish Type I and II fibers. Muscle sections stained for succinate dehydrogenase (C,G) and α-glycerophosphate dehydrogenase (D,H) were used to distinguish glycolytic (gl), oxidative (o) and intermediate (i) fibers.

Previous studies of the locomotor muscles of deep-diving marine mammals have demonstrated that these species share a suite of adaptations that increase onboard oxygen stores while slowing the rate at which these stores are utilized, thus extending ADL. Their locomotor muscles display elevated myoglobin concentrations and are composed predominantly of large Type I fibers. Vmt are also lower in deep divers than in shallow divers or athletic terrestrial species. The results of this study indicate that beaked whales and short-finned pilot whales do not uniformly display these characteristics and that each possesses a novel fiber profile compared with those of other deep divers.

The phylogeny of Cetartiodactyla: The importance of dense taxon sampling, missing data, and the remarkable promise of cytochrome b to provide reliable species-level phylogenies

Ingi Agnarsson, Laura J. May-Collado
Molecular Phylogenetics and Evolution 48 (2008) 964–985
http://dx.doi.org:/10.1016/j.ympev.2008.05.046

We perform Bayesian phylogenetic analyses on cytochrome b sequences from 264 of the 290 extant cetartiodactyl mammals (whales plus even-toed ungulates) and two recently extinct species, the ‘Mouse Goat’ and the ‘Irish Elk’. Previous primary analyses have included only a small portion of the species diversity within Cetartiodactyla, while a complete supertree analysis lacks resolution and branch lengths limiting its utility for comparative studies. The benefits of using a single-gene approach include rapid phylogenetic estimates for a large number of species. However, single-gene phylogenies often differ dramatically from studies involving multiple datasets suggesting that they often are unreliable. However, based on recovery of benchmark clades—clades supported in prior studies based on multiple independent datasets—and recovery of undisputed traditional taxonomic groups, Cytb performs extraordinarily well in resolving cetartiodactyl phylogeny when taxon sampling is dense. Missing data, however, (taxa with partial sequences) can compromise phylogenetic accuracy, suggesting a tradeoff between the benefits of adding taxa and introducing question marks. In the full data, a few species with a short sequences appear misplaced, however, sequence length alone seems a poor predictor of this phenomenon as other taxa.

The mammalian superorder Cetartiodactyla (whales and eventoed ungulates) contains nearly 300 species including many of immense commercial importance (cow, pig, and sheep) and of conservation interest and aesthetic value (antelopes, deer, giraffe, dolphins, and whales) (MacDonald, 2006). Certain members of this superorder count among the best studied organisms on earth, whether speaking morphologically, behaviorally, physiologically or genetically. Understanding the interrelationships among cetartiodactyl species, therefore, is of obvious importance with equally short sequences were not conspicuously misplaced. Although we recommend awaiting a better supported phylogeny based on more character data to reconsider classification and taxonomy within Cetartiodactyla, the new phylogenetic hypotheses provided here represent the currently best available tool for comparative species-level studies within this group. Cytb has been sequenced for a large percentage of mammals and appears to be a reliable phylogenetic marker as long as taxon sampling is dense. Therefore, an opportunity exists now to reconstruct detailed phylogenies of most of the major mammalian clades to rapidly provide much needed tools for species-level comparative studies.

Our results support the following relationship among the four major cetartiodactylan lineages (((Tylopoda ((Cetancodonta (Ruminantia + Suina))), with variable support. This arrangement has not been suggested previously, to our knowledge (see review in O’Leary and Gatesy, 2008 and discussion).

Relationships among clades within Cetancodonta are identical to those found by May-Collado and Agnarsson (2006).

Within Ruminantia all our analyzes suggest the following relationships among families: (((((Tragulidae((((Antilocapridae(((Giraffidae(( Cervidae(Moschidae + Bovidae))))) with relatively high support, supporting the subdivision of Ruminantia into Tragulina and Pecora.
In the rare cases where our results are inconsistent with benchmark clades, ad hoc explanations seem reasonable. The placement of M. meminna (Tragulidae) within Bovidae is likely an artifact of missing data, although remarkably it is the only conspicuous misplacement of a species across the whole phylogeny at the family level (while three species appear to be misplaced at the subfamily level within Cervidae in the full analysis, see Fig. 5a). This is supported by the fact that the placement of Moschiola receives low support, and the removal of Moschiola prior to analysis increases dramatically the support for clades close to where it nested (not shown, analysis available from authors), suggesting it had a tendency to ‘jump around’. Two other possibilities cannot be ruled out, however. One, that possibly the available sequence in Genbank may be mislabeled. And second, it should be kept in mind that the validity of Tragulidae has never been tested with molecular data including more than two species.

Oxygen and carbon dioxide fluctuations in burrows of subterranean blind mole rats indicate tolerance to hypoxic–hypercapnic stresses

Imad Shams, Aaron Avivi, Eviatar Nevo
Comparative Biochemistry and Physiology, Part A 142 (2005) 376 – 382
http://dx.doi.org:/10.1016/j.cbpa.2005.09.003

The composition of oxygen (O2), carbon dioxide (CO2), and soil humidity in the underground burrows from three species of the Israeli subterranean mole rat Spalax ehrenbergi superspecies were studied in their natural habitat. Two geographically close populations of each species from contrasting soil types were probed. Maximal CO2 levels (6.1%) and minimal O2 levels (7.2%) were recorded in northern Israel in the breeding mounds of S. carmeli in a flooded, poor drained field of heavy clay soil with very high volumetric water content. The patterns of gas fluctuations during the measurement period among the different Spalax species studied were similar. The more significant differentiation in gas levels was not among species, but between neighboring populations inhabiting heavy soils or light soils: O2 was lower and CO2 was higher in the heavy soils (clay and basaltic) compared to the relatively light soils (terra rossa and rendzina). The extreme values of gas concentration, which occurred during the rainy season, seemed to fluctuate with partial flooding of the tunnels, animal digging activity, and over-crowded breeding mounds inhabited by a nursing female and her offspring. The gas composition and soil water content in neighboring sites with different soil types indicated large differences in the levels of hypoxic–hypercapnic stress in different populations of the same species. A growing number of genes associated with hypoxic stress have been shown to exhibit structural and functional differences between the subterranean Spalax and the aboveground rat (Rattus norvegicus), probably reflecting the molecular adaptations that Spalax went through during 40 million years of evolution to survive efficiently in the severe fluctuations in gas composition in the underground habitat.

map of the studied sites

Schematic map of the studied sites: S. galili (2n =52): 1— Rehania (chalk); 2— Dalton (basaltic); S. golani (2n =54): 3— Majdal Shams (terra tossa); 4—Masa’ada (basaltic soils); S. carmeli (2n =58): 5— Al-Maker (heavy clay); 6— Muhraqa (terra rossa).

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli

Comparison of gas composition (O2 and CO2) and water content between light and heavy soils inhabited by S. carmeli, Al-Maker (heavy soil) and Muhraqa (light soil). AverageTSD of measurements in the burrows of approximately 10 animals at a given date is presented. **p <0.01, T-test and Mann– Whitney test).

Subterranean mammals, which live in closed underground burrow systems, experience an atmosphere that is different from the atmosphere above-ground. Gas exchange between these two atmospheres depends on diffusion through the soil, which in turn, depends on soil particle size, water content, and burrow depth. Heavy soils (clay and basaltic), hold water and have little air space for gas diffusion. A large deviation from external gas composition is found in the burrows of Spalax living in these soil types. The maximal measured concentration of CO2 was 6.1% in Spalax breeding mounds, which is one of the highest concentrations among studied mammals in natural conditions. At the same time 7.2% O2 was measured in water saturated heavy clay soil

seasonal variation from August to March in mean O2, CO2, and soil water content

Example of seasonal variation from August to March in mean O2, CO2, and soil water content (VWC) in the Al-Maker population (2n =58, heavy soil). Values are presented as mean TSD.

In this study new data were presented for a wild mammal that survives in an extreme hypoxic–hypercapnic environment. Interestingly, the very low concentrations of O2 experienced by Spalax are correlated with the expression pattern of hypoxia related genes. So far, we have shown higher and longer-term mRNA expression of erythropoietin, the main factor that regulates the level of circulating red blood cells, in subterranean Spalax compared to the above-ground rat in response to hypoxic stress, as well as differences in the response of erythropoietin to hypoxia in different populations of Spalax experiencing different hypoxic stress in nature. We also demonstrated that erythropoietin pattern of expression is different in Spalax than in Rattus throughout development, a pattern suggesting more efficient hypoxic tolerance in Spalax starting as early as in the embryonic stages. Furthermore, vascular endothelial growth factor (VEGF), which is a critical angiogenic factor that responds to hypoxia, is constitutively expressed at maximal levels in Spalax muscles, the most energy consuming tissue during digging. This level is 1.6-fold higher than in Rattus muscles and is correlated with significantly higher blood vessel concentration in the Spalax muscles compared to the Rattus muscles. Likewise, myoglobin the globin involved in oxygen homeostasis in skeletal muscles, exhibits different expression pattern under normoxia and in response to hypoxia in Spalax muscles compared to rat muscles as well as between different populations of Spalax exposed to different hypoxic stress in nature (unpublished results). Similarly, neuroglobin, a brain-specific globin involved in reversible oxygen binding, i.e., presumably in cellular homeostasis, is expressed differently in the Spalax brain compared to Rattus brain. Like erythropoietin and myoglobin also neuroglobin is expressed differently in Spalax populations experiencing different oxygen supply (unpublished results). Furthermore, Spalax p53 harbors two amino acid substitutions in its binding domain, which are identical to mutations found in p53 of human cancer cells. These substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53 and favor activation of DNA repair genes over apoptotic genes. The study of specific tumoral variants indicates that such preference of growth arrest over apoptosis possibly results as a response to the hypoxic environmental stress known in tumors. Differences in the structure of other molecules related to homeostasis, namely, hemoglobin, haptoglobin (Nevo, 1999), and cytoglobin (unpublished) were also observed in Spalax.

Stress, adaptation, and speciation in the evolution of the blind mole rat, Spalax, in Israel

Eviatar Nevo
Molecular Phylogenetics and Evolution 66 (2013) 515–525
http://dx.doi.org/10.1016/j.ympev.2012.09.008

Environmental stress played a major role in the evolution of the blind mole rat superspecies Spalax ehrenbergi, affecting its adaptive evolution and ecological speciation underground. Spalax is safeguarded all of its life underground from aboveground climatic fluctuations and predators. However, it encounters multiple stresses in its underground burrows including darkness, energetics, hypoxia, hypercapnia, food scarcity, and pathogenicity. Consequently, it evolved adaptive genomic, proteomic, and phenomic complexes to cope with those stresses. Here I describe some of these adaptive complexes, and their theoretical and applied perspectives. Spalax mosaic molecular and organismal evolution involves reductions or regressions coupled with expansions or progressions caused by evolutionary tinkering and natural genetic engineering. Speciation of Spalax in Israel occurred in the Pleistocene, during the last 2.00–2.35 Mya, generating four species associated intimately with four climatic regimes with increasing aridity stress southwards and eastwards representing an ecological speciational adaptive trend: (Spalax golani, 2n = 54?S. galili, 2n = 52?S. carmeli, 2n = 58?S. judaei, 2n = 60). Darwinian ecological speciation occurred gradually with relatively little genetic change by Robertsonian chromosomal and genic mutations. Spalax genome sequencing has just been completed. It involves multiple adaptive complexes to life underground and is an evolutionary model to a few hundred underground mammals. It involves great promise in the future for medicine, space flight, and deep-sea diving.

Stress is a major driving force of evolution (Parsons, 2005; Nevo, 2011). Parsons defined stress as the ‘‘environmental factor causing potential injurious changes to biological systems with a potential for impacts on evolutionary processes’’. The global climatic transition from the middle Eocene to the early Oligocene (45–35 Ma = Million years ago) led to extensive convergent evolution underground of small subterranean mammals across the planet (Nevo, 1999; Lacey et al., 2000; Bennett and Faulkes, 2000; Begall et al., 2007). The subterranean ecotope provided small mammals with shelter from predators and extreme aboveground climatic stressful fluctuations of temperature and humidity. However, they had to evolve genomic adaptive complexes for the immense underground stresses of darkness, energy for burrowing in solid soil, low productivity and food scarcity, hypoxia, hypercapnia, and high infectivity. These stresses have been described in Nevo (1999, 2011) and Nevo et al. (2001); and Nevo list of Spalax publication at http://evolution.haifa.ac.il with many cited references relevant to these stresses).

blind subterranean mole rat of the Spalax ehrenbergi superspecies

The blind subterranean mole rat of the Spalax ehrenbergi superspecies in Israel. An extreme example of adaptation to life underground

Circadian rhythm and genes

adaptive circadian genes. We identified the circadian rhythm of Spalax
(Nevo et al., 1982) and described, cloned, sequenced, and expressed several circadian genes in Spalax. These include Clock, MOP3, three Period (Per), and cryptochromes (Avivi et al., 2001, 2002, 2003). The Spalax circadian genes are differentially conserved, yet characterized by a significant number of amino acid substitutions. The glutamine-rich area of Clock, which is assumed to function in circadian rhythmicity, is expanded in Spalax compared with that of mice and humans and is different in amino acid composition from that of rats. All three Per genes of Spalax oscillate with a periodicity of 24 h in the suprachaismatic nucleus, eye, and Harderian gland and are expressed in peripheral organs. Per genes are involved in clock resetting. Spalax Per 3 is unique in mammals though its function is still unresolved. The Spalax Per genes contribute to the unique adaptive circadian rhythm to life underground. The cryptochrome (Cry) genes, found in animals and plants, act both as photoreceptors and as ingredients of the negative feedback mechanism of the biological Clock. The CRY 1 protein is significantly closer to the human homolog than to that of mice, as was also shown in parts of the immunogenetic system. Both Cry 1 and Cry 2 mRNAs were found in the SCN, eye, harderian gland, and in peripheral tissues. Remarkably, the distinctly hypertrophied harderian gland is central in Spalax’s unique underground circadian rhythmicity (Pevet et al., 1984).

Spalax eye mosaic evolution
Gene expression in the eye of Spalax
Brain evolution in Spalax to underground stresses
Spalax: four species in Israel

The morphological, physiological, and behavioral Spalax eye patterns are underlain by gene expression representing regressive and progressive associated transcripts. Regressive transcripts involve B-2 microglobulin, transketolase, four keratins, alpha enolase, and different heat shock proteins. Several proteins may be involved in eye degeneration. These include heat shock protein 90alpha (hsp90alpha), found also in the blind fish Astyanax mexicanus, two transcripts of programmed cell death proteins, oculospanin, and peripherin 2, both belonging to the Tetraspanin family, in which 60 different mutations cause eye degeneration in humans. Several progressive transcripts in the Spalax eye are found in the retina of many mammals involving gluthatione, peroxidase 4, B spectrin, and Ankyrin; the last two characterize rod cells in the retina. Some transcripts are involved in metabolic processing of retinal, a vertebrate key component in phototransduction, and a relative of vitamin A.

cross section of the developing eye of the mole rat

Light micrographs showing cross section of the developing eye of the mole rat Spalax ehrenbergi. (A) Optic cup and lens vesicle initially develop normally (x100). (B) Eye at a later embryonic stage. Note appearance of iris-ciliary body rudiment (arrows), and development of the lens nucleus (L). ON, optic nerve (x100). (C) Eye at a still later fetal stage. Note massive growth of the iris-ciliary body complex colobomatous opening (arrow) (x100). (D) Early postnatal stage. The iris-ciliary body complex completely fills the chamber. The lens is vascularized and vacuolated (x100). (E) Adult eye. Eyelids are completely closed and pupil is absent. Note atrophic appearance of the optic disc region (arrow) (x65). (F) Higher magnification of the adult retina. The different retinal layers are retained: PE, pigment epithelium: RE, receptor layer; ON, outer nuclear layer: IN, inner nuclear layer; GC, ganglion cell layer (x500) (from Sanyal et al., 1990, Fig. 1).

The brains of subterranean mammals underwent dramatic evolution in accordance with underground stresses for digging and photoperiodic perception associated with vibrational, tactile, vocal, olfactory, and magnetic communication systems replacing sight, as is seen in Spalax. The brain of Spalax is twice as large as that of the laboratory rat of the same body size. The somatosensory region in the isocortex of Spalax is 1.7 times, the thalamic nuclei 1.3 times, and the motor cortex 3.1 times larger than in the sighted laboratory rat Rattus norvegicus matched to body size.

The ecological stress determinant in Spalax brain evolution is highlighted by the four species of the Spalax ehrenbergi superspecies in Israel. They differentiated chromosomally (by means of Robertsonian mutations and fission), allopatrically, and clinally southwards into four species associated with different climatic regimes, following the gradient of increasing aridity stress and decreasing predictability southwards towards the desert: Spalax galili (2n = 52) ->S. golani (2n = 54)->S. carmeli (2n = 58)->S. judaei (2n = 60), and eastwards S. galili ->S. golani (2n = 52–>54) (Fig. 2). This chromosomal speciation trend southwards is associated with the regional aridity stress southwards (and eastwards) in Israel, budding new species adapted genomically, proteomically, and phenomically (i.e., in morphology, physiology, and behavior) to increasing stresses of higher solar radiation, temperature, and drought southwards (Nevo, 1999; Nevo et al., 2001; Nevo
list of Spalax at http://evolution.haifa.ac.il). A uniquely recent discovery of incipient sympatric ecological speciation at a microscale in Spalax triggered by local stresses occurs within Spalax galili.

retinal input to primary visual structures in Spalax

Relative degree of retinal input to primary visual structures in Spalax, hamster, rat, and Spalacopus cyanus (South American Octodontidae, ‘‘coruro’’). These rodents are of similar body size (120–140 g). B. Relative degree of change in the proportions of retinal input to different primary visual structures in Spalax compared with measures obtained in other rodents. A relative progressive development in Spalax is seen in structures involved in photoperiodic and neuroendocrine functions (SCN, BNST).The main regressive feature is the drastic relative reduction of retinal input to the superior colliculus. The main regressive feature is the drastic reduction of retinal input to the superior colliculus. The relative size of other visual structures in Spalax is modified compared to that of the other species. c. Comparison of the absolute size (volume, mm³ x 10^-4) of visual structures in Spalax and other rodents. The size of the SCN is equivalent in all species. The vLGN and dLGN are reduced by 87–93% in Spalax. The retino-recipient layers of the superior colliculus are reduced by 97%. Abbreviations: SCN: suprachiasmatic nucleus; BNST: bed nucleus of the stria terminalis; dLGN: dorsal lateral geniculate nucleus; SC: superior colliculus [From Cooper et al., 1993 (Fig 3)].

Subterranean life has a high energetic cost if an animal has to burrow in order to obtain its food. For a 150 g Thomomys bottae, burrowing 1 m may be 360–3400 times more expensive energetically than moving the same distance on the surface (Vleck, 1979). Mean rates of oxygen consumption during burrowing at 22 ^oC are from 2.8 to 7.1 times the RMR. Vleck developed a model examining the energetics of foraging by burrowing and found that, in the desert, Thomomys adjusts the burrow segment length to minimize the cost of burrowing. Since burrowing becomes less economic as body size increases, Vleck (1981) predicted that the maximum possible body size that a subterranean mammal can attain depends on a balance between habitat productivity and the cost of burrowing in local soils. Vleck’s cost of burrowing hypothesis has been verified in multiple cases. Heth (1989) demonstrated longer burrows in the rendzina soil and shorter ones in the terra rossa soil, associating lower productivity in the former for Spalax.

Food is a limiting factor for subterranean mammals. The abundance and distribution of food explain some of the ecological, physiological, and behavioral characteristics of subterranean mammals. In a field test of Spalax foraging strategy, we concluded that Spalax was a generalist due to the constraints of the subterranean ecotope. Restricted foraging time primarily during the winter when soil is wet, and the high energetic investment of tunneling to get to food items is significantly reduced than in summertime.
We also identified a decrease in the basic metabolic rate towards the desert, i.e., economizing energetics. The maintenance of adequate O2 transport in a subterranean mammal confronting hypoxia requires adaptation along the O2 transport system, achieved by increasing the flow of O2 in the convection systems (ventilation and perfusion) and by reduction of oxygen pressure (PO2) gradients at the diffusion barriers (lung blood, blood-tissue (Arieli, 1990). The PO2 gradient between blood capillaries and respiring mitochondria capillaries is large, and any adaptation at this level could be significant for O2 transport. Reduction of diffusion distance in a muscle can be achieved, like in Spalax, by increasing the number of capillaries that surround muscle fiber or by reducing fiber areas.

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies

Geographic distribution in Israel of the four chromosomal species belonging to the S. ehrenbergi superspecies that are separated by narrow hybrid zones (2n = 52, 54, 58, and 60, now named as S. galili, S. golani, S. carmeli, and S. judaei, respectively; see Nevo et al., 2001).

Spalacid evolution, based on mtDNA, is driven by climatic oscillations and stresses. The underground ecotope provided subterranean mammals with shelter from extreme climate (temperature and humidity) fluctuations, and predators. However, they had to extensively and intensively adapt to the multiple underground stresses (darkness, energetic, low productivity and
food scarcity, hypoxia, hypercapnia, and high infectivity). All subterranean mammals, including spalacids as an extreme case, share convergent molecular and organismal adaptations to their shared unique underground ecotope. Evolution underground, as exemplified here in spalacids, led to mosaic molecular and organismal evolutionary syndromes to cope with multiple stresses.

Speciation involves all rates – from gradual to rapid. Subterranean mammals, with the spalacid example discussed above, provide uniquely rich evolutionary global tests of speciation and adaptation, convergence, regression, progression, and mosaic evolutionary processes. Adaptation and speciation underground was one of the most dramatic natural experiments verifying Darwinian evolution.

The Spalax genome sequencing has just been completed. It is being analyzed and will soon be published in 2012. This will be a milestone in understanding how numerous mammals across the globe, who found underground shelter from climatic fluctuations and stresses above ground, cope with the new suite of stresses they encountered underground, demanding a new engineering overhaul on all organizational levels, selecting for adaptive complexes to cope with the new underground stresses. The main current and future challenges are to compare and contrast genome sequences and identify the genomic basis of adaptation and speciation.

This global Cenozoic experiment could answer the following open questions: How heterozygous is the whole genome? How prevalent are retrotransposons and what is their functional role? How many genes are involved in the Spalax genome and how are they regulated? What are the genic and regulatory networks resisting the multiple stresses underground? How much of the Spalax genome is conserved and how much is reorganized to cope with the underground stresses? How is the solitary blind mole rat, Spalax, different from the social naked mole rat Heterocephalus? How are the processes of reduction, expansion, and genetic tinkering and engineering reflected across the genome? How effective is copy number variation in regulation? Is there similarity in the transcriptomes of subterranean mammals? How could we harness the rich genome repertoire of Spalax to revolutionize medicine, especially in the realm of hypoxia tolerance and the related major diseases of the western world, e.g., cancer, stroke, and cardiovascular diseases? What is the phylogenetic origin of Spalax? How much of the Spalax genome represents its phylogenetic roots and how much of coding and noncoding genomic regions are shared with other subterranean mammals across the globe in adapting to life underground?

The Atmospheric Environment of the Fossorial Mole Rat (Spalax Ehrenbergi): Effects of Season, Soil Texture, Rain, Temperature and Activity

Arieli
Comp Biochen Physiol. 1978; 63A:569-5151. The fossorial mole rat (Spalax ehrenbergi) may inhabit heavy soil with low gas permeability.
Air composition in burrows in heavy soil deviates from atmospheric air more than that of burrows in light soil.
In winter and spring O2 and CO2 concentrations in breeding mounds were 16.5% O2 and 2.5-3x CO2 and the extreme values measured were 14.0% O2 and 4.8% Cot.
Hypoxia and hypercapnia in the burrow develop shortly after rain and when ambient temperature drops.
Composition of the burrows air is influenced by the solubility of CO2 in soil water and by faster penetration of oxygen than outflowing of CO2.

Hypo-osmotic stress-induced physiological and ion-osmoregulatory responses in European sea bass (Dicentrarchus labrax) are modulated differentially by nutritional status

Amit Kumar Sinha, AF Dasan, R Rasoloniriana, N Pipralia, R Blust, G De Boeck
Comparative Biochemistry and Physiology, Part A 181 (2015) 87–99
http://dx.doi.org/10.1016/j.cbpa.2014.11.024

We investigated the impact of nutritional status on the physiological, metabolic and ion-osmoregulatory performance of European sea bass (Dicentrarchus labrax)when acclimated to seawater (32 ppt), brackishwater (20 and 10 ppt) and hyposaline water (2.5 ppt) for 2 weeks. Following acclimation to different salinities, fish were either fed or fasted (unfed for 14 days). Plasma osmolality, [Na+], [Cl−] and muscle water contentwere severely altered in fasted fish acclimated to 10 and 2.5 ppt in comparison to normal seawater-acclimated fish, suggesting ion regulation and acid–base balance disturbances. In contrast to feed-deprived fish, fed fish were able to avoid osmotic perturbation more effectively. This was accompanied by an increase in Na+/K+-ATPase expression and activity, transitory activation of H+-ATPase (only at 2.5 ppt) and down-regulation of Na+/K+/2Cl− gene expression. Ammonia excretion rate was inhibited to a larger extent in fasted fish acclimated to low salinities while fed fish were able to excrete efficiently. Consequently, the build-up of ammonia in the plasma of fed fish was relatively lower. Energy stores, especially glycogen and lipid, dropped in the fasted fish at low salinities and progression towards the anaerobic metabolic pathway became evident by an increase in plasma lactate level. Overall, the results indicate no osmotic stress in both feeding treatments within the salinity range of 32 to 20 ppt. However, at lower salinities (10–2.5 ppt) feed deprivation tends to reduce physiological, metabolic, ion-osmo-regulatory and molecular compensatory mechanisms and thus limits the fish’s abilities to adapt to a hypo-osmotic environment.

The absence of ion-regulatory suppression in the gills of the aquatic air-breathing fish Trichogaster lalius during oxygen stress

Chun-Yen Huang, Hsueh-Hsi Lin, Cheng-Huang Lin, Hui-Chen Lin
Comparative Biochemistry and Physiology, Part A 179 (2015) 7–16
http://dx.doi.org/10.1016/j.cbpa.2014.08.017

The strategy for most teleost to survive in hypoxic or anoxic conditions is to conserve energy expenditure, which can be achieved by suppressing energy-consuming activities such as ion regulation. However, an air-breathing fish can cope with hypoxic stress using a similar adjustment or by enhancing gas exchange ability, both behaviorally and physiologically. This study examined Trichogaster lalius, an air-breathing fish without apparent gill modification, for their gill ion-regulatory abilities and glycogen utilization under a hypoxic treatment. We recorded air-breathing frequency, branchial morphology, and the expression of ion-regulatory proteins (Na+/K+-ATPase and vacuolar-type H+-ATPase) in the 1st and 4th gills and labyrinth organ (LO), and the expression of glycogen utilization (GP, glycogen phosphorylase protein expression and glycogen content) and other protein responses (catalase, CAT; carbonic anhydrase II, CAII; heat shock protein 70, HSP70; hypoxia-inducible factor-1α, HIF-1α; proliferating cell nuclear antigen, PCNA; superoxidase dismutase, SOD) in the gills of T. lalius after 3 days in hypoxic and restricted conditions. No morphological modification of the 1st and 4th gills was observed. The air breathing behavior of the fish and CAII protein expression both increased under hypoxia. Ion-regulatory abilities were not suppressed in the hypoxic or restricted groups, but glycogen utilization was enhanced within the groups. The expression of HIF-1α, HSP70 and PCNA did not vary among the treatments. Regarding the antioxidant system, decreased CAT enzyme activity was observed among the groups. In conclusion, during hypoxic stress, T. lalius did not significantly reduce energy consumption but enhanced gas exchange ability and glycogen expenditure.

The combined effect of hypoxia and nutritional status on metabolic and ionoregulatory responses of common carp (Cyprinus carpio)

Sofie Moyson, HJ Liew, M Diricx, AK Sinha, R Blusta, G De Boeck
Comparative Biochemistry and Physiology, Part A 179 (2015) 133–143
http://dx.doi.org/10.1016/j.cbpa.2014.09.017

In the present study, the combined effects of hypoxia and nutritional status were examined in common carp (Cyprinus carpio), a relatively hypoxia tolerant cyprinid. Fish were either fed or fasted and were exposed to hypoxia (1.5–1.8mgO2 L−1) at or slightly above their critical oxygen concentration during 1, 3 or 7 days followed by a 7 day recovery period. Ventilation initially increased during hypoxia, but fasted fish had lower ventilation frequencies than fed fish. In fed fish, ventilation returned to control levels during hypoxia, while in fasted fish recovery only occurred after reoxygenation. Due to this, C. carpio managed, at least in part, to maintain aerobic metabolism during hypoxia: muscle and plasma lactate levels remained relatively stable although they tended to be higher in fed fish (despite higher ventilation rates). However, during recovery, compensatory responses differed greatly between both feeding regimes: plasma lactate in fed fish increased with a simultaneous breakdown of liver glycogen indicating increased energy use, while fasted fish seemed to economize energy and recycle decreasing plasma lactate levels into increasing liver glycogen levels. Protein was used under both feeding regimes during hypoxia and subsequent recovery: protein levels reduced mainly in liver for fed fish and in muscle for fasted fish. Overall, nutritional status had a greater impact on energy reserves than the lack of oxygen with a lower hepatosomatic index and lower glycogen stores in fasted fish. Fasted fish transiently increased Na+/K+-ATPase activity under hypoxia, but in general ionoregulatory balance proved to be only slightly disturbed, showing that sufficient energy was left for ion regulation.

The effect of temperature and body size on metabolic scope of activity in juvenile Atlantic cod Gadus morhua L.

Bjørn Tirsgaard, Jane W. Behrens, John F. Steffensen
Comparative Biochemistry and Physiology, Part A 179 (2015) 89–94
http://dx.doi.org/10.1016/j.cbpa.2014.09.033

Changes in ambient temperature affect the physiology and metabolism and thus the distribution of fish. In this study we used intermittent flow respirometry to determine the effect of temperature (2, 5, 10, 15 and 20 °C) and wet body mass (BM) (~30–460 g) on standard metabolic rate (SMR, mg O2 h−1), maximum metabolic rate (MMR, mg O2 h−1) and metabolic scope (MS, mg O2 h−1) of juvenile Atlantic cod. SMR increased with BM irrespectively of temperature, resulting in an average scaling exponent of 0.87 (0.82–0.92). Q10 values were 1.8–2.1 at temperatures between 5 and 15 °C but higher (2.6–4.3) between 2 and 5 °C and lower (1.6–1.4) between 15 and 20 °C in 200 and 450 g cod. MMR increased with temperature in the smallest cod (50 g) but in the larger cod MMR plateaued between 10, 15 and 20 °C. This resulted in a negative correlation between the optimal temperature for MS (T_opt) and BM, T_opt being respectively 14.5, 11.8 and 10.9 °C in a 50, 200 and 450 g cod. Irrespective of BM cold water temperatures resulted in a reduction (30–35%) of MS whereas the reduction of MS at warm temperatures was only evident for larger fish (200 and 450 g), caused by plateauing of MMR at 10 °C and above. Warm temperatures thus seem favorable for smaller (50 g) juvenile cod, but not for larger conspecifics (200 and 450 g).

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Altitude Adaptation

Posted in Amino acids, Biochemical pathways, Biological Networks, Ca2+ triggered activation, Cardiovascular Research, Cell Biology, Curation, Developmental biology, Enzymes and isoenzymes, Fatty acids, Gene Regulation and Evolution, Hematology, Hematopoiesis, Lipid metabolism, Liver & Digestive Diseases Research, Metabolism, Mutant Gene Expression, Myocardial Metabolism, Nitric Oxide in Health and Disease, Proteins, Proteomics, Signaling, Signaling & Cell Circuits, tagged acute high altitude sickness, altitude adaptation, Andeas, cewrebral edema, chronic altitude sickness, cognitive effects, Evolution, hemoglobin gene substitutions, hemoglobins, high elevation plants, Himalayan Plateau, mammals, mountain antelope, Mountain sickness, pulmonary edema, Tibetan goat, Tibetan sheep on February 24, 2015| Leave a Comment »

Altitude Adaptation

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

Land adapted animals depend on respiration for oxygen supply, but have adapted to altitudes that have difference oxygen contents. In this discussion we explore how animals have adapted to oxygen supply in different terrestrial habitats, and also how humans adjust to short term changes in high and extreme altitudes.

High-altitude adaptation is an evolutionary modification in animals, most notably in birds and mammals, by which species are subjected to considerable physiological changes to survive in extremely high mountainous environments. As opposed to short-term adaptation, or more properly acclimatization (which is basically an immediate physiological response to changing environment), the term “high-altitude adaptation” has strictly developed into the description of an irreversible, long-term physiological responses to high-altitude environments, associated with heritable behavioral and genetic changes. Perhaps, the phenomenon is most conspicuous, at least best documented, in human populations such as the Tibetans, the South Americans and the Ethiopians, who live in the otherwise uninhabitable high mountains of the Himalayas, Andes and Ethiopia respectively; and this represents one of the finest examples of natural selection in action.

Oxygen, essential for animal life, is proportionally abundant in the atmosphere with height from the sea level; hence, the highest mountain ranges of the world are considered unsuitable for habitation. Surprisingly, some 140 million people live permanently at high altitudes (>2,500 m) in North, Central and South America, East Africa, and Asia, and flourish very well for millennia in the exceptionally high mountains, without any apparent complications. This has become a recognized instance of the process of Darwinian evolution in humans acting on favorable characters such as enhanced respiratory mechanisms. As a matter of fact, this adaptation is so far the fastest case of evolution in humans that is scientifically documented. Among animals only few mammals (such as yak, ibex, Tibetan gazelle, vicunas, llamas, mountain goats, etc.) and certain birds are known to have completely adapted to high-altitude environments.

These adaptations are an example of convergent evolution, with adaptations occurring simultaneously on three continents. Tibetan humans and Tibetan domestic dogs found the genetic mutation in both species, EPAS1. This mutation has not been seen in Andean humans, showing the effect of a shared environment on evolution.

At elevation higher than 8,000 metres (26,000 ft), which is called the “death zone” in mountaineering, the available oxygen in the air is so low that it is considered insufficient to support life. And higher than 7,600 m is seriously lethal. Yet, there are Tibetans, Ethiopians and Americans who habitually live at places higher than 2,500 m from the sea level. For normal human population, even a brief stay at these places means mountain sickness, which is a syndrome of hypoxia or severe lack of oxygen, with complications such as fatigue, dizziness, breathlessness, headaches, insomnia, malaise, nausea, vomiting, body pain, loss of appetite, ear-ringing, blistering and purpling and of the hands and feet, and dilated veins. Amazingly for the native highlanders, there are no adverse effects; in fact, they are perfectly normal in all respects. Basically, the physiological and genetic adaptations in these people involve massive modification in the oxygen transport system of the blood, especially molecular changes in the structure and functions hemoglobin, a protein for carrying oxygen in the body. This is to compensate for perpetual low oxygen environment. This adaptation is associated with better developmental patterns such as high birth weight, increased lung volumes, increased breathing, and higher resting metabolism.

http://en.wikipedia.org/wiki/High-altitude_adaptation

Acute Mountain Sickness: Pathophysiology, Prevention, and Treatment

Chris Imraya, Alex Wright, Andrew Subudhie,, Robert Roache
Progress in Cardiovascular Diseases 52 (2010) 467–484
http://dx.doi.org:/10.1016/j.pcad.2010.02.003

Barometric pressure falls with increasing altitude and consequently there is a reduction in the partial pressure of oxygen resulting in a hypoxic challenge to any individual ascending to altitude. A spectrum of high altitude illnesses can occur when the hypoxic stress outstrips the subject’s ability to acclimatize. Acute altitude-related problems consist of the common syndrome of acute mountain sickness, which is relatively benign and usually self-limiting, and the rarer, more serious syndromes of high-altitude cerebral edema and high-altitude pulmonary edema. A common feature of acute altitude illness is rapid ascent by otherwise fit individuals to altitudes above 3000 m without sufficient time to acclimatize. The susceptibility of an individual to high altitude syndromes is variable but generally reproducible. Prevention of altitude-related illness by slow ascent is the best approach, but this is not always practical. The immediate management of serious illness requires oxygen (if available) and descent of more than 300 m as soon as possible. In this article, we describe the setting and clinical features of acute mountain sickness and high altitude cerebral edema, including an overview of the known pathophysiology, and explain contemporary practices for both prevention and treatment exploring the comprehensive evidence base for the various interventions.

Acute mountain sickness (AMS) and high-altitude cerebral edema (HACE) strike people who travel too fast to high altitudes that lie beyond their current level of acclimatization. Understanding AMS and HACE is important because AMS can sharply limit recreation and work at high altitude. The syndromes can be identified early and reliably without sophisticated instruments, and when AMS and HACE are recognized early, most cases respond rapidly with complete recovery in a few hours (AMS) to days (HACE).

High-altitude headache (HAH) is the primary symptom of AMS. High-altitude headache in AMS usually occurs with some combination of other symptoms.
These are – insomnia, fatigue (beyond that expected from the day’s activities), dizziness, anorexia, and nausea. The headache often worsens during the night and with exertion. Insomnia is the next most frequent complaint. Poor sleep can occur secondary to periodic breathing, severe headache, dizziness, and shortness of breath, among other causes. Anorexia and nausea are common, with vomiting reported less frequently in trekkers to 4243 m.

AMS is distinguished only by symptoms. The progression of AMS to HACE is marked by altered mental status, including impaired mental capacity, drowsiness, stupor, and ataxia. Coma may develop as soon as 24 hours after the onset of ataxia or change in mental status. The severity of AMS can be scored using the Lake Louise Questionnaire, or the more detailed Environmental Symptoms Questionnaire, or by the use of a simple analogue scale. Today, more than 100 years after the first clear clinical descriptions of AMS and HACE, we have advanced our understanding of the physiology of acclimatization to high altitude, and the pathophysiology of AMS and HACE.

As altitude increases, barometric pressure falls (see Fig ). This fall in barometric pressure causes a corresponding drop in the partial pressure of oxygen (21% of barometric pressure) resulting in hypobaric hypoxia. Hypoxia is the major challenge humans face at high altitude, and the primary cause of AMS and HACE. It follows that oxygen partial pressure is more important than
geographic altitude, as exemplified near the poles where the atmosphere is thinner and, thus, barometric pressure is lower. Lower barometric pressure at the poles can result in oxygen partial pressures that are physiologically equivalent to altitudes 100 to 200 m higher at more moderate latitudes. We define altitude regions as high altitude (1500-3500 m), very high altitude (3500-5500 m), and extreme altitude (>5500 m).

Neurological consequences of increasing altitude

Neurological consequences of increasing altitude: The relation among altitude (classified as high [1500–3500 m], very high [3500-5500 m] and extreme [>5500 m]), the partial pressure of oxygen, and the neurological consequences of acute and gradual exposure to these pressure changes. Neurological consequences will vary greatly from person to person and with rate of ascent. HACE is far more common at higher altitudes, although there are case reports of HACE at 2500 m.

It is important for any discussion of AMS and HACE to have as a starting point an understanding of acclimatization. The process of acclimatization involves a series of adjustments by the body to meet the challenge of hypoxemia. While we have a general understanding of systemic changes associated with acclimatization, the underlying molecular and cellular processes are not yet fully described. Recent findings suggest that the process may be initiated by widespread molecular up-regulation of hypoxia inducible factor-1. Downstream processes ultimately act to offset hypoxemia, including elevated ventilation leading to a rise in arterial oxygen saturation (SaO2), a mild diuresis and contraction of plasma volume such that more oxygen is carried per unit of blood, elevated blood flow and oxygen delivery, and eventually a greater circulating hemoglobin mass. Acclimatization can be viewed as the end-stage process of how humans can best adjust to hypoxia. But optimal acclimatization takes from days to weeks, or perhaps even months.

The initial and immediate strategy to protect the body from hypoxia is to increase ventilation. This compensatory mechanism is triggered by stimulation of the carotid bodies, which sense hypoxemia (low arterial PO2), and increase central respiratory drive. This is a fast response, occurring within minutes of exposure to hypoxia persisting throughout high altitude exposure. This is why one cautions against the use of respiratory depressants such as alcohol and some sleeping medications, which can depress the hypoxic drive to breathe and may thus worsen hypoxemia. Pharmacological simulation of this natural process by acetazolamide, a respiratory stimulant and mild diuretic, largely protects from AMS and HACE by stimulating acclimatization. Circulatory responses are key to improving oxygen delivery, and are likely regulated by marked elevations in sympathetic activity. Field experience suggests that a marked elevation in early morning resting heart rate is a sign of challenges to acclimatization, perhaps secondary to increased hypoxemia, or dehydration. For the pathophysiology of AMS and HACE responses of the cerebral circulation are especially important. Maintenance of cerebral oxygen delivery is a critical factor for survival at high altitude. The balance between hypoxic vasodilation and hypocapnia-induced vasoconstriction determines overall cerebral blood flow (CBF). In a classic study, CBF increased 24% on abrupt ascent to 3810 m, and then returned to normal over 3 to 5 days. Recent studies, largely using regional transcranial Doppler measures of CBF velocity as a proxy for CBF, report discernible individual variation in the CBF response to hypoxia. All advanced brain imaging studies to date have shown both elevations in CBF in hypoxic humans and striking heterogeneity of CBF distribution in the hypoxic brain, with CBF rising up to 33% in the hypothalamus, and 20% in the thalamus with no other significant changes. Also, it is becoming clear that cerebral autoregulation, the process by which cerebral perfusion is maintained as blood pressure varies, is impaired in hypoxia. Thus, hypoxia modulates cerebral autoregulation and raises interesting questions about the importance of this process in AMS and acclimatization, since it appears to be a uniform response in all humans made hypoxemic. Further, hematocrit and hemoglobin concentration are elevated after 12 to 24 hours of hypoxic exposure due to a fall in plasma volume, but after several weeks, plasma volume returns to near sea level values. Normalization of plasma volume coupled with an increase in red cell mass secondary to the hypoxia stimulated erythropoiesis leads to an increase in total blood volume after several weeks of acclimatization. Adequate iron stores are required for adequate hematologic acclimatization to high altitude. Acclimatization, then, is a series of physiological responses to hypoxia that serve to offset hypoxemia, improve systemic oxygen delivery, and avoid AMS and HACE. When acclimatization fails, or the challenge of hypoxia is too great, AMS and HACE can develop.

AMS occurs in susceptible individuals when ascent to high altitude outpaces the ability to acclimatize. For example, most people ascending very rapidly to high altitude will get AMS. The symptoms, although often initially incapacitating, usually resolve in 24 to 48 hrs. The incidence and severity of AMS depend on the rate of ascent and the altitude attained, the length of time at altitude, the degree of physical exertion, and the individual’s physiological susceptibility. The chief significance of AMS is that planned activities may be impossible to complete during the first few days at a new altitude due to symptoms. In addition, in a few individuals, AMS may progress to life-threatening HACE or HAPE. At 4000 m and above, the incidence of AMS ranges from 50% to 65% depending on the rate and mode of ascent, altitude reached, and sleeping altitude. A survey of 3158 travelers visiting resorts in the Rocky Mountains of Colorado revealed that 25% developed AMS, and most decreased their daily activity because of their symptoms.

Singh et al. proposed that the high-altitude syndromes are secondary to the body’s responses to hypobaric hypoxia, not due simply to hypoxemia. They based this conclusion on 2 observations:

there is a delay between the onset of hypoxia and the onset of symptoms after ascent (from hours to days), and
not all symptoms are immediately reversed with oxygen.

On the other hand, scientists have long assumed that AMS and HACE are due solely to hypoxia, based largely on 2 reports:

the pioneering experiments of Paul Bert and
the Glass House experiment of Barcroft.

When these assumptions were tested in a laboratory setting to study symptom responses to hypobaric hypoxia (simulated high altitude), hypoxia alone, and hypobaric normoxia, AMS occurred soonest and with greater severity with simulated altitude, compared with either normobaric hypoxia or normoxic hypobaria. In 2 studies, one in normobaric hypoxia found no MRI signs of vasogenic edema but suggested that AMS was associated with “cytotoxic edema”, whereas a comparable study in hypobaric hypoxia found combined vasogenic and intracellular edema. The conclusions from the 2 studies have very different implications for refining a theory of the pathophysiology of AMS. Although the studies were not designed for a direct comparison between hypobaria and hypoxia, the discrepancy points out an assumption about normobaric hypoxia and the pathophysiology of AMS that may warrant further investigation.

Our central hypothesis regarding the pathophysiology of AMS, and by extension of HACE, is that it is centered on dysfunction within the brain. This is not a new idea, but it is one of current intense interest thanks to advances in brain imaging and neuroscience techniques. Barcroft, writing in 1924, argued that the brain’s response to hypoxia was central to understanding the pathophysiology of mountain sickness.

A low ventilatory response to hypoxia coupled with increased symptoms of AMS led to intensive investigation of a link between the chemical control of ventilation and the pathogenesis of AMS. The results of these investigations suggest that for most people, the ventilatory response to hypoxia has little predictive value for AMS risk. Only if the extremes of ventilator responsiveness are contrasted can accurate predictions be made, where those with extremely low ventilatory drives being more likely to suffer AMS. At the extreme end of the distribution (i.e., for very high responses), the protective role of a brisk hypoxic ventilatory response may be due to increased arterial oxygen content and cerebral oxygen delivery despite mild hypocapnic cerebral vasoconstriction.

Hansen and Evans were the first to publish a comprehensive hypothesis of the pathophysiology of AMS centered on the brain. Their theory posited that compression of the brain, either by increased cerebral venous volume, reduced absorption of cerebral spinal fluid, or increased brain-tissue hydration (edema), initiates the development of the symptoms and signs of AMS and HACE. Ross built on these ideas with his “tight fit hypothesis,” published in 1985, and others have developed these ideas into a series of testable hypotheses congruent with today’s knowledge of AMS and HACE. The tight fit hypothesis states that expanded intracranial volume (due to the reasons put forth by Hansen and Evans, or other causes) plus the volume available for intracranial buffering of that expanded volume would predict who would get AMS. Greater buffering capacity leads to AMS resistance, lower buffering capacity, or a ‘tight fit’ of the brain in the cranial vault, would lead to greater AMS susceptibility. Overall, it is clear that brain volume increases in humans on exposure to hypoxia. It is less certain whether this elevation in brain volume plays a role in AMS.

Hackett’s pioneering MRI study in HACE, with marked white matter edema suggestive of a vasogenic origin, has led to a decade of studies looking for a similar finding in AMS. In moderate to severe AMS, all imaging studies have shown some degree of cerebral edema. But in mild to moderate AMS, admittedly an arbitrary and subjective distinction, brain edema is present in some MRI studies of AMS subjects, but not in all. It seems reasonable to conclude from the available data that the increase in brain volume observed is at least partially due to brain edema, and that earlier studies missed the edema more for technical than physiological reasons. It is less clear whether the brain edema is largely of intracellular or vasogenic origin, and what role if any it plays in the pathophysiology of AMS.

Although we support transcranial doppler for many investigations in integrative physiology, the complex interplay of hypoxia and hypocapnia that is present in acutely hypoxic humans may present a situation where whole brain imaging is a more reliable and accurate tool to discern the role of CBF in the onset of AMS. To date, no brain imaging studies have addressed global cerebral perfusion in AMS.

The management of AMS and HACE is based on our current understanding of the physiological and pathophysiological responses to hypoxia. Hypoxia itself, however, does not immediately lead to AMS as there is a delay of several hours after arrival at high altitude before symptoms develop. Increased knowledge of hypoxic inducible factor and cytokines that alter capillary permeability may lead to the discovery of new drugs for the prevention and alleviation of AMS and HACE.

Much work has focused on the role of vascular endothelial growth factor (VEGF), a potent permeability factor up-regulated by hypoxia. Some studies have found no evidence of an association of changes in plasma concentrations of VEGF and AMS, whereas others support the hypothesis that VEGF contributes to the pathogensis of AMS. Clearly a better understanding of the mechanisms of increased capillary permeability of cerebral capillaries will greatly enhance the management of AMS and HACE.

Flying high: A theoretical analysis of the factors limiting exercise performance in birds at altitude

Graham R. Scott, William K. Milsom
Respiratory Physiology & Neurobiology 154 (2006) 284–301
http://dx.doi.org:/10.1016/j.resp.2006.02.012

The ability of some bird species to fly at extreme altitude has fascinated comparative respiratory physiologists for decades, yet there is still no consensus about what adaptations enable high altitude flight. Using a theoretical model of O2 transport, we performed a sensitivity analysis of the factors that might limit exercise performance in birds. We found that the influence of individual physiological traits on oxygen consumption (˙VO2 ) during exercise differed between sea level, moderate altitude, and extreme altitude. At extreme altitude, hemoglobin (Hb) O2 affinity, total ventilation, and tissue diffusion capacity for O2 (DTO2) had the greatest influences on VO2; increasing these variables should therefore have the greatest adaptive benefit for high altitude flight. There was a beneficial interaction between DTO2 and the P50 of Hb, such that increasing DTO2 had a greater influence on VO2 when P50 was low. Increases in the temperature effect on P50 could also be beneficial for high flying birds, provided that cold inspired air at extreme altitude causes a substantial difference in temperature between blood in the lungs and in the tissues. Changes in lung diffusion capacity for O2, cardiac output, blood Hb concentration, the Bohr coefficient, or the Hill coefficient likely have less adaptive significance at high altitude. Our sensitivity analysis provides theoretical suggestions of the adaptations most likely to promote high altitude flight in birds and provides direction for future in vivo studies.

The bird lung is unique among the lungs of air-breathing vertebrates, with a blood flow that is crosscurrent to gas flow, and a gas flow that occurs unidirectionally through rigid parabronchioles. As such, bird lungs are inherently more efficient than the lungs of other air-breathing vertebrates (Piiper and Scheid, 1972, 1975). While this may partially account for the greater hypoxia tolerance of birds in general when compared to mammals (cf. Scheid, 1990), its presence in all birds excludes the crosscurrent lung as a possible adaptation specific to high altitude fliers. Similarly, an extremely small diffusion distance across the blood–gas interface compared to other air breathers seems to be a characteristic of all bird lungs, and not just those of high fliers (Maina and King, 1982; Powell and Mazzone, 1983; Shams and Scheid, 1989). Partly because of this small diffusion distance, the inherent O2 diffusion capacity across the gas–blood interface (DLO2) is generally high in birds. Interestingly, pulmonary vasoconstriction does not appear to increase during hypoxia in bar-headed geese (Faraci et al., 1984a). This may be a significant advantage during combined exercise and severe hypoxia, and suggests that regulation of lung blood flow could be important in high altitude birds. In addition, the CO2/pH sensitivity of ventilation is commonly assessed by comparing the isocapnic and poikilocapnic hypoxic ventilatory responses; however, the isocapnic ventilatory responses to hypoxia of both low and high altitude birds have not been compared. In this regard, the ventilator response in high altitude birds may also depend on their capacity to maintain intracellular pH during alkalosis, or to buffer changes in extracellular pH due to hyperventilation. It therefore remains to be conclusively determined whether high altitude fliers have a greater capacity to increase ventilation during severe hypoxia.

After diffusing into the blood in the lungs, oxygen is primarily circulated throughout the body bound to hemoglobin. A high cardiac output is therefore important for exercise at high altitude to supply the working muscle with adequate amounts of O2. Indeed, animals selectively bred for exercise performance have higher maximum cardiac outputs, as do species that have evolved for exercise performance. Whether cardiac output limits exercise performance per se, however, is less clear; other factors may limit intense exercise, and in more athletic species (or individuals) cardiac output may be higher simply out of necessity. Excessive cardiac output may even be detrimental if blood transit times in the lungs or tissues are substantially reduced. Unfortunately, very little is known about cardiac performance in high flying birds. Both the high altitude bar-headed goose and the low altitude pekin duck can increase cardiac output at least five-fold during hypoxia at rest (Black and Tenney, 1980), but no comparison of maximum cardiac performance has been made between high and low altitude birds.

Once oxygenated blood is circulated to the tissues, O2 moves to the tissue mitochondria, the site of oxidative phosphorylation and oxygen consumption. Transport of oxygen from the blood to the mitochondria involves several steps. Oxygen must first dissociate from Hb and diffuse through the various compartments of the blood, but in both birds and mammals the conductances of these steps are high, and are unlikely to impose much of a limitation to O2 transport. In contrast, diffusion across the vascular wall and through the extracellular spaces is thought to provide the most sizeable limitation to O2 transport. Consequently, the size of the capillary–muscle fiber interface is an extremely important determinant of a muscle’s aerobic capacity. Finally, oxygen diffuses across the muscle fiber membrane and moves through the cytoplasm until it associates with cytochrome c oxidase, the O2 acceptor in the mitochondrial electron transport chain. Myoglobin probably assists intracellular O2 transport, so diffusion through the muscle likely provides very little resistance to O2 flux.

It is obvious that the ability of some bird species to fly at extreme altitudes is poorly understood. The adaptive benefit of high hemoglobin oxygen affinity is well established, but its relative importance is unknown. Some evidence suggests that traits increasing oxygen diffusion capacity in flight muscle are adaptive in high fliers as well, but the adaptive significance of differences in the respiratory and cardiovascular systems of high altitude fliers is not clear. The remainder of this study assesses these possibilities using theoretical sensitivity analysis, and explores potential adaptations for high altitude flight in birds.

Oxygen transport in birds

Oxygen transport in birds. The crosscurrent parabronchial lung is unidirectionally ventilated by air sacs, and oxygen diffuses into blood capillaries from air capillaries (not shown) all along the length of the parabronchi. Oxygen is then circulated in the blood, and diffuses to mitochondria in the tissues. The rate of oxygen transport at both the lungs and tissues can be calculated using the Fick equation, and the amount of O2 transferred from the lungs into the blood can be calculated using an oxygen conservation equation.

Oxygen tensions in the lung

Oxygen tensions in the lung (A) and tissue (B) capillaries during normoxia. In the crosscurrent avian lung, PO2 varies in two dimensions: PO2 increases along the path of blood flow through the lungs, but does not increase by as much at the end of the parabronchi as at the start (gas PO2 decreases along the length of the parabronchi). In the tissues, blood PO2 decreases continuously along the capillary length as O2 diffuses to tissue mitochondria. To reach a solution, our model iterates between gas transport calculations in the lungs (A) and tissues (B) until a stable result is reached.

varying different biochemical features of hemoglobin (Hb) on oxygen consumption

The effects of varying different biochemical features of hemoglobin (Hb) on oxygen consumption during exercise in normoxia (PIO2 of 150 Torr; red), moderate hypoxia (84 Torr; green dashed), and severe hypoxia (30 Torr; dark blue). (A) P50, the PO2 at 50% Hb saturation; (B and C) Bohr coefficient (φ); and (D and E) Hill coefficient (n) (see Section 2 for a mathematical description of each). In (B)–(E), the effects of each variable were assessed at the P50 of pekin ducks (40 Torr; B and D) as well as the P50 of bar-headed geese (25 Torr; C and E).

Unlike in vivo studies, theoretical sensitivity analyses allow individual physiological variables to be altered independently so their individual effects on oxygen consumption can be assessed. By applying this analysis to hypoxia in birds, we feel we can predict which factors most likely limit oxygen consumption and exercise performance. As a consequence, our analysis identifies which steps in the oxygen cascade can provide the basis for adaptive change in birds that evolved for high altitude flight, namely ventilation and tissue diffusion capacity.

Since our interest was in the factors limiting exercise performance at altitude, the starting data for our model were obtained from previous studies on pekin ducks near maximal oxygen consumption. These ducks were exercising on a treadmill, however, and were not flying. Unfortunately, to the best of our knowledge only one previous study has made all the required measurements for this analysis during flight, and this was only done in normoxia (in pigeons, Butler et al., 1977). Pekin ducks are the only species for which we could find all the required measurements for our analysis during exercise in both normoxia and hypoxia. Only the lung and tissue diffusion capacities remained to be calculated in our analysis, but previous experimental determinations of DLO2 in pekin ducks were similar to the values calculated in this study (Scheid et al., 1977). Similar values for DTO2 are not available.

The physiological variables limiting exercise performance in birds during moderate hypoxia are similar to those limiting performance in normoxia. DTO2 continues to pose the greatest limitation, and limitations imposed by the circulation (˙Q and CHb) are still greater at a lower P50. Unlike normoxia, however, ˙VO2 in moderate hypoxia appears to be limited less by the circulation and more by respiratory variables, as is also the case in humans (Wagner, 1996). The most substantial difference between severe hypoxia and normoxia/moderate hypoxia is in the effects of altering ventilation. Ventilation appears to become a major limitation to exercise performance at extreme altitude. DTO2 also appears to limit ˙VO2 in severe hypoxia, but only at lower P50 values. This is not entirely unsurprising: in severe hypoxia the venous blood of pekin ducks (a species which has a higher P50) is almost completely deoxygenated in vivo, so there are no possible benefits of increasing DTO2 . At the lower P50, there is a substantially higher arterial oxygen content, so more oxygen can be removed, and increasing DTO2 can have a greater influence. In humans during severe hypoxia, DTO2, DLO2, and ˙V have the greatest influence on exercise performance.

Tissue diffusion capacity should also be adaptive in high altitude birds with a high hemoglobin O2 affinity. In the present study, a simultaneous decrease in P50 (from 40 to 25 Torr) and increase in DTO2 (twofold) increased ˙VO2 by 51%. Thus, in high flying birds that are known to have a low P50, such as the barheaded goose and Ruppell’s griffon (Gyps rueppellii), increases in flight muscle diffusion capacity should be of extreme importance. This suggestion is supported by research demonstrating greater muscle capillarization in bar-headed geese than in low altitude fliers, as the size of the capillary–muscle fiber interface is known to be the primary structural determinant of O2 flux into the muscle.

Our analysis suggests that an enhanced capacity to increase ventilation should also benefit birds significantly in severe hypoxia, and could therefore be an important source of adaptation for high altitude flight. This is likely true regardless of P50; although there is a small amount of interaction between P50 and ventilation, increasing ˙V always had a substantial effect on oxygen consumption. Data from the literature addressing this possibility have unfortunately been inconclusive. Both bar-headed geese and pekin ducks can effectively increase ventilation, thus reducing the inspired-arterial O2 difference, during severe poikilocapnic hypoxia at rest, as well as during moderate poikilocapnic hypoxia and running exercise.

oxyhemoglobin dissociation curve

In contrast to the Bohr effect and Hill coefficient, the temperature effect on Hb-O2 binding affinity may have a substantial effect on oxygen consumption, and may therefore be a source of adaptive change for high altitude flight. An effect of temperature on ˙VO2 may arise if hyperventilation during flight at extreme altitude cools the pulmonary blood. This would reduce the P50 of Hb in the lungs, and thus facilitate oxygen uptake. When this blood enters the exercising muscles it would then be rewarmed to body temperature, and oxygen would be released from Hb. Our modelling suggests that a temperature effect on Hb could significantly enhance ˙VO2 . The greater the difference in temperature between blood in the lungs and in the muscles, and the greater the temperature effect on Hb-O2 binding, the greater the increase in ˙VO2 . At normal levels of temperature sensitivity, the increase in ˙VO2 was approximately 5% for every 1 ◦C difference. It could be adaptive at high altitude to alter the magnitude of the temperature effect on Hb while allowing lung temperature to fall. At present, however, it is unknown whether the Hb of high altitude birds has a heightened sensitivity to temperature, or whether pulmonary blood is actually cooled during high altitude flight.

Using a theoretical sensitivity analysis that allows individual physiological variables to be altered independently, we have identified the factors most likely to limit oxygen consumption and exercise performance in birds, and by extension, the physiological changes that are likely adaptive for high altitude flight. The adaptive benefits of some of these changes, in particular hemoglobin oxygen affinity, are already well established for high flying birds. For other traits, such as an enhanced hypoxic ventilatory response or O2 diffusion capacity of flight muscle, adaptive differences have not been conclusively recognized in studies in vivo. Furthermore, the beneficial interaction between increasing DTO2 and decreasing hemoglobin P50 has not yet been demonstrated in vivo. Our theoretical analysis suggests that changes in these respiratory processes could also adapt birds to environmental extremes, and future studies should explore these findings.

Adaptation and Convergent Evolution within the Jamesonia-Eriosorus Complex in High-Elevation Biodiverse Andean Hotspots

Patricia Sanchez-Baracaldo, Gavin H. Thomas
PLoS ONE 9(10): e110618. http://dx.doi.org:/10.1371/journal.pone.0110618

The recent uplift of the tropical Andes (since the late Pliocene or early Pleistocene) provided extensive ecological opportunity for evolutionary radiations. We test for phylogenetic and morphological evidence of adaptive radiation and convergent evolution to novel habitats (exposed, high-altitude paramo habitats) in the Andean fern genera Jamesonia and Eriosorus. We construct time-calibrated phylogenies for the Jamesonia-Eriosorus clade. We then use recent phylogenetic comparative methods to test for evolutionary transitions among habitats, associations between habitat and leaf morphology, and ecologically driven variation in the rate of morphological evolution. Paramo species (Jamesonia) display morphological adaptations consistent with convergent evolution in response to the demands of a highly exposed environment but these adaptations are associated with microhabitat use rather than the paramo per se. Species that are associated with exposed microhabitats (including Jamesonia and Eriorsorus) are characterized by many but short pinnae per frond whereas species occupying sheltered microhabitats (primarily Eriosorus) have few but long pinnae per frond. Pinnae length declines more rapidly with altitude in sheltered species. Rates of speciation are significantly higher among paramo than non-paramo lineages supporting the hypothesis of adaptation and divergence in the unique Pa´ramo biodiversity hotspot.

AltitudeOmics: Rapid Hemoglobin Mass Alterations with Early Acclimatization to and De-Acclimatization from 5,260 m in Healthy Humans

Benjamin J. Ryan, NB Wachsmuth, WF Schmidt, WC Byrnes, et al.
PLoS ONE 9(10): e108788. http://dx.doi.org:/10.1371/journal.pone.0108788

It is classically thought that increases in hemoglobin mass (Hb mass) take several weeks to develop upon ascent to high altitude and are lost gradually following descent. However, the early time course of these erythropoietic adaptations has not been thoroughly investigated and data are lacking at elevations greater than 5,000 m, where the hypoxic stimulus is dramatically increased. As part of the AltitudeOmics project, we examined Hb mass in healthy men and women at sea level (SL) and 5,260 m following 1, 7, and 16 days of high altitude exposure (ALT1/ALT7/ALT16). Subjects were also studied upon return to 5,260 m following descent to 1,525 m for either 7 or 21 days. Compared to SL, absolute Hb mass was not different at ALT1 but increased by 3.7-5.8% (mean 6 SD; n = 20; p<0.01) at ALT7 and 7.6-6.6% (n = 21; p=0.001) at ALT16. Following descent to 1,525 m, Hb mass was reduced compared to ALT16 (-6.0+3.7%; n = 20; p = 0.001) and not different compared to SL, with no difference in the loss in Hb mass between groups that descended for 7 (-6.3+3.0%; n = 13) versus 21 days (-5.7+5.0; n = 7). The loss in Hb mass following 7 days at 1,525 m was correlated with an increase in serum ferritin
(r =20.64; n = 13; p,0.05), suggesting increased red blood cell destruction. Our novel findings demonstrate that Hb mass increases within 7 days of ascent to 5,260 m but that the altitude-induced Hb mass adaptation is lost within 7 days of descent to 1,525 m. The rapid time course of these adaptations contrasts with the classical dogma, suggesting the need to further examine mechanisms responsible for Hb mass adaptations in response to severe hypoxia.

Cardiovascular adjustments for life at high altitude

Roger Hainsworth, Mark J. Drinkhill
Respiratory Physiology & Neurobiology 158 (2007) 204–211
http://dx.doi.org:/10.1016/j.resp.2007.05.006

The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. There are increases in sympathetic activity resulting in increases in systemic vascular resistance, blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. Systemic vasoconstriction may also occur as a reflex response to the high pulmonary arterial pressures. Many communities live permanently at high altitude and most dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Despite living all their lives at altitude, some dwellers, particularly Andeans, may develop a maladaptation syndrome known as chronic mountain sickness. The most prominent characteristic of this is excessive polycythemia, the cause of which has been attributed to peripheral chemoreceptor dysfunction. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.

High altitude places are not only destinations of adventurous travelers, many people are born, live their lives and die in these cold and hypoxic regions. According to WHO, in 1996 there were approximately 140 million people living at altitudes over 2,500m and there are several areas of permanent habitation at over 4,000 m. These are in three main regions of the world: the Andes of South America, the highlands of Eastern Africa, and the Himalayas of South-Central Asia. This review is concerned with the effects of exposure to high altitude on the cardiovascular system and its autonomic control, in visitors, and the means by which the permanent high altitude dwellers have adapted to their environment.

For visitors the period of initial adaptation, i.e. the first days and weeks following arrival at attitude, is a critical time since it is during this period that acute mountain sickness and/or pulmonary edema may occur. The processes of adaptation occurring during this initial period may well determine the individual’s ability to continue to function normally. Recent studies in animals and man have highlighted the role of the autonomic nervous system in adaptation and in particular the importance of sympathetic activation of the cardiovascular system following high altitude exposure.

An increase in resting heart rate in response to acute hypoxia has been
described in several species including man. Vogel and Harris (1967)
investigated the effects of simulated exposure to high altitude in man
at pressures equivalent to 600, 3,400 and 4,600m using a hypobaric
chamber. Each level of chamber pressure was developed over a 30 min
period andwas maintained for 48 h in an attempt to simulate expedition
conditions. After 10 h at the equivalent of 3,400 m resting
heart rate was significantly increased and by 40 h it had increased by
16% from the resting value at 600 m. At 4,600 m it increased by 34%.
Similar findings, an increase in heart rate of 18%, were shown following
ascent to 4,300 m for periods up to 5 weeks. However, this study also
demonstrated that the rate of ascent also influenced the magnitude of
the heart rate increase. A gradual increase in altitude over a period
of 2 weeks resulted in the resting heart rate increasing by 25%
compared with an abrupt ascent which resulted in an increase of
only 9%. As subjects acclimatize at altitudes up to about 4,500 m
much of the increase in heart rate is lost and resting heart rates
return towards their sea level values. Acute hypoxia also causes
increases in cardiac output both at rest and for given levels of
exercise compared with values during normoxia.

The effect of hypoxia on the pulmonary circulation is dramatic
resulting in pulmonary hypertension caused by an increase in
pulmonary vascular resistance. The onset has been shown in man
to be very rapid, reaching a maximum within 5 min. Zhao et al.
(2001) demonstrated that breathing 11% oxygen for 30 min
increased mean pulmonary artery pressure by 56%, from 16 to
25 mmHg. The effect of hypoxia on the pulmonary circulation is
even more pronounced during exercise, as demonstrated in studies
carried out on subjects of Operation Everest II. Resting pulmonary
artery pressure increased from 15 mmHg at sea level to 34 mmHg
at the equivalent of 8,840 m. During near maximal exercise at
8,840 m it increased from the sea level value of 33–54 mm Hg.
In the short term the mechanism of this pulmonary artery vaso-
constriction has been shown to involve inhibition of O2 sensitive
K+ channels leading to depolarization of pulmonary artery smooth
muscle cells and activation of voltage gated Ca2+ channels. This
causes Ca2+ influx and vasocon-striction. This process is
immediately reversed by breathing oxygen.

Healthy high altitude residents show excellent adaptation to their
environment. These adaptations are likely to be associated with
altered gene expression as the expression of genes associated with
vascular control and reactions to hypoxia have been found to be high
in altitude dwellers. Different communities, however, seem to adopt
different adaptation strategies. For example Andeans hyperventilate
to decrease end-tidal and arterial CO2 levels to as low as 25 mmHg
and have hemoglobin levels well above those in sea-level people.
Tibetans Hyperventilate but have normal hemoglobin levels below
4,000 m. Ethiopian highlanders, on the other hand, have CO2 and
hemoglobin levels similar to those of sea-level dwellers.

Blood volumes are larger in high altitude dwellers. In Andeans this
is due to large packed cell volumes whereas in Ethiopians it was the
plasma volumes that were large. Probably as the result of the large
blood volumes, tolerance to orthostatic stress was greater than that
in sea-level residents.

CMS is a condition frequently found in long term residents of high
altitudes, particularly in the Andes where it is a major public health
problem. It also occurs in residents on the Tibetan plateau, although
not in Ethiopians. Patients with CMS develop excessive polycythemia
and various clinical features including dyspnea, palpitations, insomnia,
dizziness, headaches, confusion, loss of appetite, lack of mental
concentration and memory alterations. Patients may also complain
of decreased exercise tolerance, bone pains, acral paresthesia and
occasionally hemoptysis. The impairment of mental function may
be reversed by phlebotomy. Physical examination reveals cyanosis,
due to the combination of polycythemia and low oxygen saturation,
and a marked pigmentation of the skin exposed to the sun.
Hyperemia of conjunctivae is characteristic and the retinal vessels
are also dilated and engorged. The second heart sound is frequently
accentuated and there is an increased cardiac size, mainly due to
right ventricular hypertrophy. As the condition progresses, overt
congestive heart failure becomes evident, characterized by dyspnea
at rest and during mild effort, peripheral edema, distension of
superficial veins, and progressive cardiac dilation.

The major mechanism for the control of blood pressure is through
regulation of peripheral vascular resistance, but most studies have
examined only the control of heart rate. We have recently studied
the responses of forearm vascular resistance to carotid baroreceptor
stimulation in high altitude residents with and without CMS, both at
their resident altitude and shortly after descent to sea level. Results
showed that baroreflex “set point” was higher in CMS, but only at
altitude. At sea level, values were similar.

The chronic hypoxia at high altitude stresses many of the body’s
homeostatic mechanisms. There have been many investigations
which have examined the effects on respiration. However, cardio-
vascular effects are no less important and it is largely through effects
on the cardiovascular system that both acute and chronic mountain
sickness are caused. The hypoxia exerts both direct and reflex effects.
In the lung it causes vasoconstriction and pulmonary hypertension.
The sympathetic nervous system is excited partly through a central
effect of the hypoxia, through stimulation of chemoreceptors and
possibly pulmonary arterial baroreceptors and altered systemic
baroreceptor function. In some individuals the excessive hemopoiesis
causes increased blood viscosity and tissue hypoperfusion leading
to the syndrome of chronic mountain sickness.

New Insights in the Pathogenesis of High-Altitude Pulmonary Edema

Urs Scherrer, Emrush Rexhaj, Pierre-Yves Jayet, et al.
Progress in Cardiovascular Diseases 52 (2010) 485–492
http://dx.doi.org:/10.1016/j.pcad.2010.02.004

High-altitude pulmonary edema is a life-threatening condition occurring in predisposed but otherwise healthy individuals. It therefore permits the study of underlying mechanisms of pulmonary edema in the absence of confounding factors such as coexisting cardiovascular or pulmonary disease, and/or drug therapy. There is evidence that some degree of asymptomatic alveolar fluid accumulation may represent a normal phenomenon in healthy humans shortly after arrival at high altitude. Two fundamental mechanisms then determine whether this fluid accumulation is cleared or whether it progresses to HAPE: the quantity of liquid escaping from the pulmonary vasculature and the rate of its clearance by the alveolar respiratory epithelium. The former is directly related to the degree of hypoxia induced pulmonary hypertension, whereas the latter is determined by the alveolar epithelial sodium transport. Here, we will review evidence that, in HAPE-prone subjects, impaired pulmonary endothelial and epithelial NO synthesis and/or bioavailability may represent a central underlying defect predisposing to exaggerated hypoxic pulmonary vasoconstriction and, in turn, capillary stress failure and alveolar fluid flooding. We will then demonstrate that exaggerated pulmonary hypertension, although possibly a condition sine qua non, may not always be sufficient to induce HAPE and how defective alveolar fluid clearance may represent a second important pathogenic mechanism.

Cerebral Blood Flow at High Altitude

Philip N. Ainslie and Andrew W. Subudhi
High Altitude Medicine & Biology 2014; 15(2): 133–140
http://dx.doi.org:/10.1089/ham.2013.1138

This brief review traces the last 50 years of research related to cerebral blood flow (CBF) in humans exposed to high altitude. The increase in CBF within the first 12 hours at high altitude and its return to near sea level values after 3–5 days of acclimatization was first documented with use of the Kety-Schmidt technique in 1964. The degree of change in CBF at high altitude is influenced by many variables, including arterial oxygen and carbon dioxide tensions, oxygen content, cerebral spinal fluid pH, and hematocrit, but can be collectively summarized in terms of the relative strengths of four key integrated reflexes:

hypoxic cerebral vasodilatation;
2) hypocapnic cerebral vasoconstriction;
3) hypoxic ventilatory response; and
4) hypercapnic ventilatory response.

Understanding the mechanisms underlying these reflexes and their interactions with one another is critical to advance our understanding of global and regional CBF regulation. Whether high altitude populations exhibit cerebrovascular adaptations to chronic levels of hypoxia or if changes in CBF are related to the development of acute mountain sickness are currently unknown; yet overall, the integrated CBF response to high altitude appears to be sufficient to meet the brain’s large and consistent demand for oxygen.

Relative to its size, the brain is the most oxygen dependent organ in the body, but many pathophysiological and environmental processes may either cause or result in an interruption to its oxygen supply. As such, studying the brain at high altitude is an appropriate model to investigate both acute and chronic effects of hypoxemia on cerebrovascular function. The cerebrovascular responses to high altitude are complex, involving mechanistic interactions of physiological, metabolic, and biochemical processes.

This short review is organized as follows: An historical overview of the earliest CBF measurements collected at high altitude introduces a summary of reported CBF changes at altitude over the last 50 years in both lowlanders and high-altitude natives. The most tenable candidate mechanism(s) regulating CBF at altitude are summarized with a focus on available data in humans, and a role for these mechanisms in the pathophysiology of AMS is considered. Finally, suggestions for future directions are provided.

Angelo Mosso (1846–1910) is undoubtedly the forefather of high altitude cerebrovascular physiology. In order to pursue his principal curiosity of the physiological effects of hypobaria, Mosso built barometric chambers and was reported to expose himself pressures as low as 192 mmHg (equivalent to > 10,000 m). He was also responsible for the building of the Capanna Margherita laboratory on Monta Rosa at 4,559 m. In both settings, Mosso utilized his hydrosphygmomanometer to measure changes in ‘‘brain pulsations’’ in patients that had suffered removal of skull sections, due to illness or trauma. Indicative of changes in CBF, these recordings preceded the next estimates of CBF in humans by some 50 years.

At sea level, Kety and Schmidt (1945) were the first to quantify human CBF using an inert tracer (nitrous oxide, N2O) combined with arterial and jugular venous sampling. This method for the measurement of global CBF is based on the Fick principle, whereby the integrated difference of multiple arterial and venous blood samples during the first 10 or more minutes after the sudden introduction into the lung of a soluble gas tracer is inversely proportional to cerebral blood flow. In 1948, they showed that breathing 10% oxygen increased CBF by 35%; however, it was not until 1964 that the first measurements of CBF were made in humans at high altitude. The motivation for these high altitude experiments was stimulated, in part, from the earlier discovery of the brain’s ventral medullary cerebrospinal fluid (CSF) pH sensors in animals. Following the location of these central chemoreceptors, Severinghaus and colleagues examined in humans the role of CSF pH and bicarbonate in acclimatization to high altitude (3,810 m) at the White Mountain (California, USA) laboratories (Severinghaus et al., 1963). A year later, at the same location, John Severinghaus performed his seminal study of CBF at high altitude. He was joined by Tom Hornbein—shortly after his first ascent of Everest by the West Ridge—who was part of the research team and also volunteered for the study (Fig.). The results showed clear time dependent changes in CBF during acclimatization to high altitude (HA).

the Kety-Schmidt nitrous oxide method of measuring CBF

From left to right, Larry Saidman (administering the gas), Tom Hornbien (volunteer), Ed Munson (drawing jugular venous blood samples), and John Severinghaus. Here (1964) the Kety-Schmidt nitrous oxide method of measuring CBF is used. The subject breathed about 15% N2O for 15 min while arterial and jugular venous blood was frequently sampled. (B) Results from Severinghaus et al. (1966). Graphs shows that CBF as estimated by cerebral A-VO2 differences from sea level controls increased about 24% within hours of arrival at 3810 m, and fell over 4 days to about 13% above control. CBF by the N2O method was increased by 40% on day 1, and returned to 6% above control on day 4. However, the N2O method data had greater variance. Acute normoxia on day 1 and day 4 returned CBF to sea level values within 15 min. Photograph courtesy of Dr. John W Severinghaus.

Native Tibetan (or Himalayan) and Andean populations arrived approximately 25,000 and 11,000 years ago, suggesting that these populations either carried traits that allowed them to thrive at high altitude or were able to adapt to the environment. The physiological and genetic traits associated with native high-altitude populations have been elegantly reviewed (Beall, 2007; Erzurum et al., 2007; Frisancho, 2013). As such, this topic is briefly summarized here with the focus on CBF at altitude in context of Andean and Tibetan high-altitude residents.

In general, native Andeans have lower CBF values compared to sea level natives. The first evidence suggesting lower flow was reported in 8 Peruvian natives living at 4300m altitude in Cerro de Pasco (Milledge and Sørensen, 1972). The authors found the mean arterial–venous oxygen content difference across the brain was 7.9 – 1 vol%, about 20% higher than the published sea level mean of 6.5 vol%. They suggested that CBF probably was proportionately about 20% below sea level normal values, assuming that brain metabolic rate was normal, and postulated that the mechanism might be high blood viscosity given the high hematocrit (58 – 6%) in these subjects. However, since the cerebral metabolic rate for oxygen (CMRO2) is constant even in severe hypoxia (Kety and Schmidt 1948b; Ainslie et al. 2013), the inverse linear relationship between CBF and arterial–venous oxygen content differences could also explain the reduction in CBF, as less flow would be needed to match the oxygen demand of the brain when arterial content is elevated. A similar study (Sørensen et al., 1974), using arterio-venous differences combined (in a subgroup) with a modified version of Kety–Schmidt method (krypton instead of N2O,) conducted in high-altitude residents in La Paz in Bolivia at 3800 m, also reported a 15%–20% reduction in CBF (with a reported average hematocrit of 50%) compared to a sea level control group.

Percent changes in cerebral blood flow

Percent changes in cerebral blood flow (D%CBF, graph A), arterial oxygen content (Cao2, graph B), and cerebral oxygen delivery (CDO2, graph C) with time at high-altitude from seven studies at various altitudes and durations. Severinghaus et al. (1966) studied CBF using the Kety-Schmidt technique in five subjects brought rapidly by car to 3810 m. Using the Xe133 method, Jensen et al. (1990) measured CBF in 12 subjects at 3475 m. Huang et al. (1987) measured ICA and VA blood velocities as a metric of CBF on Pikes Peak (4300 m). Baumgartner et al. (1994) studied 24 subjects who rapidly ascended to 3200m by cable car, slept one night at 3600 m, and ascended by foot to 4559m the next day. Cerebral blood flow was estimated by transcranial Doppler ultrasound. About two-thirds of the subjects developed symptoms of AMS, data included are the mean of all subjects. Lucas et al. (2011) employed an 8–9 day ascent to 5050m and estimated changes in CBF by transcranial Doppler ultrasound of the middle cerebral artery. Willie et al. (2013) following the same ascent measured flow (Duplex ultrasound; and TCCD) in the ICA and VA and estimated global flow from: 2*ICA + 2* VA. The same methodological approach was used time Subudhi upon rapid ascent via car and oxygen breathing to 5240 m (Subudhi et al. 2013). Cao2 was calculated from: (1.39 · [Hb] · SaO2) + Pao2 *0.003. In some studies [Hb] data were not available, and typical data from previous studies over comparable time at related elevation were used. In other studies, Pao2 was not always reported; therefore, Sao2 was used to estimate Pao2 via (Severinghaus, 1979).

Only two studies have measured serial changes in CBF during progressive ascent to high altitude, but the findings may help explain small discrepancies between studies. In 2011, Wilson et al. (2011) measured diameter and velocity in the MCA (using transcranial color-coded Duplex-ultrasound, TCCD) following partial acclimation to 5300m (n = 24), 6400 m (n = 14), and 7950m (n = 5). Remarkable elevations (200%) in flow in the MCA occurred at 7950 m. Notably, the authors estimated *24% dilation of the MCA occurred at 6400 m. Dilation of the MCA further increased to *90% at 7950m (Fig.) and was rapidly reversed with oxygen supplementation (Fig.). Cerebral oxygen delivery and oxygenation were maintained by commensurate elevations of CBF even at these extreme altitudes. In another recent study, CBF and MCA diameter were measured at 1338 m, 3440 m, 4371 m, and over time at 5050 m (Willie et al., 2013). Dilation of the MCA was observed upon arrival at 5050 m with subsequent normalization of CBF and MCA diameter by days 10–12. Such findings are consistent with unchanged diameter following 17 days at 5400m (Wilson et al., 2011). It is important to note that according to Poiseuille’s Law, flow is proportional to radius raised to the fourth power. Therefore, consistent with previous concerns about TCD (Giller, 2003), that the MCA dilates at such levels of hypoxemia indicates that previous studies using TCD at altitude may have underestimated flow (see previous Fig.) and thus may explain differences between studies. These findings are particularly important because they suggest regional regulation of CBF occurs in both large and small cerebral arteries.

Changes in blood flow in the middle cerebral artery (MCA) upon progressive ascent to 7950 m

Changes in blood flow in the middle cerebral artery (MCA) upon progressive ascent to 7950 m. Data were collected following partial acclimation to 5300 m (n = 24), at 6400 m (n = 14), and at 7950 m (n = 5). Remarkable elevations (200%) in flow in the MCA occurred at 7950 m following removal of breathing supplementary oxygen and breathing air for 20 min. Dilation (*24%) of the MCA occurred at 6400 m, which was further increased to 90% at 7950 m. Oxygen supplementation at this highest altitude rapidly reversed the observed MCA vessel dilation (denoted by blue triangle). Elevations in CBF via cerebral vasodilation were adequate to maintain oxygen delivery, even at these extreme altitudes. Modified from Wilson et al. (2011).

Summary of the major factors acting to increase ( plus) and decrease (minus) CBF during exposure to hypoxia

Summary of the major factors acting to increase ( plus) and decrease (minus) CBF during exposure to hypoxia. Cao2, arterial oxygen content; CBV, cerebral blood volume; EDHF, endothelium-derived hyperpolarizing factor; ET-1, endothelin-1; HCT, hematocrit; NO, nitric oxide; O2-, superoxide; PGE, prostaglandins; SNA, sympathetic nerve activity; VAH, ventilatory acclimatization to hypoxia/altitude. Modified from Ainslie and Ogoh (2010); Ainslie et al. (2014).

It is clear that many aspects of CBF regulation and brain function at high altitude warrant further investigation. Indeed, several questions remain. For example, over the period of ventilatory acclimatization (weeks to months), how do interactions between the hypoxic ventilatory response, hypercapnic ventilatoy response, hypoxic cerebral vasodilatation, and hypocapnic cerebral vasoconstriction interact to alter CBF? Furthermore, what is the role of NO and/or adenosine in mediating cerebral vasodilation at high altitude? And last, what is the time-course of recovery in CBF following descent to sea level?

Cognitive Impairments at High Altitudes and Adaptation

Xiaodan Yan
High Alt Med Biol. 15:141–145, 2014
http://dx.doi.org:/10.1089/ham.2014.1009

High altitude hypoxia has been shown to have significant impact on cognitive performance. This article reviews the aspects in which, and the conditions under which, decreased cognitive performance has been observed at high altitudes. Neural changes related to high altitude hypoxia are also reviewed with respect to their possible contributions to cognitive impairments. In addition, potential adaptation mechanisms are reviewed among indigenous high altitude residents and long-term immigrant residents, with discussions about methodological concerns related to these studies.

The amount of cognitive impairments at high altitudes is related to the chronicity of exposure. Acute exposure usually refers to a duration of several weeks, whereas chronic exposure usually refer to ‘‘extended permanence’’ in the high altitude environment (Virue´s-Ortega and others, 2004). The altitude of ascending or residence is another factor affecting the severity of impairments. This review will first summarize the cognitive impairments in acute exposure, then talk about impairments in chronic exposure, with discussions about the effect of altitudes in corresponding sections.

High altitude-related neurocognitive impairments with ascending altitudes

High altitude-related neurocognitive impairments with ascending altitudes in acute high altitude exposure (Wilson and others, 2009).

human brain consumes about 20% of the total oxygen intake

The human brain consumes about 20% of the total oxygen intake, which is disproportional to its size (about 2% of the total body weight). In this figure, oxygen consumption is reflected from glucose consumption in positron emission tomography (PET) (Alavi and Reivich, 2002).

The possibility of adaptation to high altitude hypoxia has always been an intriguing issue. In the acute cases, the human body does have some capacity for acclimatization, which varies significantly for different individuals. The question is, in chronic cases, for example, does growing up at high altitude regions guarantee sufficient adaption to occur to compensate for the risk of cognitive impairments? Existing research tends to suggest that, although some level of adaptation does occur, neural and cognitive impairments are still observed in these populations who are native or long-term residents at high altitude.

Although multiple studies have suggested that growing up at high altitudes is associated with cognitive impairments, it is not to say that adaptation does not happen with prolonged chronic exposure to high altitudes. One study has revealed that as a function of the length of low altitude residence (across the range of 1–5 years), some neuroimaging parameters of original highlanders who grew up at high altitude regions had shown the trend of converging towards the patterns of original low altitude residents, although such changes were not accompanied by statistically significant changes in cognitive performance (Yan and others, 2010). It is possible that, given sufficiently long time for normoxia adaptation, the neural and cognitive impairments associated with high altitude hypoxia may be alleviated to a certain extent.

In summary, various cognitive impairments associated with high altitude hypoxia have been reported from existing studies, which are accompanied by findings about neural impairments, suggesting that these cognitive impairments have legitimate neural basis. The specific relationships between physiological symptoms and cognitive impairments appear to be complicated and require further elucidation. There are cognitive impairments associated with both acute and chronic exposure to high altitudes; however, particular caution should be taken when interpreting the findings about cognitive impairments among native high altitude residents because of the differences
in cultural and socioeconomic factors. Existing studies have suggested that there can be some level of adaptation to high altitudes, in spite of the fact that some neuronal impairment may be irreversible.

Exercise Capacity and Selected Physiological Factors by Ancestry and Residential Altitude: Cross-Sectional Studies of 9–10-Year-Old Children in Tibet

Bianba, Sveinung Berntsen, Lars Bo Andersen, Hein Stigum, et al.
High Alt Med Biol. 2014; 15:162–169
http://dx.doi.org:/10.1089/ham.2013.1084

Aim: Several physiological compensatory mechanisms have enabled Tibetans to live and work at high altitude, including increased ventilation and pulmonary diffusion capacity, both of which serve to increase oxygen transport in the blood. The aim of the present study was to compare exercise capacity (maximal power output) and selected physiological factors (arterial oxygen saturation and heart rate at rest and during maximal exercise, resting hemoglobin concentration, and forced vital capacity) in groups of native Tibetan children living at different residential altitudes (3700 vs. 4300 m above sea level) and across ancestry (native Tibetan vs. Han Chinese children living at the same altitude of 3700 m). Methods: A total of 430 9–10-year-old native Tibetan children from Tingri (4300 m) and 406 native Tibetan and 406 Han Chinese immigrants (77% lowland-born and 33% highland-born) from Lhasa (3700 m) participated in two cross-sectional studies. The maximal power output (Wmax) was assessed using an ergometer cycle. Results: Lhasa Tibetan children had a 20% higher maximal power output (watts/kg) than Tingri Tibetan and 4% higher than Lhasa Han Chinese. Maximal heart rate, arterial oxygen saturation at rest, lung volume, and arterial oxygen saturation were significantly associated with exercise capacity at a given altitude, but could not fully account for the differences in exercise capacity observed between ancestry groups or altitudes. Conclusions: The superior exercise capacity in native Tibetans vs. Han Chinese may reflect a better adaptation to life at high altitude. Tibetans at the lower residential altitude of 3700 m demonstrated a better exercise capacity than residents at a higher altitude of 4300m when measured at their respective residential altitudes. Such altitude- or ancestry-related difference could not be fully attributed to the physiological factors measured.

Group size effects on foraging and vigilance in migratory Tibetan antelope

Xinming Lian, Tongzuo Zhang, Yifan Cao, Jianping Su, Simon Thirgood
Behavioural Processes 76 (2007) 192–197
http://dx.doi.org:/10.1016/j.beproc.2007.05.001

Large group sizes have been hypothesized to decrease predation risk and increase food competition. We investigated group size effects on vigilance and foraging behavior during the migratory period in female Tibetan antelope Pantholops hodgsoni, in the Kekexili Nature Reserve of Qinghai Province, China. During June to August, adult female antelope and yearling females gather in large migratory groups and cross the Qinghai–Tibet highway to calving grounds within the Nature Reserve and return to Qumalai county after calving. Large groups of antelope aggregate in the migratory corridor where they compete for limited food resources and attract the attention of mammalian and avian predators and scavengers. We restricted our sampling to groups of less than 30 antelopes and thus limit our inference accordingly. Focal-animal sampling was used to record the behavior of the free-ranging antelope except for those with lambs. Tibetan antelope spent more time foraging in larger groups but frequency of foraging bouts was not affected by group size. Conversely, the time spent vigilant and frequency of vigilance bouts decreased with increased group size. We suggest that these results are best explained by competition for food and risk of predation.

High altitude exposure alters gene expression levels of DNA repair enzymes, and modulates fatty acid metabolism by SIRT4 induction in human skeletal muscle

Zoltan Acsa, Zoltan Boria, Masaki Takedaa, Peter Osvatha, et al.
Respiratory Physiology & Neurobiology 196 (2014) 33–37
http://dx.doi.org/10.1016/j.resp.2014.02.006

We hypothesized that high altitude exposure and physical activity associated with the attack to Mt Everest could alter mRNA levels of DNA repair and metabolic enzymes and cause oxidative stress-related challenges in human skeletal muscle. Therefore, we have tested eight male mountaineers (25–40 years old) before and after five weeks of exposure to high altitude, which included attacks to peaks above 8000 m. Data gained from biopsy samples from vastus lateralis revealed increased mRNA levels of both cytosolic and mitochondrial superoxide dismutase. On the other hand 8-oxoguanine DNA glycosylase(OGG1) mRNA levels tended to decrease while Ku70 mRNA levels and SIRT6 decreased with altitude exposure. The levels of SIRT1 and SIRT3 mRNA did not change significantly. But SIRT4 mRNA level increased significantly, which could indicate decreases in fatty acid metabolism, since SIRT4 is one of the important regulators of this process. Within the limitations of this human study, data suggest that combined effects of high altitude exposure and physical activity climbing to Mt. Everest, could jeopardize the integrity of the particular chromosome.

High-altitude adaptations in vertebrate hemoglobins

Roy E. Weber
Respiratory Physiology & Neurobiology 158 (2007) 132–142
http://dx.doi.org:/10.1016/j.resp.2007.05.001

Vertebrates at high altitude are subjected to hypoxic conditions that challenge aerobic metabolism. O2 transport from the respiratory surfaces to tissues requires matching between theO2 loading and unloading tensions and theO2-affinity of blood, which is an integrated function of hemoglobin’s intrinsic O2-affinity and its allosteric interaction with cellular effectors (organic phosphates, protons and chloride). Whereas short-term altitudinal adaptations predominantly involve adjustments in allosteric interactions, long-term, genetically-coded adaptations typically involve changes in the structure of the hemoglobin molecules. The latter commonly comprise substitutions of amino acid residues at the effector binding sites, the heme protein contacts, or at inter-subunit contacts that stabilize either the low-affinity (‘Tense’) or the high-affinity (‘Relaxed’) structures of the molecules. Molecular heterogeneity (multiple iso-Hbs with differentiated oxygenation properties) can further broaden the range of physico-chemical conditions where Hb functions under altitudinal hypoxia. This treatise reviews the molecular and cellular mechanisms that adapt hemoglobin-oxygen affinities in mammals, birds and ectothermic vertebrates at high altitude.

Vertebrate animals display remarkable ability to tolerate high altitudes and cope with the concomitant decreases in O2 tension that potentially constrain aerobic life (Monge and Leon-Velarde, 1991;Weber, 1995; Samaja et al., 2003). Compared to an ambient PO2 of approximately 160 mm Hg at sea level, inspired tension approximates only 95 mm Hg for llamas and frogs from Andean habitats above 4000 m, 45 mm Hg for bar-headed geese that fly across the Himalayas, and 33 mm Hg for Ruppell’s griffon that soars at 11,300 m over Africa’s Ivory Coast. Apart from the distinct adaptations manifest in blood’s O2-transporting properties, tolerance to decreased O2 availability may entail reconfigurations at the organ and cellular levels that include a switch to partial anaerobiosis. Driven by needs to reduce aerobic metabolic rate and maintain functional integrity (Ramirez et al., 2007), these pertain to a core triad of adaptations:

metabolic suppression,
tolerance to metabolite (e.g. lactate) accumulation, and
defenses against increased free radicals associated with return to high O2 tensions (Bickler and Buck, 2007).

The response to oxygen lack comprises two phases

defense, which includes metabolic arrest (a suppression of ATP-demand and ATP-supply) and channel arrest (decreases cell membrane permeability), and
rescue, which commonly involves preferential expression of proteins that are implicated in extending metabolic down-regulation (Hochachka et al., 1996).

These responses vary greatly in different species and different tissues. Thus, although mixed-venous lactate concentrations increase strongly in sea-level as well as high-altitude acclimated pigeons that are exposed to altitude (from 1–2 mM at sea level to 5–7 mM at 9000 m) (Weinstein et al., 1985), and humans performing submaximal work at high altitude show a transient ‘lactate paradox’ (lower peak lactate levels that humans living at sea level (Lundby et al., 2000)), many species do not exhibit altitude-related changes in anaerobic metabolism.

Organismic adaptations to survive and perform physical exercise at extreme altitudinal hypoxia are diverse. In birds the undisputed high-altitude champions, where flapping flight may raise the energy demand 10–20-fold compared to resting levels (Scott et al., 2006), a highly efficient “cross-current” ventilation perfusion arrangement in the lungs may increase arterial O2 tensions above the tensions in expired air (Scheid, 1979) and drastically reduce the difference between inhalant and arterial O2 tensions (to 1 mm Hg in bar-headed geese subjected to simulated altitude of 11580 m) (Black and Tenney, 1980). The Andean frog Telmatobius culeus has a highly ‘oversized’ (folded) and vascularized skin that is ventilated by ‘bobbing’ behavior to support water(=skin) breathing. Manifold organismic adaptations moreover include combinations of increased muscle Mb concentrations (Reynafarje and Morrison, 1962) increased muscle capillarization (manifest in mammals and birds (cf. Monge et al., 1991)) and decreased red cell size (seen in amphibians but not high-altitude reptiles (Ruiz et al., 1989; Ruiz et al., 1993)). Amphibians exhibit an interspecific correlation between erythrocyte count and the degree of vascularization of respiratory surfaces and muscle tissues (Hutchison and Szarski, 1965), that reflect differences in their ability to tolerate altitudinal hypoxia.

A sensitivity analysis of the factors that may limit exercise performance identifies high Hb-O2 affinity, together with high total ventilation and high tissue diffusion capacity as the physiological traits that have greatest adaptive benefit for bird flight at extreme high altitude (Scott and Milsom, 2006). Blood O2 affinity is a combination of the intrinsic O2 affinity of the ‘stripped’ (purified) Hb molecules and the interaction of allosteric effectors (like organic phosphates, protons and chloride ions) that decrease Hb-O2 affinity inside the rbcs (Weber and Fago, 2004). Short-term adaptations in O2 affinity are commonly mediated by changes in erythrocytic effectors such as organic phosphates (2,3-diphosphoglycerate, DPG, in mammals, inositol pentaphosphate, IPP, in birds, ATP in reptiles, and ATP and DPG in amphibians), whereas long-term adaptations (that include interspecific ones that are genetically determined) commonly involve changes in Hb structure (amino acid exchanges) that alter Hb’s intrinsic O2 affinity or its sensitivity to allosteric effectors.

Vertebrate Hbs are tetrameric molecules composed of two α (or α-like) chains and two β (or β-like) chains, which in humans consist of 141 and 146 amino acid residues, respectively. Each subunit exhibits a highly characteristic “globin fold” comprised of seven or eight α-helices (labelled A, B, C, etc.) linked by nonhelical (EF, FG) segments, and N- and C-terminal extensions termed NA and HC, respectively. Individual amino acid residues are identified by their sequential positions in chain or/and the helix; thus α1131(H14)-Ser refers to Serine that is the 131^st residue of α1 chain and the 14th of the H. During (de-) oxygenation Hb switches between two major structural states:

the high affinity oxygenated R (relaxed) state that prevails at the respiratory surfaces, and
the low affinity, deoxygenated T (tense) state that occurs predominantly in the tissues and is constrained by additional hydrogen bonds and salt bridges.

The Hbs exhibit cooperative homotropic interactions between the O2 binding heme groups (that cause the S-shaped O2 equilibrium curves and increase O2 loading and unloading for a given change in O2 tension) as well as inhibitory, heterotropic interactions between the hemes and the binding sites of effectors that decrease O2 affinity (increase the half-saturation O2 loading tension, P50) and facilitate O2 unloading.

A comparison of Hbs from different species (cf. Perutz, 1983) reveals that variation in the sensitivities to effectors correlates generally with exchanges of very few of the approximately 287 amino acid residues that comprise each αβ dimer. Thus in adult human Hb (HbA) at physiological pH, the majority of the Bohr effect (pH dependence of Hb-O2 affinity that facilitates O2 release in relatively acid working muscles) results from proton binding at the C-terminal residues of the β-chains (β146-His) (cf. Lukin and Ho, 2004). Correspondingly DPG binds to only four β-chain residues (β1-Val, β2-His, β82-Lys and β143-His), CO2 binding (carbamate formation) occurs at the uncharged amino-termini of both chains (α1-Val and β1-Val), and monovalent anions like chloride are considered to bind at one α-chain site (between α1-Val and α131–Ser) and one β-chain site (between β82-Lys and β1-Val) (cf. Riggs, 1988).

The small number of sites that primarily determine Hb-O2 affinity and its sensitivity to effectors aligns with the neutral theory of molecular evolution (Kimura, 1979), which holds that the majority of amino acid substitutions are non-adaptive and harmless—and facilitates identification of key molecular mechanisms implicated in adaptations at altitude.

The role of effectors in altitude adaptation is aptly illustrated in humans where Hb structure (intrinsic O2 affinity) remains unchanged. Newcomers and permanent residents at moderate altitude (e.g. 2000 m) show increased DPG levels, resulting in a decreased O2 affinity that positions arterial and mixed venous O2 tensions on the steep part of the O2 equilibrium curve, increasing O2 capacitance ([1]bO2) and O2 transport, without materially compromising O2 loading (Turek et al., 1973; Mairbaurl, 1994). The increased DPG correlates with erythropoietin-mediated formation of new rbcs that have higher glycolytic rates and higher DPG and ATP levels than old rbcs. However, faster increases in P50 than in DPG level indicate contributions from other factors, such as chloride and ATP, and Mg ions that neutralize the anionic effectors (Mairbaurl et al., 1993). At higher altitudes (4559 m) increased hyperventilation that drives off CO2 causes respiratory alkalosis (Mairbaurl, 1994). The higher pH increases O2 affinity via the Bohr effect and, offsetting the effect of increased DPG, leads to a similar O2 affinity and arterio-venous O2 saturation difference as at sea level (Fig.). O2 unloading in the tissues is moreover enhanced by metabolic acidification of capillary blood (Fig.).

Obviously right-shifted curves (that favor O2 unloading) becomes counterproductive at extreme altitudes where O2 loading becomes compromised, predicting that decreased O2 affinity becomes maladaptive under severe hypoxic stress. This is consistent with the observation that a carbamylation-induced increase in blood O2 affinity of rats (that lowers P50 from 27 to 15 mm Hg), increases survival under hypobaric hypoxia equivalent to 9200 meters’ altitude (Eaton et al., 1974). The altitude limit where increased affinity rather than a decreased affinity optimizes tissue O2 supply < 5000 m in man (Samaja et al., 2003)] depends on organismic adaptations (e.g. efficiency of gas exchange) and thus will vary between species. Mammals that permanently inhabit high altitudes and show high blood O2 affinities include the Andean rodent Chinchilla brevicaudata living at 3000–5000 m (blood P50 = 23 mm Hg compared to 38 mm Hg in the rat) (Ostojic et al., 2002). The deer mouse, Peromyscus maniculatus that occurs continuously from sea level to altitudes above 4300 m shows a strong correlation between blood O2 affinity and native altitude (Snyder et al., 1988). That genetically based differences in cofactor levels may contribute to this relationship follows from lower DPG/Hb ratios found in specimens resident, and native to, high altitude than in those from low altitude, after long-term acclimation of both groups to low altitude (Snyder, 1982).

O2 equilibrium curves of human blood illustrating the effects of increases in red cell DPG and pH at high-altitude

O2 equilibrium curves of human blood illustrating the effects of increases in red cell DPG and pH at high-altitude (4559 m). Solid curves refer to arterial blood (P50 = 26 mm,upper section) and cubical venous blood (P50 = 27.5 mm Hg, lower section); their displacement reflects the Bohr effect. The broken curves depict effects of increased DPG levels (↑DPG) at unchanged pH, increased pH (↑pH) at unchanged DPG, and of decreased tissue pH (↓pH) resulting from higher degrees of metabolic acidification in the tissues. Open and shaded vertical columns indicate O2 unloaded at sea level and 4559 m, respectively, for venous O2 tensions (PvO2) of 25 and 15 mm Hg,respectively [Modified after (Mairbaurl, 1994)].

Camelids. The high blood-O2 affinities in Andean camelids (llama, vicunia, alpaca and guanaco) whose natural habitats exceed 3000 m (Bartels et al., 1963) compared to those of similarly-sized lowland mammals are well-established. In the camelids a β2His→Asn substitution deletes two of the seven DPG contacts in the tetrameric Hb, which increases blood O2 affinity by reducing the DPG effect. Although the intrinsic Hb-O2 affinity is lower in llama than in the related, lowland camel (Bauer et al., 1980), llama blood has a higher O2 affinity due to a three-fold lower DPG-binding than in camel Hb that has the same DPG binding sites as humans (Bauer et al., 1980). In vicunia, a higher O2 affinity than in llama (that has identical β-chains), correlates with the α130Ala→Thr substitution, which introduces a hydroxyl polar group that predictably reduces the chloride binding at adjacent α131Asn residue .

Sheep and goats commonly express two isoforms, HbA and HbB. The heterogeneity is controlled by two autosomal alleles with codominant expression. Whereas individuals expressing HbA have higher blood-O2 affinity than those that express HbB, heterozygotes that express both forms at equimolar concentrations in the same erythrocytes show intermediate affinity. Anemic blood loss induces switching from HbA to HbC that has a similarly high affinity. Hbs A, B and C have identical α-chains but different β[1]-chains. It appears unknown whether altitudinal exposure (which like anemia, induces tissue hypoxia) modulates Hb heterogeneity via selective expression of specific β-chains.

Compared to most mammals that possess one major adult and one major fetal Hb, yak, Poephagus (=Bos) grunniens, a native to altitudes of 3000–6000 m in Tibet, Nepal and Bhutan, has two or four major adult Hbs and two major fetal Hbs. These Hbs exhibit higher intrinsic affinities than closely-related bovine Hb, marked DPG sensitivities and, exceptional amongst mammals, differentiated O2 affinities that indicates an extended range of ambient O2 tensions (and altitudes) in which the composite Hb functions.

(Not shown). Representation of interchain contacts considered to underly differentiated O2 affinities in Rueppell’s griffon isoHbs A, A , D and D that have identical β- chains but different α- chains. Accordingly the van der Waal’s contact between β134Ile and β₁125-Asp in Hbs A , D and D stabilizes the low-affinity, T-state less strongly than the H-bond between Thr 134 and β₁125-Asp and thus increases O2 affinity in Hbs A, D and D. Analogously, the hydrogen bonds between α₁38-β₂97/99 that stabilize the high-affinity oxystructure (raising O2 affinity in isoHbs D and D) cannot form in HbA and HbA that have Pro at α₁38.

Ostriches, the largest extant birds, exhibit a β2His→Gln exchange (that reduces phosphate interaction). They moreover ‘use’ ITP (inositol phosphate) that carries fewer negative charges, and predictably has lesser allosteric effect, than IPP (Isaacks et al., 1977), predicting a high blood O2 affinity that is compatible with ‘scaling’ and (as in elephants) increases high altitude tolerance.

Whereas some adult birds express one major iso-Hb (HbA), the majority of species, reportedly all that fly at high altitudes (Hiebl et al., 1987), also express a less abundant HbD. HbD has the same β-chains as HbA but different α-chains (α^D) and exhibits higher O2 affinities (Huisman et al., 1964). There is no consistent evidence for hypoxia-induced changes in HbD expression.

An example of how “molecular anatomy is just as key to understanding molecular adaptation as phylogeny and physiological ecology” (Golding and Dean, 1998) is Hb of the high-altitude tolerant bar-headed goose that has a sharply higher blood O2 affinity than that of the closely related graylag goose that is restricted to lower altitudes (P50 = 29.7 and 39.5mmHg at 37 ◦C and pH 7.4). The Hbs differ by only four (greylag→bar-headed) amino acid exchanges: α18Gly→Ser, α63Ala→Val, β125Glu→Asp and α119Pro→Ala. The last mentioned exchange that is unique in birds, predictably increases O2 affinity, by deleting a contact between α₁119 and β₁55 that destabilizes the T-structure (Perutz, 1983). Moreover, Andean ‘goose’ Hb that also has high blood O2 affinity shows β55 Leu→Ser that deletes the same contact. Significantly, two human Hb mutants (α₁19Pro–Ala and β₁55Met→Ser) engineered by site-directed mutagenesis to mimic the mutations found in bar-headed and Andean geese possess markedly higher O2 affinities than native HbA.

Although “the study of molecular adaptation has long been fraught with difficulties not the least of which is identifying out the hundreds of amino acid replacements, those few directly responsible for major adaptations” Hb’s adaptations to high altitude are a prime example of how “an amino acid replacement of modest effect at the molecular level causes a dramatic expansion in an ecological niche” [quotations from (Golding et al., 1998)].

However, the pathway of molecular O2 from the respiratory medium to the cellular combustion sites via the Hb molecules is regulated by a symphony of supplementary adaptations that span different levels of biological organization, each of which (according to the principle of symmorphosis) may become maximally recruited in extreme cases (as in birds actively flying above 10,000 m). Apart from hyperventilation, that appears to occur ubiquitously (and increases blood O2 affinity via increased pH), different species subjected to less extreme hypoxic stress utilize different adaptations among the arsenal of organismic, cellular and molecular strategies that favor efficient aerobic utilization of the scarce O2 available at high altitude. No clear correlations exist between the adaptive strategies recruited by different animals on the one hand, and their phylogenetic position, mode of life or ecological niches on the other. An overall limitation is that short-term adaptive adjustments in O2 affinity (that may occur within individual animals) necessarily involves rapid adaptive responses, such as changes in the levels of erythrocytic effectors, whereas the long-term acclimations that have accumulated in permanent high-altitude dwellers during evolutionary development.

Genetic Diversity of Microsatellite DNA Loci of Tibetan Antelope (Chiru, Pantholops hodgsonii) in Hoh Xil National Nature Reserve, Qinghai, China

Hui Zhou, Diqiang Li, Yuguang Zhang, Tao Yang, Yi Liu
J Genetics and Genomics (Formerly Acta Genetica Sinica) 2007; 34(7): 600-607

The Tibetan antelope (Pantholops hodgsonii), indigenous to China, became an endangered species because of considerable reduction both in number and distribution during the 20th century. Presently, it is listed as an AppendixⅠspecies by CITES and as CategoryⅠ by the Key Protected Wildlife List of China. Understanding the genetic diversity and population structure of the Tibetan antelope is significant for the development of effective conservation plans that will ensure the recovery and future persistence of this species. Twenty-five microsatellites were selected to obtain loci with sufficient levels of polymorphism that can provide in-formation for the analysis of population structure. Among the 25 loci that were examined, nine of them showed high levels of genetic diversity. The nine variable loci (MCM38, MNS64, IOBT395, MCMAI, TGLA68, BM1329, BMS1341, BM3501, and MB066) were used to examine the genetic diversity of the Tibetan antelope (n = 75) in Hoh Xil National Nature Reserve(HXNNR), Qinghai, China. The results obtained by estimating the number of population suggested that all the 75 Tibetan antelope samples were from the same population. The mean number of alleles per locus was 9.4 ± 0.5300 (range, 7–12) and the mean effective number of alleles was 6.519 ± 0.5271 (range, 4.676–9.169). The observed mean and expected heterozygosity were 0.844 ± 0.0133 (range, 0.791–0.897) and 0.838 ± 0.0132 (range, 0.786–0.891), respectively. Mean Polymorphism Information Content (PIC) was 0.818 ± 0.0158 (range, 0.753–0.881). The value of Fixation index (Fis) ranged from −0.269 to −0.097 with the mean of −0.163 ± 0.0197. Mean Shannon’s information index was 1.990 ± 0.0719 among nine loci (range, 1.660–2.315). These results provide baseline data for the evaluation of the level of genetic variation in Tibetan antelope, which will be important for the development of conservation strategies in future.

Expression profiling of abundant genes in pulmonary and cardiac muscle tissues of Tibetan Antelope (Pantholops hodgsonii)

Xiaomei Tong, Yingzhong Yang, Weiwei Wang, Zenzhong Bai, et al.
Gene 523 (2013) 187–191
http://dx.doi.org/10.1016/j.gene.2013.03.011

The Tibetan Antelope (TA), which has lived at high altitude for millions of years, was selected as the model species of high hypoxia-tolerant adaptation. Here we constructed two cDNA libraries from lung and cardiac muscle tissues, obtained EST sequences from the libraries, and acquired extensive expression data related energy metabolism genes. Comparative analyses of synonymous (Ks) and nonsynonymous (Ka) substitution rates of nucleus-encoded mitochondrial unigenes among different species revealed that many antelope genes have undergone rapid evolution. Surfactant-associated protein A (SP-A) and surfactant-associated protein B (SP-B) genes in the AT lineage experienced accelerated evolution compared to goat and sheep, and these two genes are highly expressed in the lung tissue. This study suggests that many specific genes of lung and cardiac muscle tissues showed unique expression profiles and may undergo fast adaptive evolution in TA. These data provide useful information for studying on molecular adaptation to high-altitude in humans as well as other mammals.

Exogenous Sphingosine-1-Phosphate Boosts Acclimatization in Rats Exposed to Acute Hypobaric Hypoxia: Assessment of Haematological and Metabolic Effects

Sonam Chawla, Babita Rahar, Mrinalini Singh, Anju Bansal, et al.
PLoS ONE 9(6): e98025. http://dx.doi.org:/10.1371/journal.pone.0098025

Background: The physiological challenges posed by hypobaric hypoxia warrant exploration of pharmacological entities to improve acclimatization to hypoxia. The present study investigates the preclinical efficacy of sphingosine-1-phosphate (S1P) to improve acclimatization to simulated hypobaric hypoxia. Experimental Approach: Efficacy of intravenously administered S1P in improving hematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620 m for 6 hours) following S1P pre-treatment for three days. Major Findings: Altitude exposure of the control rats caused systemic hypoxia, hypocapnia (plausible sign of hyperventilation) and respiratory alkalosis due to suboptimal renal compensation indicated by an overt alkaline pH of the mixed venous blood. This was associated with pronounced energy deficit in the hepatic tissue along with systemic oxidative stress and inflammation. S1P pre-treatment improved blood oxygen-carrying-capacity by increasing hemoglobin, hematocrit, and RBC count, probably as an outcome of hypoxia inducible factor-1a mediated erythropoiesis and renal S1P receptor 1 mediated hemoconcentation. The improved partial pressure of oxygen in the blood could further restore aerobic respiration and increase ATP content in the hepatic tissue of S1P treated animals. S1P could also protect the animals from hypoxia mediated oxidative stress and inflammation. Conclusion: The study findings highlight S1P’s merits as a preconditioning agent for improving acclimatization to acute hypobaric hypoxia exposure. The results may have long term clinical application for improving physiological acclimatization of subjects venturing into high altitude for occupational or recreational purposes.

S1P Stabilizes HIF-1a and Boosts HIF-1a Mediated Hypoxia Adaptive Responses

S1P pre-conditioning led to 1.9 fold higher HIF-1a level in the kidney tissue (p<0.001) and 1.3 fold higher HIF-1a level in the liver tissue (p<0.001) in 1 mg/kg b.w. S1P group than in hypoxia control group. However, the hypoxia control group also had 1.3 folds higher HIF-1a levels in both liver and kidney tissues than in normoxia control groups, indicating a non-hypoxic boost of HIF-1a in S1P treated animals (Figure 1a and b). Further, plasma Epo levels were also observed to be significantly higher following S1P pre-treatment compared to the hypoxia control groups (p=0.05) (Figure 1a). Epo being primarily secreted by the kidneys and its expression being under regulation of HIF-1a, the raised plasma Epo level could be attributed to higher HIF-1a level in the kidney.

Figure 1. (not shown) Effect of S1P treatment on HIF-1a accumulation and downstream gene expression. a) Renal HIF-1a accumulation and Epo accumulation in plasma. HIF-1a accumulation in the renal tissue homogenate and build-up of erythropoietin in plasma was quantified. b) Hepatic HIF-1a accumulation. c) Effect S1P pre-treatment on circulatory VEGF. Vascular endothelial growth factor (VEGF) was quantified in plasma of experimental animals. These estimations were carried out using sandwich ELISA, and were carried out in triplicates for each experimental animal. Values are representative of mean 6 SD (n = 6). Statistical significance was calculated using ANOVA/post hoc Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 mg S1P/kg b.w., 10: 10 mg S1P/kg b.w., 100: 100 mg S1P/kg b.w., p<0.05 compared with the normoxic control, p<0.01 compared with the normoxic control, p<0.001 compared with the normoxic control, p<0.05 compared with the hypoxic control, p<0.01 compared with the hypoxic control, p<0.001 compared with the hypoxic control. http://dx.doi.org:/10.1371/journal.pone.0098025.g001

Figure 2.(not shown) Effect of S1P treatment on S1P1 expression in renal tissue. Representative immune-blot of S1P1. Densitometric analysis of blot normalized against the loading control (α-tubulin). Values are representative of mean 6 SD (n = 6). Statistical significance was calculated using ANOVA/post hoc Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 mg S1P/kg b.w., 10: 10 mg S1P/kg b.w., 100: 100 mg S1P/kg b.w., p<0.05 compared with the normoxic control, p<0.01 compared with the normoxic control, p<0.001 compared with the normoxic control, p< 0.05 compared with the hypoxic control, p<0.01 compared with the hypoxic control, p<0.001 compared with the hypoxic control. http://dx.doi.org:/10.1371/journal.pone.0098025.g002

Cloning of hypoxia-inducible factor 1α cDNA from a high hypoxia tolerant mammal—plateau pika (Ochotona curzoniae)

T.B. Zhao, H.X. Ning, S.S. Zhu, P. Sun, S.X. Xu, Z.J. Chang, and X.Q. Zhao
Biochemical and Biophysical Research Communications 316 (2004) 565–572
http://dx.doi.org:/10.1016/j.bbrc.2004.02.087

Hypoxia-inducible factor 1 is a transcription factor composed of HIF-1α and HIF-1β. It plays an important role in the signal transduction of cell response to hypoxia. Plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant and cold adaptation species living only at 3000–5000m above sea level on the Qinghai-Tibet Plateau. In this study, HIF-1α cDNA of plateau pika was cloned and its expression in various tissues was studied. The results indicated that plateau pika HIF-1α cDNA was highly identical to those of the human (82%), bovine (89%), mouse (82%), and Norway rat (77%). The deduced amino acid sequence (822 bp) showed 90%, 92%, 86%, and 86% identities with those of the human, bovine, house mouse, and Norway rat, respectively. Northern blot analyses detected two isoforms named pLHIF-1α and pSHIF-1α. The HIF-1α mRNA was highly expressed in the brain and kidney, and much less in the heart, lung, liver, muscle, and spleen, which was quite different from the expression pattern of mouse mRNA. Meanwhile, a new variant of plateau pika HIF-1α mRNA was identified by RT-PCR and characterized. The deduced protein, composed of 536 amino acids, lacks a part of the oxygen-dependent degradation domain (ODD), both transactivation domains (TADs), and the nuclear localization signal motif (NLS). Our results suggest that HIF-1α may play an important role in the pika’s adaptation to hypoxia, especially in brain and kidney, and pika HIF-1α function pattern may be different from that of mouse HIF-1α. Furthermore, for the high ratio of HIF-1α homology among the animals, the HIF-1α gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa.

Comparative Proteomics Analyses of Kobresia pygmaea Adaptation to Environment along an Elevational Gradient on the Central Tibetan Plateau

Xiong Li, Yunqiang Yang, Lan Ma, Xudong Sun, et al.
PLoS ONE 9(6): e98410. http://dx.doi.org:/10.1371/journal.pone.0098410

Variations in elevation limit the growth and distribution of alpine plants because multiple environmental stresses impact plant growth, including sharp temperature shifts, strong ultraviolet radiation exposure, low oxygen content, etc. Alpine plants have developed special strategies to help survive the harsh environments of high mountains, but the internal mechanisms remain undefined. Kobresia pygmaea, the dominant species of alpine meadows, is widely distributed in the Southeastern Tibet Plateau, Tibet Autonomous Region, China. In this study, we mainly used comparative proteomics analyses to investigate the dynamic protein patterns for K. pygmaea located at four different elevations (4600, 4800, 4950 and 5100 m). A total of 58 differentially expressed proteins were successfully detected and functionally characterized. The proteins were divided into various functional categories, including material and energy metabolism, protein synthesis and degradation, redox process, defense response, photosynthesis, and protein kinase. Our study confirmed that increasing levels of antioxidant and heat shock proteins and the accumulation of primary metabolites, such as proline and abscisic acid, conferred K. pygmaea with tolerance to the alpine environment. In addition, the various methods K. pygmaea used to regulate material and energy metabolism played important roles in the development of tolerance to environmental stress. Our results also showed that the way in which K. pygmaea mediated stomatal characteristics and photosynthetic pigments constitutes an enhanced adaptation to alpine environmental stress. According to these findings, we concluded that K. pygmaea adapted to the high-elevation environment on the Tibetan Plateau by aggressively accumulating abiotic stress related metabolites and proteins and by the various life events mediated by proteins. Based on the species flexible physiological and biochemical processes, we surmised that environment change has only a slight impact on K. pygmaea except for possible impacts to populations on vulnerable edges of the species’ range
Altered mitochondrial biogenesis and its fusion gene expression is involved in the high-altitude adaptation of rat lung

Loganathan Chitra, Rathanam Boopathy
Respiratory Physiology & Neurobiology 192 (2014) 74– 84
http://dx.doi.org/10.1016/j.resp.2013.12.007

Intermittent hypobaric hypoxia-induced preconditioning (IHH-PC) of rat favored the adaption of lungs to severe HH conditions, possibly through stabilization of mitochondrial function. This is based on the data generated on regulatory coordination of nuclear DNA-encoded mitochondrial biogenesis; dynamics,and mitochondrial DNA (mtDNA)-encoded oxidative phosphorylation (mt-OXPHOS) genes expression. At16th day after start of IHH-PC (equivalent to 5,000 m, 6 h/d, 2 w of treatment), rats were exposed to severe HH stimulation at 9142 m for 6 h. The IHH-PC significantly counteracted the HH-induced effect of increased lung: water content; tissue damage; and oxidant injury. Further, IHH-PC significantly increased the mitochondrial number, mtDNA content and mt- OXPHOS complex activity in the lung tissues. This observation is due to an increased expression of genes involved in mitochondrial biogenesis (PGC-1α,ERRα, NRF1, NRF2 and TFAM), fusion (Mfn1 and Mfn2) and mt OXPHOS. Thus, the regulatory pathway formed by PGC-1α/ERRα/Mfn2 axes is required for the mitochondrial adaptation provoked by IHH-PC regimen to counteract subsequent HH stress.

Molecular characteristics of Tibetan antelope (Pantholops hodgsonii) mitochondrial DNA control region and phylogenetic inferences with related species

Feng, B. Fan, K. Li, Q.D. Zhang, et al.
Small Ruminant Research 75 (2008) 236–242
http://dx.doi.org:/10.1016/j.smallrumres.2007.06.011

Although Tibetan antelope (Pantholops hodgsonii) is a distinctive wild species inhabiting the Tibet-Qinghai Plateau, its taxonomic classification within the Bovidae is still unclear and little molecular information has been reported to date. In this study of Tibetan antelope, the complete control regions of mtDNA were sequenced and compared to those of Tibetan sheep (Ovis aries) and goat (Capra hircus). The length of the control region in Tibetan antelope, sheep and goat is 1067, 1181/1106 and 1121 bp, respectively. A 75-bp repeat sequence was found near the 5’ end of the control region of Tibetan antelope and sheep, the repeat numbers of which were two in Tibetan antelope and three or four in sheep. Three major domain regions, including HVI, HVII and central domain, in Tibetan antelope, sheep and goat were outlined, as well as other less conserved blocks, such as CSB-1, CSB-2, ETAS-1 and ETAS-2. NJ cluster analysis of the three species revealed that Tibetan antelope was more closely related to Tibetan sheep than Tibetan goat. These results were further confirmed by phylogenetic analysis using the partial control region sequences of these and 13 other antelope species. Tibetan antelope is better assigned to the Caprinae rather than the Antilopinae subfamily of the Bovidae.

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Neonatal Pathophysiology

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Neonatal Pathophysiology

Writer and Curator: Larry H. Bernstein, MD, FCAP

Introduction

This curation deals with a large and specialized branch of medicine that grew since the mid 20th century in concert with the developments in genetics and as a result of a growing population, with large urban populations, increasing problems of premature deliveries. The problems of prematurity grew very preterm to very low birth weight babies with special problems. While there were nurseries, the need for intensive care nurseries became evident in the 1960s, and the need for perinatal care of pregnant mothers also grew as a result of metabolic problems of the mother, intrauterine positioning of the fetus, and increasing numbers of teen age pregnancies as well as nutritional problems of the mother. There was also a period when the manufacturers of nutritional products displaced the customary use of breast feeding, which was consequential. This discussion is quite comprehensive, as it involves a consideration of the heart, the lungs, the brain, and the liver, to a large extent, and also the kidneys and skeletal development.

It is possible to outline, with a proportionate emphasis based on frequency and severity, this as follows:

Genetic and metabolic diseases
Nervous system
Cardiovascular
Pulmonary
Skeletal – bone and muscle
Hematological
Liver
Esophagus, stomach, and intestines
Kidneys
Immune system

Fetal Development

Gestation is the period of time between conception and birth when a baby grows and develops inside the mother’s womb. Because it’s impossible to know exactly when conception occurs, gestational age is measured from the first day of the mother’s last menstrual cycle to the current date. It is measured in weeks. A normal gestation lasts anywhere from 37 to 41 weeks.

Week 5 is the start of the “embryonic period.” This is when all the baby’s major systems and structures develop. The embryo’s cells multiply and start to take on specific functions. This is called differentiation. Blood cells, kidney cells, and nerve cells all develop. The embryo grows rapidly, and the baby’s external features begin to form.

Week 6-9: Brain forms into five different areas. Some cranial nerves are visible. Eyes and ears begin to form. Tissue grows that will the baby’s spine and other bones. Baby’s heart continues to grow and now beats at a regular rhythm. Blood pumps through the main vessels. Your baby’s brain continues to grow. The lungs start to form. Limbs look like paddles. Essential organs begin to grow.

Weeks 11-18: Limbs extended. Baby makes sucking motion. Movement of limbs. Liver and pancreas produce secretions. Muscle and bones developing.

Week 19-21: Baby can hear. Mom feels baby – and quickening.

http://www.nlm.nih.gov/medlineplus/ency/article/002398.htm

fetal-development

https://polination.files.wordpress.com/2014/02/abortion-new-research-into-fetal-development.jpg

Inherited Metabolic Disorders

The original cause of most genetic metabolic disorders is a gene mutation that occurred many, many generations ago. The gene mutation is passed along through the generations, ensuring its preservation.

Each inherited metabolic disorder is quite rare in the general population. Considered all together, inherited metabolic disorders may affect about 1 in 1,000 to 2,500 newborns. In certain ethnic populations, such as Ashkenazi Jews (Jews of central and eastern European ancestry), the rate of inherited metabolic disorders is higher.

Hundreds of inherited metabolic disorders have been identified, and new ones continue to be discovered. Some of the more common and important genetic metabolic disorders include:

Lysosomal storage disorders : Lysosomes are spaces inside cells that break down waste products of metabolism. Various enzyme deficiencies inside lysosomes can result in buildup of toxic substances, causing metabolic disorders including:

Hurler syndrome (abnormal bone structure and developmental delay)
Niemann-Pick disease (babies develop liver enlargement, difficulty feeding, and nerve damage)
Tay-Sachs disease (progressive weakness in a months-old child, progressing to severe nerve damage; the child usually lives only until age 4 or 5)
Gauchers disease and others

Galactosemia: Impaired breakdown of the sugar galactose leads to jaundice, vomiting, and liver enlargement after breast or formula feeding by a newborn.

Maple syrup urine disease: Deficiency of an enzyme called BCKD causes buildup of amino acids in the body. Nerve damage results, and the urine smells like syrup.

Phenylketonuria (PKU): Deficiency of the enzyme PAH results in high levels of phenylalanine in the blood. Mental retardation results if the condition is not recognized.

Glycogen storage diseases: Problems with sugar storage lead to low blood sugar levels, muscle pain, and weakness.

Metal metabolism disorders: Levels of trace metals in the blood are controlled by special proteins. Inherited metabolic disorders can result in protein malfunction and toxic accumulation of metal in the body:

Wilson disease (toxic copper levels accumulate in the liver, brain, and other organs)

Hemochromatosis (the intestines absorb excessive iron, which builds up in the liver, pancreas, joints, and heart, causing damage)

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

Red cell enzyme disorders – glucose-6-phosphate dehydrogenase, pyruvate kinase

This list is by no means complete.

http://www.webmd.com/a-to-z-guides/inherited-metabolic-disorder-types-and-treatments

New variations in the galactose-1-phosphate uridyltransferase (GALT) gene

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: Structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene

E Viggiano, A Marabotti, AP Burlina, C Cazzorla, MR D’Apice, et al.
Gene 559 (2015) 112–118
http://dx.doi.org/10.1016/j.gene.2015.01.013
Galactosemia (OMIM 230400) is a rare autosomal recessive inherited disorder caused by deficiency of galactose-1-phosphate uridyltransferase (GALT; OMIM 606999) activity. The incidence of galactosemia is 1 in 30,000–60,000, with a prevalence of 1 in 47,000 in the white population. Neonates with galactosemia can present acute symptoms, such as severe hepatic and renal failure, cataract and sepsis after milk introduction. Dietary restriction of galactose determines the clinical improvement in these patients. However, despite early diagnosis by neonatal screening and dietary treatment, a high percentage of patients develop long-term complications such as cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction.

With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30 years.

No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications.

A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of < 1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype–phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Diagnosis and Management of Hereditary Hemochromatosis

Reena J. Salgia, Kimberly Brown
Clin Liver Dis 19 (2015) 187–198
http://dx.doi.org/10.1016/j.cld.2014.09.011

Hereditary hemochromatosis (HH) is a diagnosis most commonly made in patients with elevated iron indices (transferrin saturation and ferritin), and HFE genetic mutation testing showing C282Y homozygosity.

The HFE mutation is believed to result in clinical iron overload through altering hepcidin levels resulting in increased iron absorption.

The most common clinical complications of HH include cirrhosis, diabetes, nonischemic cardiomyopathy, and hepatocellular carcinoma.

Liver biopsy should be performed in patients with HH if the liver enzymes are elevated or serum ferritin is greater than 1000 mg/L. This is useful to determine the degree of iron overload and stage the fibrosis.

Treatment of HH with clinical iron overload involves a combination of phlebotomy and/or chelation therapy. Liver transplantation should be considered for patients with HH-related decompensated cirrhosis.

Health economic evaluation of plasma oxysterol screening in the diagnosis of Niemann–Pick Type C disease among intellectually disabled using discrete event simulation

CDM van Karnebeek, Tima Mohammadi, Nicole Tsaod, Graham Sinclair, et al.
Molecular Genetics and Metabolism 114 (2015) 226–232
http://dx.doi.org/10.1016/j.ymgme.2014.07.004

Background: Recently a less invasive method of screening and diagnosing Niemann–Pick C (NP-C) disease has emerged. This approach involves the use of a metabolic screening test (oxysterol assay) instead of the current practice of clinical assessment of patients suspected of NP-C (review of medical history, family history and clinical examination for the signs and symptoms). Our objective is to compare costs and outcomes of plasma oxysterol screening versus current practice in diagnosis of NP-C disease among intellectually disabled (ID) patients using decision-analytic methods.
Methods: A discrete event simulation model was conducted to follow ID patients through the diagnosis and treatment of NP-C, forecast the costs and effectiveness for a cohort of ID patients and compare the outcomes and costs in two different arms of the model: plasma oxysterol screening and routine diagnosis procedure (anno 2013) over 5 years of follow up. Data from published sources and clinical trials were used in simulation model. Unit costs and quality-adjusted life-years (QALYs) were discounted at a 3% annual rate in the base case analysis. Deterministic and probabilistic sensitivity analyses were conducted.
Results: The outcomes of the base case model showed that using plasma oxysterol screening for diagnosis of NP-C disease among ID patients is a dominant strategy. It would result in lower total cost and would slightly improve patients’ quality of life. The average amount of cost saving was $3642 CAD and the incremental QALYs per each individual ID patient in oxysterol screening arm versus current practice of diagnosis NP-C was 0.0022 QALYs. Results of sensitivity analysis demonstrated robustness of the outcomes over the wide range of changes in model inputs.
Conclusion: Whilst acknowledging the limitations of this study, we conclude that screening ID children and adolescents with oxysterol tests compared to current practice for the diagnosis of NP-C is a dominant strategy with clinical and economic benefits. The less costly, more sensitive and specific oxysterol test has potential to save costs to the healthcare system while improving patients’ quality of life and may be considered as a routine tool in the NP-C diagnosis armamentarium for ID. Further research is needed to elucidate its effectiveness in patients presenting characteristics other than ID in childhood and adolescence.

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

Maria Bondesson, Ruixin Hao, Chin-Yo Lin, Cecilia Williams, Jan-Åke Gustafsson
Biochimica et Biophysica Acta 1849 (2015) 142–151
http://dx.doi.org/10.1016/j.bbagrm.2014.06.005

Estrogen receptors are expressed and their cognate ligands produced in all vertebrates, indicative of important and conserved functions. Through evolution estrogen has been involved in controlling reproduction, affectingboth the development of reproductive organs and reproductive behavior. This review broadly describes the synthesis of estrogens and the expression patterns of aromatase and the estrogen receptors, in relation to estrogen functions in the developing fetus and child. We focus on the role of estrogens for the development of reproductive tissues, as well as non-reproductive effects on the developing brain. We collate data from human, rodent, bird and fish studies and highlight common and species-specific effects of estrogen signaling on fetal development. Morphological malformations originating from perturbed estrogen signaling in estrogen receptor and aromatase knockout mice are discussed, as well as the clinical manifestations of rare estrogen receptor alpha and aromatase gene mutations in humans. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Memory function and hippocampal volumes in preterm born very-low-birth-weight (VLBW) young adults

Synne Aanes, Knut Jørgen Bjuland, Jon Skranes, Gro C.C. Løhaugen
NeuroImage 105 (2015) 76–83
http://dx.doi.org/10.1016/j.neuroimage.2014.10.023

The hippocampi are regarded as core structures for learning and memory functions, which is important for daily functioning and educational achievements. Previous studies have linked reduction in hippocampal volume to working memory problems in very low birth weight (VLBW; ≤1500 g) children and reduced general cognitive ability in VLBW adolescents. However, the relationship between memory function and hippocampal volume has not been described in VLBW subjects reaching adulthood. The aim of the study was to investigate memory function and hippocampal volume in VLBW young adults, both in relation to perinatal risk factors and compared to term born controls, and to look for structure–function relationships. Using Wechsler Memory Scale-III and MRI, we included 42 non-disabled VLBW and 61 control individuals at age 19–20 years, and related our findings to perinatal risk factors in the VLBW-group. The VLBW young adults achieved lower scores on several subtests of the Wechsler Memory Scale-III, resulting in lower results in the immediate memory indices (visual and auditory), the working memory index, and in the visual delayed and general memory delayed indices, but not in the auditory delayed and auditory recognition delayed indices. The VLBW group had smaller absolute and relative hippocampal volumes than the controls. In the VLBW group inferior memory function, especially for the working memory index, was related to smaller hippocampal volume, and both correlated with lower birth weight and more days in the neonatal intensive care unit (NICU). Our results may indicate a structural–functional relationship in the VLBW group due to aberrant hippocampal development and functioning after preterm birth.

The relation of infant attachment to attachment and cognitive and behavioural outcomes in early childhood

Yan-hua Ding, Xiu Xua, Zheng-yan Wang, Hui-rong Li, Wei-ping Wang
Early Human Development 90 (2014) 459–464
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.004

Background: In China, research on the relation of mother–infant attachment to children’s development is scarce.
Aims: This study sought to investigate the relation of mother–infant attachment to attachment, cognitive and behavioral development in young children. Study design: This study used a longitudinal study design.
Subjects: The subjects included healthy infants (n=160) aged 12 to 18 months.
Outcome measures: Ainsworth’s “Strange Situation Procedure” was used to evaluate mother–infant attachment types. The attachment Q-set (AQS) was used to evaluate the attachment between young children and their mothers. The Bayley scale of infant development-second edition (BSID-II) was used to evaluate cognitive developmental level in early childhood. Achenbach’s child behavior checklist (CBCL) for 2- to 3-year-oldswas used to investigate behavioral problems.
Results: In total, 118 young children (73.8%) completed the follow-up; 89.7% of infants with secure attachment and 85.0% of infants with insecure attachment still demonstrated this type of attachment in early childhood (κ = 0.738, p b 0.05). Infants with insecure attachment collectively exhibited a significantly lower mental development index (MDI) in early childhood than did infants with secure attachment, especially the resistant type. In addition, resistant infants were reported to have greater social withdrawal, sleep problems and aggressive behavior in early childhood.
Conclusion: There is a high consistency in attachment development from infancy to early childhood. Secure mother–infant attachment predicts a better cognitive and behavioral outcome; whereas insecure attachment, especially the resistant attachment, may lead to a lower cognitive level and greater behavioral problems in early childhood.

representations of the HPA axis

representations of limbic stress-integrative pathways from the prefrontal cortex, amygdala and hippocampus

Fetal programming of schizophrenia: Select mechanisms

Monojit Debnatha, Ganesan Venkatasubramanian, Michael Berk
Neuroscience and Biobehavioral Reviews 49 (2015) 90–104
http://dx.doi.org/10.1016/j.neubiorev.2014.12.003

Mounting evidence indicates that schizophrenia is associated with adverse intrauterine experiences. An adverse or suboptimal fetal environment can cause irreversible changes in brain that can subsequently exert long-lasting effects through resetting a diverse array of biological systems including endocrine, immune and nervous. It is evident from animal and imaging studies that subtle variations in the intrauterine environment can cause recognizable differences in brain structure and cognitive functions in the offspring. A wide variety of environmental factors may play a role in precipitating the emergent developmental dysregulation and the consequent evolution of psychiatric traits in early adulthood by inducing inflammatory, oxidative and nitrosative stress (IO&NS) pathways, mitochondrial dysfunction, apoptosis, and epigenetic dysregulation. However, the precise mechanisms behind such relationships and the specificity of the risk factors for schizophrenia remain exploratory. Considering the paucity of knowledge on fetal programming of schizophrenia, it is timely to consolidate the recent advances in the field and put forward an integrated overview of the mechanisms associated with fetal origin of schizophrenia.

NMDA receptor dysfunction in autism spectrum disorders

Eun-Jae Lee, Su Yeon Choi and Eunjoon Kim
Current Opinion in Pharmacology 2015, 20:8–13
http://dx.doi.org/10.1016/j.coph.2014.10.007

Autism spectrum disorders (ASDs) represent neurodevelopmental disorders characterized by two core symptoms;

(1) impaired social interaction and communication, and
(2) restricted and repetitive behaviors, interests, and activities.

ASDs affect ~ 1% of the population, and are considered to be highly genetic in nature. A large number (~600) of ASD-related genetic variations have been identified (sfari.org), and target gene functions are apparently quite diverse. However, some fall onto common pathways, including synaptic function and chromosome remodeling, suggesting that core mechanisms may exist.

Abnormalities and imbalances in neuronal excitatory and inhibitory synapses have been implicated in diverse neuropsychiatric disorders including autism spectrum disorders (ASDs). Increasing evidence indicates that dysfunction of NMDA receptors (NMDARs) at excitatory synapses is associated with ASDs. In support of this, human ASD-associated genetic variations are found in genes encoding NMDAR subunits. Pharmacological enhancement or suppression of NMDAR function ameliorates ASD symptoms in humans. Animal models of ASD display bidirectional NMDAR dysfunction, and correcting this deficit rescues ASD-like behaviors. These findings suggest that deviation of NMDAR function in either direction contributes to the development of ASDs, and that correcting NMDAR dysfunction has therapeutic potential for ASDs.

Among known synaptic proteins implicated in ASD are metabotropic glutamate receptors (mGluRs). Functional enhancement and suppression of mGluR5 are associated with fragile X syndrome and tuberous sclerosis, respectively, which share autism as a common phenotype. More recently, ionotropic glutamate receptors, namely NMDA receptors (NMDARs) and AMPA receptors (AMPARs), have also been implicated in ASDs. In this review, we will focus on NMDA receptors and summarize evidence supporting the hypothesis that NMDAR dysfunction contributes to ASDs, and, by extension, that correcting NMDAR dysfunction has therapeutic potential for ASDs. ASD-related human NMDAR genetic variants.

Chemokines roles within the hippocampus

IL-1 mediates stress-induced activation of the HPA axis

A systemic model of the beneficial role of immune processes in behavioral and neural plasticity

Three Classes of Glutamate Receptors

Clinical studies on ASDs have identified genetic variants of NMDAR subunit genes. Specifically, de novo mutations have been identified in the GRIN2B gene, encoding the GluN2B subunit. In addition, SNP analyses have linked both GRIN2A (GluN2A subunit) and GRIN2B with ASDs. Because assembled NMDARs contain four subunits, each with distinct properties, ASD-related GRIN2A/ GRIN2B variants likely alter the functional properties of NMDARs and/or NMDAR-dependent plasticity.

Pharmacological modulation of NMDAR function can improve ASD symptoms. D-cycloserine (DCS), an NMDAR agonist, significantly ameliorates social withdrawal and repetitive behavior in individuals with ASD. These results suggest that reduced NMDAR function may contribute to the development of ASDs in humans.

We can divide animal studies into two groups. The first group consists of animals in which NMDAR modulators were shown to normalize both NMDAR dysfunction and ASD-like behaviors, establishing strong association between NMDARs and ASD phenotypes (Fig.). In the second group, NMDAR modulators were shown to rescue ASD-like behaviors, but NMDAR dysfunction and its correction have not been demonstrated.

ASD models with data showing rescue of both NMDAR dysfunction and ASD like behaviors Mice lacking neuroligin-1, an excitatory postsynaptic adhesion molecule, show reduced NMDAR function in the hippocampus and striatum, as evidenced by a decrease in NMDA/AMPA ratio and long-term potentiation (LTP). Neuroligin-1 is thought to enhance synaptic NMDAR function, by directly interacting with and promoting synaptic localization of NMDARs.

Fig not shown.

Bidirectional NMDAR dysfunction in animal models of ASD. Animal models of ASD with bidirectional NMDAR dysfunction can be positioned on either side of an NMDAR function curve. Model animals were divided into two groups.

Group 1: NMDAR modulators normalize both NMDAR dysfunction and ASD-like behaviors (green).

Group 2: NMDAR modulators rescue ASD-like behaviors, but NMDAR dysfunction and its rescue have not been demonstrated (orange). Note that Group 2 animals are tentatively placed on the left-hand side of the slope based on the observed DCS rescue of their ASD-like phenotypes, but the directions of their NMDAR dysfunctions remain to be experimentally determined.

ASD models with data showing rescue of ASD-like behaviors but no demonstrated NMDAR dysfunction

Tbr1 is a transcriptional regulator, one of whose targets is the gene encoding the GluN2B subunit of NMDARs. Mice haploinsufficient for Tbr1 (Tbr1+/-) show structural abnormalities in the amygdala and limited GluN2B induction upon behavioral stimulation. Both systemic injection and local amygdalar infusion of DCS rescue social deficits and impaired associative memory in Tbr1+/- mice. However, reduced NMDAR function and its DCS-dependent correction have not been demonstrated.

Spatial working memory and attention skills are predicted by maternal stress during pregnancy

André Plamondon, Emis Akbari, Leslie Atkinson, Meir Steiner
Early Human Development 91 (2015) 23–29
http://dx.doi.org/10.1016/j.earlhumdev.2014.11.004

Introduction: Experimental evidence in rodents shows that maternal stress during pregnancy (MSDP) negatively impacts spatial learning and memory in the offspring. We aim to investigate the association between MSDP (i.e., life events) and spatial working memory, as well as attention skills (attention shifting and attention focusing), in humans. The moderating roles of child sex, maternal anxiety during pregnancy and postnatal care are also investigated. Methods: Participants were 236mother–child dyads that were followed from the second trimester of pregnancy until 4 years postpartum. Measurements included questionnaires and independent observations.
Results: MSDP was negatively associated with attention shifting at 18monthswhen concurrent maternal anxiety was low. MSDP was associated with poorer spatial working memory at 4 years of age, but only for boys who experienced poorer postnatal care.
Conclusion: Consistent with results observed in rodents, MSDP was found to be associated with spatial working memory and attention skills. These results point to postnatal care and maternal anxiety during pregnancy as potential targets for interventions that aim to buffer children from the detrimental effects of MSDP.

Acute and massive bleeding from placenta previa and infants’ brain damage

Ken Furuta, Shuichi Tokunaga, Seishi Furukawa, Hiroshi Sameshima
Early Human Development 90 (2014) 455–458
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.002

Background: Among the causes of third trimester bleeding, the impact of placenta previa on cerebral palsy is not well known.
Aims: To clarify the effect ofmaternal bleeding fromplacenta previa on cerebral palsy, and in particular when and how it occurs.
Study design: A descriptive study.
Subjects: Sixty infants born to mothers with placenta previa in our regional population-based study of 160,000 deliveries from 1998 to 2012. Premature deliveries occurring atb26 weeks of gestation and placenta accrete were excluded.
Outcome measures: Prevalence of cystic periventricular leukomalacia (PVL) and cerebral palsy (CP).
Results: Five infants had PVL and 4 of these infants developed CP (1/40,000 deliveries). Acute and massive bleeding (>500 g) within 8 h) occurred at around 30–31 weeks of gestation, and was severe enough to deliver the fetus. None of the 5 infants with PVL underwent antenatal corticosteroid treatment, and 1 infant had mild neonatal hypocapnia with a PaCO2 < 25 mm Hg. However, none of the 5 PVL infants showed umbilical arterial academia with pH < 7.2, an abnormal fetal heart rate monitoring pattern, or neonatal hypotension.
Conclusions: Our descriptive study showed that acute and massive bleeding from placenta previa at around 30 weeks of gestation may be a risk factor for CP, and requires careful neonatal follow-up. The underlying process connecting massive placental bleeding and PVL requires further investigation.

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

Courtney J. Wusthoff, Irene M. Loe
Seminars in Fetal & Neonatal Medicine 20 (2015) 52e57
http://dx.doi.org/10.1016/j.siny.2014.12.003

Extreme neonatal hyperbilirubinemia has long been known to cause the clinical syndrome of kernicterus, or chronic bilirubin encephalopathy (CBE). Kernicterus most usually is characterized by choreoathetoid cerebral palsy (CP), impaired upward gaze, and sensorineural hearing loss, whereas cognition is relatively spared. The chronic condition of kernicterus may be, but is not always, preceded in the acute stage by acute bilirubin encephalopathy (ABE). This acute neonatal condition is also due to hyperbilirubinemia, and is characterized by lethargy and abnormal behavior, evolving to frank neonatal encephalopathy, opisthotonus, and seizures. Less completely defined is the syndrome of bilirubin-induced neurologic dysfunction (BIND).

Bilirubin-induced neurologic dysfunction (BIND) is the constellation of neurologic sequelae following milder degrees of neonatal hyperbilirubinemia than are associated with kernicterus. Clinically, BIND may manifest after the neonatal period as developmental delay, cognitive impairment, disordered executive function, and behavioral and psychiatric disorders. However, there is controversy regarding the relative contribution of neonatal hyperbilirubinemia versus other risk factors to the development of later neurodevelopmental disorders in children with BIND. In this review, we focus on the empiric data from the past 25 years regarding neurodevelopmental outcomes and BIND, including specific effects on developmental delay, cognition, speech and language development, executive function, and the neurobehavioral disorders, such as attention deficit/hyperactivity disorder and autism.

As noted in a technical report by the American Academy of Pediatrics Subcommittee on Hyperbilirubinemia, “it is apparent that the use of a single total serum bilirubin level to predict long-term outcomes is inadequate and will lead to conflicting results”. As described above, this has certainly been the case in research to date. To clarify how hyperbilirubinemia influences neurodevelopmental outcome, more sophisticated consideration is needed both of how to assess bilirubin exposure leading to neurotoxicity, and of those comorbid conditions which may lower the threshold for brain injury.

For example, premature infants are known to be especially susceptible to bilirubin neurotoxicity, with kernicterus reported following TB levels far lower than the threshold expected in term neonates. Similarly, among extremely preterm neonates, BBC is proportional to gestational age, meaning that the most premature infants have the highest UB, even for similar TB levels. Thus, future studies must be adequately powered to examine preterm infants separately from term infants, and should consider not just peak TB, but also BBC, as independent variables in neonates with hyperbilirubinemia. Similarly, an analysis by the NICHD NRN found that, among ELBW infants, higher UB levels were associated with a higher risk of death or NDI. However, increased TB levels were only associated with death or NDI in unstable infants. Again, UB or BBC appeared to be more useful than TB.

Are the neuromotor disabilities of bilirubin-induced neurologic dysfunction disorders related to the cerebellum and its connections?

Jon F. Watchko, Michael J. Painter, Ashok Panigrahy
Seminars in Fetal & Neonatal Medicine 20 (2015) 47e51
http://dx.doi.org/10.1016/j.siny.2014.12.004

Investigators have hypothesized a range of subcortical neuropathology in the genesis of bilirubin induced neurologic dysfunction (BIND). The current review builds on this speculation with a specific focus on the cerebellum and its connections in the development of the subtle neuromotor disabilities of BIND. The focus on the cerebellum derives from the following observations:
(i) the cerebellum is vulnerable to bilirubin-induced injury; perhaps the most vulnerable region within the central nervous system;
(ii) infants with cerebellar injury exhibit a neuromotor phenotype similar to BIND; and (iii) the cerebellum has extensive bidirectional circuitry projections to motor and non-motor regions of the brain-stem and cerebral cortex that impact a variety of neurobehaviors.
Future study using advanced magnetic resonance neuroimaging techniques have the potential to shed new insights into bilirubin’s effect on neural network topology via both structural and functional brain connectivity measurements.

Bilirubin-induced neurologic damage is most often thought of in terms of severe adverse neuromotor (dystonia with or without athetosis) and auditory (hearing impairment or deafness) sequelae. Observed together, they comprise the classic neurodevelopmental phenotype of chronic bilirubin encephalopathy or kernicterus, and may also be seen individually as motor or auditory predominant subtypes. These injuries reflect both a predilection of bilirubin toxicity for neurons (relative to glial cells) and the regional topography of bilirubin-induced neuronal damage characterized by prominent involvement of the globus pallidus, subthalamic nucleus, VIII cranial nerve, and cochlear nucleus.

It is also asserted that bilirubin neurotoxicity may be associated with other less severe neurodevelopmental disabilities, a condition termed “subtle kernicterus” or “bilirubin-induced neurologic dysfunction” (BIND). BIND is defined by a constellation of “subtle neurodevelopmental disabilities without the classical findings of kernicterus that, after careful evaluation and exclusion of other possible etiologies, appear to be due to bilirubin neurotoxicity”. These purportedly include:

(i) mild-to-moderate disorders of movement (e.g., incoordination, clumsiness, gait abnormalities, disturbances in static and dynamic balance, impaired fine motor skills, and ataxia); (ii) disturbances in muscle tone; and
(iii) altered sensorimotor integration. Isolated disturbances of central auditory processing are also included in the spectrum of BIND.

Cerebellar vulnerability to bilirubin-induced injury
Cerebellar injury phenotypes and BIND
Cerebellar projections

Transverse section of cerebellum and brainstem

Transverse section of cerebellum and brain-stem from a 34 gestational-week premature kernicteric infant formalin-fixed for two weeks. Yellow staining is evident in the cerebellar dentate nuclei (upper arrow) and vestibular nuclei at the pontomedullary junction (lower arrowhead). Photo is courtesy of Mahmdouha Ahdab-Barmada and reprinted with permission from Taylor-Francis Group (Ahdab Barmada M. The neuropathology of kernicterus: definitions and debate. In: Maisel MJ, Watchko JF editors. Neonatal jaundice. Amsterdam: Harwood Academic Publishers; 2000. p. 75e88

Whether cerebellar injury is primal or an integral part of disturbed neural circuitry in bilirubin-induced CNS damage is unclear. Movement disorders, however, are increasingly recognized to arise from abnormalities of neuronal circuitry rather than localized, circumscribed lesions. The cerebellum has extensive bidirectional circuitry projections to an array of brainstem nuclei and the cerebral cortex that modulate and refine motor activities. In this regard, the cerebellum is characteristically subdivided into three lobes based on neuroanatomic and phylogenetic criteria as well as by their primary afferent and efferent connections. They include:
(i) flocculonodular lobe (archicerebellum);
(ii) anterior lobe (paleocerebellum); and
(iii) posterior lobe (neocerebellum).

The archicerebellum, the oldest division phylogenically, receives extensive input from the vestibular system and is therefore also known as the vestibulocerebellum and is important for equilibrium control. The paleocerebellum, also a primitive region, receives extensive somatosensory input from the spinal cord, including the anterior and posterior spinocerebellar pathways that convey unconscious proprioception, and is therefore also known as the spinocerebellum. The neocerebellum is the most recently evolved region, receives most of the input from the cerebral cortex, and is thus termed the cerebrocerebellum. This area has greatly expanded in association with the extensive development of the cerebral cortex in mammals and especially primates. To cause serious longstanding dysfunction, cerebellar injury must typically involve the deep cerebellar nuclei and their projections.

Schematic of the bidirectional connectivity between the cerebellum and other

Schematic of the bidirectional connectivity between the cerebellum and other brain regions including the cerebral cortex. Most cerebro-cerebellar afferent projections pass through the basal (anterior or ventral) pontine nuclei and intermediate cerebellar peduncle, whereas most cerebello-cerebral efferent projections pass through the dentate and ventrolateral thalamic nuclei. DCN, deep cerebellar nuclei; RN, red nucleus; ATN, anterior thalamic nucleus; PFC, prefrontal cortex; MC, motor cortex; PC, parietal cortex; TC, temporal cortex; STN, subthalamic nucleus; APN, anterior pontine nuclei. Reprinted under the terms of the Creative Commons Attribution License from D’Angelo E, Casali S. Seeking a unified framework for cerebellar function and dysfunction: from circuit to cognition. Front Neural Circuits 2013; 6:116.

Given the vulnerability of the cerebellum to bilirubin-induced injury, cerebellar involvement should also be evident in classic kernicterus, contributing to neuromotor deficits observed therein. It is of interest, therefore, that cerebellar damage may play a role in the genesis of bilirubin-induced dystonia, a prominent neuromotor feature of chronic bilirubin encephalopathy in preterm and term neonates alike. This complex movement disorder is characterized by involuntary sustained muscle contractions that result in abnormal position and posture. Moreover, dystonia that is brief in duration results in chorea, and, if brief and repetitive, leads to athetosis ‒ conditions also classically observed in kernicterus. Recent evidence suggests that dystonic movements may depend on disruption of both basal ganglia and cerebellar neuronal networks, rather than isolated dysfunction of only one motor system.

Dystonia is also a prominent feature in Gunn rat pups and neonatal Ugt1‒/‒-deficient mice both robust models of kernicterus. The former is used as an experimental model of dystonia. Although these models show basal ganglia injury, the sine qua non of bilirubin-induced murine neuropathology is cerebellar damage and resultant cerebellar hypoplasia.

Studies are needed to define more precisely the motor network abnormalities in kernicterus and BIND. Magnetic resonance imaging (MRI) has been widely used in evaluating infants at risk for bilirubin-induced brain injury using conventional structural T1-and T2-weighted imaging. Infants with chronic bilirubin encephalopathy often demonstrate abnormal bilateral, symmetric, high-signal intensity on T2-weighted MRI of the globus pallidus and subthalamic nucleus, consistent with the neuropathology of kernicterus. Early postnatal MRI of at-risk infants, although frequently showing increased T1-signal in these regions, may give false-positive findings due to the presence of myelin in these structures.

Diffusion tensor imaging and tractography could be used to delineate long-term changes involving specific white matter pathways, further elucidating the neural basis of long-term disability in infants and children with chronic bilirubin encephalopathy and BIND. It will be equally valuable to use blood oxygen level-dependent (BOLD) “resting state” functional MRI to study intrinsic connectivity in order to identify vulnerable brain networks in neonates with kernicterus and BIND. Structural networks of the CNS (connectome) and functional network topology can be characterized in infants with kernicterus and BIND to determine disease-related pattern(s) with respect to both long- and short-range connectivity. These findings have the potential to shed novel insights into the pathogenesis of these disorders and their impact on complex anatomical connections and resultant functional deficits.

Audiologic impairment associated with bilirubin-induced neurologic damage

Cristen Olds, John S. Oghalai
Seminars in Fetal & Neonatal Medicine 20 (2015) 42e46
http://dx.doi.org/10.1016/j.siny.2014.12.006

Hyperbilirubinemia affects up to 84% of term and late preterm infants in the first week of life. The elevation of total serum/plasma bilirubin (TB) levels is generally mild, transitory, and, for most children, inconsequential. However, a subset of infants experiences lifelong neurological sequelae. Although the prevalence of classic kernicterus has fallen steadily in the USA in recent years, the incidence of jaundice in term and premature infants has increased, and kernicterus remains a significant problem in the global arena. Bilirubin-induced neurologic dysfunction (BIND) is a spectrum of neurological injury due to acute or sustained exposure of the central nervous system(CNS) to bilirubin. The BIND spectrum includes kernicterus, acute bilirubin encephalopathy, and isolated neural pathway dysfunction.

Animal studies have shown that unconjugated bilirubin passively diffuses across cell membranes and the blood‒brain barrier (BBB), and bilirubin not removed by organic anion efflux pumps accumulates within the cytoplasm and becomes toxic. Exposure of neurons to bilirubin results in increased oxidative stress and decreased neuronal proliferation and presynaptic neuro-degeneration at central glutaminergic synapses. Furthermore, bilirubin administration results in smaller spiral ganglion cell bodies, with decreased cellular density and selective loss of large cranial nerve VIII myelinated fibers. When exposed to bilirubin, neuronal supporting cells have been found to secrete inflammatory markers, which contribute to increased BBB permeability and bilirubin loading.

The jaundiced Gunn rat is the classic animal model of bilirubin toxicity. It is homozygous for a premature stop codon within the gene for UDP-glucuronosyltransferase family 1 (UGT1). The resultant gene product has reduced bilirubin-conjugating activity, leading to a state of hyperbilirubinemia. Studies with this rat model have led to the concept that impaired calcium homeostasis is an important mechanism of neuronal toxicity, with reduced expression of calcium-binding proteins in affected cells being a sensitive index of bilirubin-induced neurotoxicity. Similarly, application of bilirubin to cultured auditory neurons from brainstem cochlear nuclei results in hyperexcitability and excitotoxicity.

The auditory pathway and normal auditory brainstem response (ABR).

The auditory pathway and normal auditory brain-stem response (ABR). The ipsilateral (green) and contralateral (blue) auditory pathways are shown, with structures that are known to be affected by hyperbilirubinemia highlighted in red. Roman numerals in parentheses indicate corresponding waves in the normal human ABR (inset). Illustration adapted from the “Ear Anatomy” series by Robert Jackler and Christine Gralapp, with permission.

Bilirubin-induced neurologic dysfunction (BIND)

Vinod K. Bhutani, Ronald Wong
Seminars in Fetal & Neonatal Medicine 20 (2015) 1
http://dx.doi.org/10.1016/j.siny.2014.12.010

Beyond the traditional recognized areas of fulminant injury to the globus pallidus as seen in infants with kernicterus, other vulnerable areas include the cerebellum, hippocampus, and subthalamic nuclear bodies as well as certain cranial nerves. The hippocampus is a brain region that is particularly affected by age related morphological changes. It is generally assumed that a loss in hippocampal volume results in functional deficits that contribute to age-related cognitive deficits. Lower grey matter volumes within the limbic-striato-thalamic circuitry are common to other etiological mechanisms of subtle neurologic injury. Lower grey matter volumes in the amygdala, caudate, frontal and medial gyrus are found in schizophrenia and in the putamen in autism. Thus, in terms of brain volumetrics, schizophrenia and autism spectrum disorders have a clear degree of overlap that may reflect shared etiological mechanisms. Overlap with injuries observed in infants with BIND raises the question about how these lesions are arrived at in the context of the impact of common etiologies.

Stress-induced perinatal and transgenerational epigenetic programming of brain development and mental health

Olena Babenko, Igor Kovalchuk, Gerlinde A.S. Metz
Neuroscience and Biobehavioral Reviews 48 (2015) 70–91
http://dx.doi.org/10.1016/j.neubiorev.2014.11.013

Research efforts during the past decades have provided intriguing evidence suggesting that stressful experiences during pregnancy exert long-term consequences on the future mental wellbeing of both the mother and her baby. Recent human epidemiological and animal studies indicate that stressful experiences in utero or during early life may increase the risk of neurological and psychiatric disorders, arguably via altered epigenetic regulation. Epigenetic mechanisms, such as miRNA expression, DNA methylation, and histone modifications are prone to changes in response to stressful experiences and hostile environmental factors. Altered epigenetic regulation may potentially influence fetal endocrine programming and brain development across several generations. Only recently, however, more attention has been paid to possible transgenerational effects of stress. In this review we discuss the evidence of transgenerational epigenetic inheritance of stress exposure in human studies and animal models. We highlight the complex interplay between prenatal stress exposure, associated changes in miRNA expression and DNA methylation in placenta and brain and possible links to greater risks of schizophrenia, attention deficit hyperactivity disorder, autism, anxiety- or depression-related disorders later in life. Based on existing evidence, we propose that prenatal stress, through the generation of epigenetic alterations, becomes one of the most powerful influences on mental health in later life. The consideration of ancestral and prenatal stress effects on lifetime health trajectories is critical for improving strategies that support healthy development and successful aging.

Sensitive time-windows for susceptibility in neurodevelopmental disorders

Rhiannon M. Meredith, Julia Dawitz and Ioannis Kramvis
Trends in Neurosciences, June 2012; 35(6): 335-344
http://dx.doi.org:/10.1016/j.tins.2012.03.005

Many neurodevelopmental disorders (NDDs) are characterized by age-dependent symptom onset and regression, particularly during early postnatal periods of life. The neurobiological mechanisms preceding and underlying these developmental cognitive and behavioral impairments are, however, not clearly understood. Recent evidence using animal models for monogenic NDDs demonstrates the existence of time-regulated windows of neuronal and synaptic impairments. We propose that these developmentally-dependent impairments can be unified into a key concept: namely, time-restricted windows for impaired synaptic phenotypes exist in NDDs, akin to critical periods during normal sensory development in the brain. Existence of sensitive time-windows has significant implications for our understanding of early brain development underlying NDDs and may indicate vulnerable periods when the brain is more susceptible to current therapeutic treatments.

Fig (not shown)

Misregulated mechanisms underlying spine morphology in NDDs. Several proteins implicated in monogenic NDDs (highlighted in red) are linked to the regulation of the synaptic cytoskeleton via F-actin through different Rho-mediated signaling pathways (highlighted in green). Mutations in OPHN1, TSC1/2, FMRP, p21-activated kinase (PAK) are directly linked to human NDDs of intellectual disability. For instance, point mutations in OPHN1 and a PAK isoform are linked to non-syndromic mental retardation, whereas mutations or altered expression of TSC1/2 and FMRP are linked to TSC and FXS, respectively. Cytoplasmic interacting protein (CYFIP) and LIM-domain kinase 1 (LIMK1) are known to interact with FMRP and PAK, respectively [105]. LIMK1 is one of many dysregulated proteins contributing to the NDD Williams syndrome. Mouse models are available for all highlighted (red) proteins and reveal specific synaptic and behavioral deficits. Local protein synthesis in synapses, dendrites and glia is also regulated by proteins such as TSC1/2 and the FMRP/CYFIP complex. Abbreviations: 4EBP, 4E binding protein; eIF4E, eukaryotic translation initiation factor 4E.

Fig (not shown)

Sensitive time-windows, synaptic phenotypes and NDD gene targets. Sensitive time-windows exist in neural circuits, during which gene targets implicated in NDDs are normally expressed. Misregulation of these genes can affect multiple synaptic phenotypes during a restricted developmental period. The effect upon synaptic phenotypes is dependent upon the temporal expression of these NDD genes and their targets. (a) Expression outside a critical period of development will have no effect upon synaptic phenotypes. (b,c) A temporal expression pattern that overlaps with the onset (b) or closure (c) of a known critical period can alter the synaptic phenotype during that developmental time-window.

Outstanding questions

(1) Can treatment at early presymptomatic stages in animal models for NDDs prevent or ease the later synaptic, neuronal, and behavioral impairments?

(2) Are all sensory critical periods equally misregulated in mouse models for a specific NDD? Are there different susceptibilities for auditory, visual and somatosensory neurocircuits that reflect the degree of impairments observed in patients?

(3) If one critical period is missed or delayed during formation of a layer-specific connection in a network, does the network overcome this misregulated connectivity or plasticity window?

(4) In monogenic NDDs, does the severity of misregulating one particular time-window for synaptic establishment during development correlate with the importance of that gene for that synaptic circuit?

(5) Why do critical periods close in brain development?

(6) What underlies the regression of some altered synaptic phenotypes in Fmr1-KO mice?

(7) Can the concept of susceptible time-windows be applied to other NDDs, including schizophrenia and Tourette’s syndrome?

Cardiovascular

Cardiac output monitoring in newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 143–148
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.032

There is an increased interest in methods of objective cardiac output measurement in critically ill patients. Several techniques are available for measurement of cardiac output in children, although this remains very complex in newborns. Cardiac output monitoring could provide essential information to guide hemodynamic management. An overview is given of various methods of cardiac output monitoring with advantages and major limitations of each technology together with a short explanation of the basic principles.

Fick principle

According to the Fick principle the volume of blood flow in a given period equals the amount of substance entering the blood stream in the same period divided by the difference in concentrations of the substrate upstream respectively downstream to the point of entry in the circulation. This substance can be oxygen (O2-Fick) or carbon dioxide (CO2-FICK), so cardiac output can be calculated by dividing measured pulmonary oxygen uptake by the arteriovenous oxygen concentration difference. The direct O2-Fick method is regarded as gold standard in cardiac output monitoring in a research setting, despite its limitations. When the Fick principle is applied for carbon dioxide (CO2 Fick), the pulmonary carbon dioxide exchange is divided by the venoarterial CO2 concentration difference to calculate cardiac output.

In the modified CO2 Fick method pulmonary CO2 exchange is measured at the endotracheal tube. Measurement of total CO2 concentration in blood is more complex and simultaneous sampling of arterial and central venous blood is required. However, frequent blood sampling will result in an unacceptable blood loss in the neonatal population.

Blood flow can be calculated if the change in concentration of a known quantity of injected indicator is measured in time distal to the point of injection, so an indicator dilution curve can be obtained. Cardiac output can then be calculated with the use of the Stewart–Hamilton equation. Several indicators are used, such as indocyanine green, Evans blue and brilliant red in dye dilution, cold solutions in thermodilution, lithium in lithium dilution, and isotonic saline in ultrasound dilution.

Cardiovascular adaptation to extra uterine life

Alice Lawford, Robert MR Tulloh
Paediatrics And Child Health 2014; 25(1): 1-6.

The adaptation to extra uterine life is of interest because of its complexity and the ability to cause significant health concerns. In this article we describe the normal changes that occur and the commoner abnormalities that are due to failure of normal development and the effect of congenital cardiac disease. Abnormal development may occur as a result of problems with the mother, or with the fetus before birth. After birth it is essential to determine whether there is an underlying abnormality of the fetal pulmonary or cardiac development and to determine the best course of management of pulmonary hypertension or congenital cardiac disease. Causes of underdevelopment, maldevelopment and maladaptation are described as are the causes of critical congenital heart disease. The methods of diagnosis and management are described to allow the neonatologist to successfully manage such newborns.

Fetal vascular structures that exist to direct blood flow

Fetal structure	Function
Arterial duct	Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale	Opening between the two atria thatdirects blood flow returning to right atrium through the septal wall into the left atrium bypassing lungs
Ductus venosus	Receives oxygenated blood fromumbilical vein and directs it to the inferior vena cava and right atrium
Umbilical arteries	Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein	Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

Maternal disorders	rubella, SLE, diabetes mellitus
Maternal drug use	Warfarin, alcohol
Chromosomal abnormality	Down, Edward, Patau, Turner, William, Noonan

Fetal and Neonatal Circulation The fetal circulation is specifically adapted to efficiently exchange gases, nutrients, and wastes through placental circulation. Upon birth, the shunts (foramen ovale, ductus arteriosus, and ductus venosus) close and the placental circulation is disrupted, producing the series circulation of blood through the lungs, left atrium, left ventricle, systemic circulation, right heart, and back to the lungs.

Clinical monitoring of systemic hemodynamics in critically ill newborns

Willem-Pieter de Boode
Early Human Development 86 (2010) 137–141
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.031

Circulatory failure is a major cause of mortality and morbidity in critically ill newborn infants. Since objective measurement of systemic blood flow remains very challenging, neonatal hemodynamics is usually assessed by the interpretation of various clinical and biochemical parameters. An overview is given about the predictive value of the most used indicators of circulatory failure, which are blood pressure, heart rate, urine output, capillary refill time, serum lactate concentration, central–peripheral temperature difference, pH, standard base excess, central venous oxygen saturation and color.

Key guidelines

➢ The clinical assessment of cardiac output by the interpretation of indirect parameters of systemic blood flow is inaccurate, irrespective of the level of experience of the clinician

➢ Using blood pressure to diagnose low systemic blood flow will consequently mean that too many patients will potentially be undertreated or overtreated, both with substantial risk of adverse effects and iatrogenic damage.

➢ Combining different clinical hemodynamic parameters enhances the predictive value in the detection of circulatory failure, although accuracy is still limited.

➢ Variation in time (trend monitoring) might possibly be more informative than individual, static values of clinical and biochemical parameters to evaluate the adequacy of neonatal circulation.

Monitoring oxygen saturation and heart rate in the early neonatal period

J.A. Dawson, C.J. Morley
Seminars in Fetal & Neonatal Medicine 15 (2010) 203e207
http://dx.doi.org:/10.1016/j.siny.2010.03.004

Pulse oximetry is commonly used to assist clinicians in assessment and management of newly born infants in the delivery room (DR). In many DRs, pulse oximetry is now the standard of care for managing high risk infants, enabling immediate and dynamic assessment of oxygenation and heart rate. However, there is little evidence that using pulse oximetry in the DR improves short and long term outcomes. We review the current literature on using pulse oximetry to measure oxygen saturation and heart rate and how to apply current evidence to management in the DR.

Practice points

Understand how SpO2 changes in the first minutes after birth.
Apply a sensor to an infant’s right wrist as soon as possible after birth.
Attach sensor to infant then to oximeter cable.
Use two second averaging and maximum sensitivity.

Using pulse oximetry assists clinicians:

Assess changes in HR in real time during transition.
Assess oxygenation and titrate the administration of oxygen to maintain oxygenation within the appropriate range for SpO2 during the first minutes after birth.

Research directions

What are the appropriate centiles to target during the minutes after birth to prevent hypoxia and hyperoxia: 25th to 75th, or 10th to 90th, or just the 50th (median)?
Can the inspired oxygen be titrated against the SpO2 to keep the SpO2 in the ‘normal range’?
Does the use of centile charts in the DR for HR and oxygen saturation reduce the rate of hyperoxia when infants are treated with oxygen.
Does the use of pulse oximetry immediately after birth improve short term outcomes, e.g. efficacy of immediate respiratory support, intubation rates in the DR, percentage of inspired oxygen, rate of use of adrenalin or chest compressions, duration of hypoxia/hyperoxia and bradycardia.
Does the use of pulse oximetry in the DR improve short term respiratory and long term neurodevelopmental outcomes for preterm infants, e.g. rate of intubation, use of surfactant, and duration of ventilation, continuous positive airway pressure, or supplemental oxygen?
Can all modern pulse oximeters be used effectively in the DR or do some have a longer delay before giving an accurate signal and more movement artefact?
Would a longer averaging time result in more stable data?

Peripheral haemodynamics in newborns: Best practice guidelines

Michael Weindling, Fauzia Paize
Early Human Development 86 (2010) 159–165
http://dx.doi.org:/10.1016/j.earlhumdev.2010.01.033

Peripheral hemodynamics refers to blood flow, which determines oxygen and nutrient delivery to the tissues. Peripheral blood flow is affected by vascular resistance and blood pressure, which in turn varies with cardiac function. Arterial oxygen content depends on the blood hemoglobin concentration (Hb) and arterial pO2; tissue oxygen delivery depends on the position of the oxygen-dissociation curve, which is determined by temperature and the amount of adult or fetal hemoglobin. Methods available to study tissue perfusion include near-infrared spectroscopy, Doppler flowmetry, orthogonal polarization spectral imaging and the peripheral perfusion index. Cardiac function, blood gases, Hb, and peripheral temperature all affect blood flow and oxygen extraction. Blood pressure appears to be less important. Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing the intrathoracic pressure.

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation. Taken from Yoxall and Weindling

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue

graphic

Schematic representation of the biphasic relationship between oxygen delivery and oxygen consumption in tissue. (a) oxygen delivery (DO2). (b) As DO2 decreases, VO2 is dependent on DO2. The slope of the line indicates the FOE, which in this case is about 0.50. (c) The slope of the line indicates the FOE in the normal situation where oxygenation is DO2 independent, usually < 0.35

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

Considerable information about the response of the peripheral circulation has been obtained using NIRS with venous occlusion. Although these measurements were validated against blood co-oximetry in human adults and infants, they can only be made intermittently by a trained operator and are thus not appropriate for general clinical use. Further research is needed to find other better measures of peripheral perfusion and oxygenation which may be easily and continuously monitored, and which could be useful in a clinical setting.

Peripheral oxygenation and management in the perinatal period

Michael Weindling
Seminars in Fetal & Neonatal Medicine 15 (2010) 208e215
http://dx.doi.org:/10.1016/j.siny.2010.03.005

The mechanisms for the adequate provision of oxygen to the peripheral tissues are complex. They involve control of the microcirculation and peripheral blood flow, the position of the oxygen dissociation curve including the proportion of fetal and adult hemoglobin, blood gases and viscosity. Systemic blood pressure appears to have little effect, at least in the non-shocked state. The adequate delivery of oxygen (DO2) depends on consumption (VO2), which is variable. The balance between VO2 and DO2 is given by fractional oxygen extraction (FOE ¼ VO2/DO2). FOE varies from organ to organ and with levels of activity. Measurements of FOE for the whole body produce a range of about 0.15-0.33, i.e. the body consumes 15-33% of oxygen transported.

Fig (not shown)

Biphasic relationship between oxygen delivery (DO2) and oxygen consumption (VO2) in tissue. Dotted lines show fractional oxygen extraction (FOE). ‘A’ indicates the normal situation when VO2 is independent ofDO2 and FOE is about 0.30. AsDO2 decreases in the direction of the arrow, VO2 remains independent of DO2 until the critical point is reached at ‘B’; in this illustration, FOE is about 0.50. The slope of the dotted line indicates the FOE (¼ VO2/DO2), which increases progressively as DO2 decreases.

$Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction$

Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction

Graphic
(A)Relationship between haemoglobin F fraction (HbF) and peripheral fractional oxygen extraction in anaemic and control infants. (From Wardle et al.) (B) HbF synthesis and concentration. (From Bard and Widness.) (C) Oxygen dissociation curve.

$Peripheral fractional oxygen extraction in babies$

Peripheral fractional oxygen extraction in babies

graphic

Peripheral fractional oxygen extraction in babies with asymptomatic or symptomatic anemia compared to controls. Bars represent the median for each group. (From Wardle et al.)

Practice points

Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:

Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
It is possible to compensate for a low Hb by increasing cardiac output and ventilation, and so the ability to compensate for anemia depends on an individual’s cardio-respiratory reserve as well as Hb.
The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

The relationship between VO2 and DO2 is complex and various factors need to be taken into account, including the position of the oxygen dissociation curve, determined by the proportion of HbA and HbF, temperature and pH. Furthermore, diffusion of oxygen from capillaries to the cell depends on the oxygen tension gradient between erythrocytes and the mitochondria, which depends on microcirculatory conditions, e.g. capillary PO2, distance of the cell from the capillary (characterized by intercapillary distances) and the surface area of open capillaries. The latter can change rapidly, for example, in septic shock where arteriovenous shunting occurs associated with tissue hypoxia in spite of high DO2 and a low FOE.

Changes in local temperature deserve particular consideration. When the blood pressure is low, there may be peripheral vasoconstriction with decreased local perfusion and DO2. However, the fall in local tissue temperature would also be expected to be associated with a decreased metabolic rate and a consequent decrease in VO2. Thus a decreased DO2 may still be appropriate for tissue needs.

Pulmonary

Accurate Measurements of Oxygen Saturation in Neonates: Paired Arterial and Venous Blood Analyses

Shyang-Yun Pamela K. Shiao
Newborn and Infant Nurs Rev, 2005; 5(4): 170–178
http://dx.doi.org:/10.1053/j.nainr.2005.09.001

Oxygen saturation (So2) measurements (functional measurement, So2; and fractional measurement, oxyhemoglobin [Hbo2]) and monitoring are commonly investigated as a method of assessing oxygenation in neonates. Differences exist between the So2 and Hbo2 when blood tests are performed, and clinical monitors indicate So2 values. Oxyhemoglobin will decrease with the increased levels of carbon monoxide hemoglobin (Hbco) and methemo-globin (MetHb), and it is the most accurate measurements of oxygen (O2) association of hemoglobin (Hb). Pulse oximeter (for pulse oximetry saturation [Spo2] measurement) is commonly used in neonates. However, it will not detect the changes of Hb variations in the blood for accurate So2 measurements. Thus, the measurements from clinical oximeters should be used with caution. In neonates, fetal hemoglobin (HbF) accounts for most of the circulating Hb in their blood. Fetal hemoglobin has a high O2 affinity, thus releases less O2 to the body tissues, presenting a left-shifted Hbo2 dissociation curve.5,6 To date, however, limited data are available with HbF correction, for accurate arterial and venous (AV) So2 measurements (arterial oxygen saturation [Sao2] and venous oxygen saturation [Svo2]) in neonates, using paired AV blood samples.

In a study of critically ill adult patients, increased pulmonary CO production and elevation in arterial Hbco but not venous Hbco were documented by inflammatory stimuli inducing pulmonary heme oxygenase–1. In normal adults, venous Hbco level might be slightly higher than or equal to arterial Hbco because of production of CO by enzyme heme oxygenase–2, which is predominantly produced in the liver and spleen. However, hypoxia or pulmonary inflammation could induce heme oxygenase–1 to increase endogenous CO, thus elevating pulmonary arterial and systemic arterial Hbco levels in adults. Both endogenous and exogenous CO can suppress proliferation of pulmonary smooth muscles, a significant consideration for the prevention of chronic lung diseases in newborns. Despite these considerations, a later study in healthy adults indicated that the AV differences in Hbco were from technical artifacts and perhaps from inadequate control of different instruments. Thus, further studies are needed to provide more definitive answers for the AV differences of Hbco for adults and neonates with acute and chronic lung diseases.

Methemoglobin is an indicator of Hb oxidation and is essential for accurate measurement of Hbo2, So2, and oxygenation status. No evidence exists to show the AV MetHb difference, although this difference was elucidated with the potential changes of MetHb with different O2 levels. Methemoglobin can be increased with nitric oxide (NO) therapy, used in respiratory distress syndrome (RDS) to reduce pulmonary hypertension and during heart surgery. Nitric oxide, in vitro, is an oxidant of Hb, with increased O2 during ischemia reperfusion. In hypoxemic conditions in vivo, nitrohemoglobin is a product generated by vessel responsiveness to nitrovasodilators. Nitro-hemoglobin can be spontaneously reversible in vivo, requiring no chemical agents or reductase. However, when O2 levels were increased experimentally in vitro following acidic conditions (pH 6.5) to simulate reperfusion conditions, MetHb levels were increased for the hemolysates (broken red cells). Nitrite-induced oxidation of Hb was associated with an increase in red blood cell membrane rigidity, thus contributing to Hb breakdown. A newer in vitro study of whole blood cells, however, concluded that MetHb formation is not dependent on increased O2 levels. Additional studies are needed to examine in vivo reperfusion of O2 and MetHb effects.

Purpose: The aim of this study was to examine the accuracy of arterial oxygen saturation (Sao2) and venous oxygen saturation (Svo2) with paired arterial and venous (AV) blood in relation to pulse oximetry saturation (Spo2) and oxyhemoglobin (Hbo2) with fetal hemoglobin determination, and their Hbo2 dissociation curves. Method: Twelve preterm neonates with gestational ages ranging from 27 to 34 weeks at birth, who had umbilical AV lines inserted, were investigated. Analyses were performed with 37 pairs of AV blood samples by using a blood volume safety protocol. Results: The mean differences between Sao2 and Svo2, and AV Hbo2 were both 6 percent (F6.9 and F6.7 percent, respectively), with higher Svo2 than those reported for adults. Biases were 2.1 – 0.49 for Sao2, 2.0 – 0.44 for Svo2, and 3.1 – 0.45 for Spo2, compared against Hbo2. With left-shifted Hbo2 dissociation curves in neonates, for the critical values of oxygen tension values between 50 and 75 millimeters of mercury, Hbo2 ranged from 92 to 93.4 percent; Sao2 ranged from 94.5 to 95.7 percent; and Spo2 ranged from 93.7 to 96.3 percent (compared to 85–94 percent in healthy adults). Conclusions: In neonates, both left-shifted Hbo2 dissociation curve and lower AV differences of oxygen saturation measurements indicated low flow of oxygen to the body tissues. These findings demonstrate the importance of accurate assessment of oxygenation statues in neonates.

In these neonates, the mean AV blood differences for both So2 and Hbo2 were about 6 percent, which was much lower than those reported for healthy adults (23 percent) for O2 supply and demand. In addition, with very high levels of HbF releasing less O2 to the body tissue, the results of blood analyses are worrisome for these critically ill neonates for low systemic oxygen states. O’Connor and Hall determined AV So2 in neonates without HbF determination. Much of the AV So2 difference is dependent on Svo2 measurement. The ranges of Svo2 spanned for 35 percent, and the ranges of Sao2 spanned 6 percent in these neonates. The greater intervals for Svo2 measurements contribute to greater sensitivity for the measurements (than Sao2 measurements) in responding to nursing care and changes of O2 demand. Thus, Svo2 measurement is essential for better assessment of oxygenation status in neonates.

The findings of this study on AV differences of So2 were limited with very small number of paired AV blood samples. However, critically ill neonates need accurate assessment of oxygenation status because of HbF, which releases less O2 to the tissues. Decreased differences of AV So2 measurements added further possibilities of lower flow of O2 to the body tissues and demonstrated the greater need to accurately assess the proper oxygenation in the neonates. The findings of this study continued to clarify the accuracy of So2 measurements for neonates. Additional studies are needed to examine So2 levels in neonates to further validate these findings by using larger sample sizes.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

A Model Analysis of Arterial Oxygen Desaturation during Apnea in Preterm Infants

Scott A. Sands, BA Edwards, VJ Kelly, MR Davidson, MH Wilkinson, PJ Berger
PLoS Comput Biol 5(12): e1000588
http://dx.doi.org:/10.1371/journal.pcbi.1000588

Rapid arterial O2 desaturation during apnea in the preterm infant has obvious clinical implications but to date no adequate explanation for why it exists. Understanding the factors influencing the rate of arterial O2 desaturation during apnea (_SSaO2 ) is complicated by the non-linear O2 dissociation curve, falling pulmonary O2 uptake, and by the fact that O2 desaturation is biphasic, exhibiting a rapid phase (stage 1) followed by a slower phase when severe desaturation develops (stage 2). Using a mathematical model incorporating pulmonary uptake dynamics, we found that elevated metabolic O2 consumption accelerates _SSaO2 throughout the entire desaturation process. By contrast, the remaining factors have a restricted temporal influence: low pre-apneic alveolar PO2 causes an early onset of desaturation, but thereafter has little impact; reduced lung volume, hemoglobin content or cardiac output, accelerates _SSaO2 during stage 1, and finally, total blood O2 capacity (blood volume and hemoglobin content) alone determines _SSaO2 during stage 2. Preterm infants with elevated metabolic rate, respiratory depression, low lung volume, impaired cardiac reserve, anemia, or hypovolemia, are at risk for rapid and profound apneic hypoxemia. Our insights provide a basic physiological framework that may guide clinical interpretation and design of interventions for preventing sudden apneic hypoxemia.

A novel approach to study oxidative stress in neonatal respiratory distress syndrome

Reena Negi, D Pande, K Karki, A Kumar, RS Khanna, HD Khanna
BBA Clinical 3 (2015) 65–69
http://dx.doi.org/10.1016/j.bbacli.2014.12.001

Oxidative stress is an imbalance between the systemic manifestation of reactive oxygen species and a biological system’s ability to readily detoxify the reactive intermediates or to repair the resulting damage. It is a physiological event in the fetal-to-neonatal transition, which is actually a great stress to the fetus. These physiological changes and processes greatly increase the production of free radicals, which must be controlled by the antioxidant defense system, the maturation of which follows the course of the gestation. This could lead to several functional alterations with important repercussions for the infants. Adequately mature and healthy infants are able to tolerate this drastic change in the oxygen concentration. A problem occurs when the intrauterine development is incomplete or abnormal. Preterm or intrauterine growth retarded (IUGR) and low birth weight neonates are typically of this kind. An oxidant/antioxidant imbalance in infants is implicated in the pathogenesis of the major complications of prematurity including respiratory distress syndrome (RDS), necrotizing enterocolitis (NEC), chronic lung disease, retinopathy of prematurity and intraventricular hemorrhage (IVH).

Background: Respiratory distress syndrome of the neonate (neonatal RDS) is still an important problem in treatment of preterm infants. It is accompanied by inflammatory processes with free radical generation and oxidative stress. The aim of study was to determine the role of oxidative stress in the development of neonatal RDS. Methods: Markers of oxidative stress and antioxidant activity in umbilical cord blood were studied in infants with neonatal respiratory distress syndrome with reference to healthy newborns. Results: Status of markers of oxidative stress (malondialdehyde, protein carbonyl and 8-hydroxy-2-deoxy guanosine) showed a significant increase with depleted levels of total antioxidant capacity in neonatal RDS when compared to healthy newborns. Conclusion: The study provides convincing evidence of oxidative damage and diminished antioxidant defenses in newborns with RDS. Neonatal RDS is characterized by damage of lipid, protein and DNA, which indicates the augmentation of oxidative stress. General significance: The identification of the potential biomarker of oxidative stress consists of a promising strategy to study the pathophysiology of neonatal RDS.

Neonatal respiratory distress syndrome represents the major lung complications of newborn babies. Preterm neonates suffer from respiratory distress syndrome (RDS) due to immature lungs and require assisted ventilation with high concentrations of oxygen. The pathogenesis of this disorder is based on the rapid formation of the oxygen reactive species, which surpasses the detoxification capacity of antioxidative defense system. The high chemical reactivity of free radical leads to damage to a variety of cellular macro molecules including proteins, lipids and nucleic acid. This results in cell injury and may induce respiratory cell death.

Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation. The present study showed increased concentration of MDA in neonates with respiratory disorders than that of control in consonance with the reported study.

Anemia, Apnea of Prematurity, and Blood Transfusions

Kelley Zagol, Douglas E. Lake, Brooke Vergales, Marion E. Moorman, et al
J Pediatr 2012;161:417-21
http://dx.doi.org:/10.1016/j.jpeds.2012.02.044

The etiology of apnea of prematurity is multifactorial; however, decreased oxygen carrying capacity may play a role. The respiratory neuronal network in neonates is immature, particularly in those born preterm, as demonstrated by their paradoxical response to hypoxemia. Although adults increase the minute ventilation in response to hypoxemia, newborns have a brief increase in ventilation followed by periodic breathing, respiratory depression, and occasionally cessation of respiratory effort. This phenomenon may be exacerbated by anemia in preterm newborns, where a decreased oxygen carrying capacity may result in decreased oxygen delivery to the central nervous system, a decreased efferent output of the respiratory neuronal network, and an increase in apnea.

Objective Compare the frequency and severity of apneic events in very low birth weight (VLBW) infants before and after blood transfusions using continuous electronic waveform analysis. Study design We continuously collected waveform, heart rate, and oxygen saturation data from patients in all 45 neonatal intensive care unit beds at the University of Virginia for 120 weeks. Central apneas were detected using continuous computer processing of chest impedance, electrocardiographic, and oximetry signals. Apnea was defined as respiratory pauses of >10, >20, and >30 seconds when accompanied by bradycardia (<100 beats per minute) and hypoxemia (<80% oxyhemoglobin saturation as detected by pulse oximetry). Times of packed red blood cell transfusions were determined from bedside charts. Two cohorts were analyzed. In the transfusion cohort, waveforms were analyzed for 3 days before and after the transfusion for all VLBW infants who received a blood transfusion while also breathing spontaneously. Mean apnea rates for the previous 12 hours were quantified and differences for 12 hours before and after transfusion were compared. In the hematocrit cohort, 1453 hematocrit values from all VLBW infants admitted and breathing spontaneously during the time period were retrieved, and the association of hematocrit and apnea in the next 12 hours was tested using logistic regression. Results Sixty-seven infants had 110 blood transfusions during times when complete monitoring data were available. Transfusion was associated with fewer computer-detected apneic events (P < .01). Probability of future apnea occurring within 12 hours increased with decreasing hematocrit values (P < .001). Conclusions Blood transfusions are associated with decreased apnea in VLBW infants, and apneas are less frequent at higher hematocrits.

Bronchopulmonary dysplasia: The earliest and perhaps the longest lasting obstructive lung disease in humans

Silvia Carraro, M Filippone, L Da Dalt, V Ferraro, M Maretti, S Bressan, et al.
Early Human Development 89 (2013) S3–S5
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.015

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy, an umbrella term for a number of different diseases that evolve as a consequence of a neonatal respiratory disorder. BPD is defined as the need for supplemental oxygen for at least 28 days after birth, and its severity is graded according to the respiratory support required at 36 post-menstrual weeks.

BPD was initially described as a chronic respiratory disease occurring in premature infants exposed to mechanical ventilation and oxygen supplementation. This respiratory disease (later named “old BPD”) occurred in relatively large premature newborn and, from a pathological standpoint, it was characterized by intense airway inflammation, disruption of normal pulmonary structures and lung fibrosis.

Bronchopulmonary dysplasia (BPD) is one of the most important sequelae of premature birth and the most common form of chronic lung disease of infancy. From a clinical standpoint BPD subjects are characterized by recurrent respiratory symptoms, which are very frequent during the first years of life and, although becoming less severe as children grow up, they remain more common than in term-born controls throughout childhood, adolescence and into adulthood. From a functional point of view BPD subjects show a significant airflow limitation that persists during adolescence and adulthood and they may experience an earlier and steeper decline in lung function during adulthood. Interestingly, patients born prematurely but not developing BPD usually fare better, but they too have airflow limitations during childhood and later on, suggesting that also prematurity per se has life-long detrimental effects on pulmonary function. For the time being, little is known about the presence and nature of pathological mechanisms underlying the clinical and functional picture presented by BPD survivors. Nonetheless, recent data suggest the presence of persistent neutrophilic airway inflammation and oxidative stress and it has been suggested that BPD may be sustained in the long term by inflammatory pathogenic mechanisms similar to those underlying COPD. This hypothesis is intriguing but more pathological data are needed. A better understanding of these pathogenetic mechanisms, in fact, may be able to orient the development of novel targeted therapies or prevention strategies to improve the overall respiratory health of BPD patients.

We have a limited understanding of the presence and nature of pathological mechanisms in the lung of BPD survivors. The possible role of asthma-like inflammation has been investigated because BPD subjects often present with recurrent wheezing and other symptoms resembling asthma during their childhood and adolescence. But BPD subjects have normal or lower than normal exhaled nitric oxide levels and exhaled air temperatures, whereas they are higher than normal in asthmatic patients.

Of all obstructive lung diseases in humans, BPD has the earliest onset and is possibly the longest lasting. Given its frequent association with other conditions related to preterm birth (e.g. growth retardation, pulmonary hypertension, neurodevelopmental delay, hearing defects, and retinopathy of prematurity), it often warrants a multidisciplinary management.

Effects of Sustained Lung Inflation, a lung recruitment maneuver in primary acute respiratory distress syndrome, in respiratory and cerebral outcomes in preterm infants

Chiara Grasso, Pietro Sciacca, Valentina Giacchi, Caterina Carpinato, et al.
Early Human Development 91 (2015) 71–75
http://dx.doi.org/10.1016/j.earlhumdev.2014.12.002

Background: Sustained Lung Inflation (SLI) is a maneuver of lung recruitment in preterm newborns at birth that can facilitate the achieving of larger inflation volumes, leading to the clearance of lung fluid and formation of functional residual capacity (FRC). Aim: To investigate if Sustained Lung Inflation (SLI) reduces the need of invasive procedures and iatrogenic risks. Study design: 78 newborns (gestational age ≤ 34 weeks, weighing ≤ 2000 g) who didn’t breathe adequately at birth and needed to receive SLI in addition to other resuscitation maneuvers (2010 guidelines). Subjects: 78 preterm infants born one after the other in our department of Neonatology of Catania University from 2010 to 2012. Outcome measures: The need of intubation and surfactant, the ventilation required, radiological signs, the incidence of intraventricular hemorrhage (IVH), periventricular leukomalacia, retinopathy in prematurity from III to IV plus grades, bronchopulmonary dysplasia, patent ductus arteriosus, pneumothorax and necrotizing enterocolitis. Results: In the SLI group infants needed less intubation in the delivery room (6% vs 21%; p b 0.01), less invasive mechanical ventilation (14% vs 55%; p ≤ 0.001) and shorter duration of ventilation (9.1 days vs 13.8 days; p ≤ 0.001). There wasn’t any difference for nasal continuous positive airway pressure (82% vs 77%; p = 0.43); but there was less surfactant administration (54% vs 85%; p ≤ 0.001) and more infants received INSURE (40% vs 29%; p=0.17). We didn’t found any differences in the outcomes, except for more mild intraventricular hemorrhage in the SLI group (23% vs 14%; p = 0.15; OR= 1.83). Conclusion: SLI is easier to perform even with a single operator, it reduces the necessity of more complicated maneuvers and surfactant without statistically evident adverse effects.

Long-term respiratory consequences of premature birth at less than 32 weeks of gestation

Anne Greenough
Early Human Development 89 (2013) S25–S27
http://dx.doi.org/10.1016/j.earlhumdev.2013.07.004

Chronic respiratory morbidity is a common adverse outcome of very premature birth, particularly in infants who had developed bronchopulmonary dysplasia (BPD). Prematurely born infants who had BPD may require supplementary oxygen at home for many months and affected infants have increased healthcare utilization until school age. Chest radiograph abnormalities are common; computed tomography of the chest gives predictive information in children with ongoing respiratory problems. Readmission to hospital is common, particularly for those who have BPD and suffer respiratory syncytial virus lower respiratory infections (RSV LRTIs). Recurrent respiratory symptoms requiring treatment are common and are associated with evidence of airways obstruction and gas trapping. Pulmonary function improves with increasing age, but children with BPD may have ongoing airflow limitation. Lung function abnormalities may be more severe in those who had RSV LRTIs, although this may partly be explained by worse premorbid lung function. Worryingly, lung function may deteriorate during the first year. Longitudinal studies are required to determine if there is catch up growth.

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

Anita Bhandari and Sharon McGrath-Morrow
Seminars in Perinatology 37 (2013)132–137
http://dx.doi.org/10.1053/j.semperi.2013.01.010

Bronchopulmonary dysplasia (BPD) is the commonest cause of chronic lung disease in infancy. The incidence of BPD has remained unchanged despite many advances in neonatal care. BPD starts in the neonatal period but its effects can persist long term. Premature infants with BPD have a greater incidence of hospitalization, and continue to have a greater respiratory morbidity and need for respiratory medications, compared to those without BPD. Lung function abnormalities, especially small airway abnormalities, often persist. Even in the absence of clinical symptoms, BPD survivors have persistent radiological abnormalities and presence of emphysema has been reported on chest computed tomography scans. Concern regarding their exercise tolerance remains. Long-term effects of BPD are still unknown, but given reports of a more rapid decline in lung function and their susceptibility to develop chronic obstructive pulmonary disease phenotype with aging, it is imperative that lung function of survivors of BPD be closely monitored.

Neonatal ventilation strategies and long-term respiratory outcomes

Sandeep Shetty, Anne Greenough
Early Human Development 90 (2014) 735–739
http://dx.doi.org/10.1016/j.earlhumdev.2014.08.020

Long-term respiratory morbidity is common, particularly in those born very prematurely and who have developed bronchopulmonary dysplasia (BPD), but it does occur in those without BPD and in infants born at term. A variety of neonatal strategies have been developed, all with short-term advantages, but meta-analyses of randomized controlled trials (RCTs) have demonstrated that only volume-targeted ventilation and prophylactic high-frequency oscillatory ventilation (HFOV) may reduce BPD. Few RCTs have incorporated long-term follow-up, but one has demonstrated that prophylactic HFOV improves respiratory and functional outcomes at school age, despite not reducing BPD. Results from other neonatal interventions have demonstrated that any impact on BPD may not translate into changes in long-term outcomes. All future neonatal ventilation RCTs should have long-term outcomes rather than BPD as their primary outcome if they are to impact on clinical practice.

Prediction of neonatal respiratory distress syndrome in term pregnancies by assessment of fetal lung volume and pulmonary artery resistance index

Mohamed Laban, GM Mansour, MSE Elsafty, AS Hassanin, SS EzzElarab
International Journal of Gynecology and Obstetrics 128 (2015) 246–250
http://dx.doi.org/10.1016/j.ijgo.2014.09.018

Objective: To develop reference cutoff values for mean fetal lung volume (FLV) and pulmonary artery resistance index (PA-RI) for prediction of neonatal respiratory distress syndrome (RDS) in low-risk term pregnancies. Methods: As part of a cross-sectional study, women aged 20–35 years were enrolled and admitted to a tertiary hospital in Cairo, Egypt, for elective repeat cesarean at 37–40 weeks of pregnancy between January 1, 2012, and July 31, 2013. FLV was calculated by virtual organ computer-aided analysis, and PA-RI was measured by Doppler ultrasonography before delivery. Results: A total of 80 women were enrolled. Neonatal RDS developed in 11 (13.8%) of the 80 newborns. Compared with neonates with RDS, healthy neonates had significantly higher FLVs (P b 0.001) and lower PA-RIs (P b 0.001). Neonatal RDS is less likely with FLV of at least 32 cm³ or PA-RI less than or equal to 0.74. Combining these two measures improved the accuracy of prediction. Conclusion: The use of either FLV or PA-RI predicted neonatal RDS. The predictive value increased when these two measures were combined

Pulmonary surfactant – a front line of lung host defense, 2003 JCI0318650.f2

Pulmonary hypertension in bronchopulmonary dysplasia

Sara K.Berkelhamer, Karen K.Mestan, and Robin H. Steinhorn
Seminars In Perinatology 37 (2013)124–131
http://dx.doi.org/10.1053/j.semperi.2013.01.009

Pulmonary hypertension (PH) is a common complication of neonatal respiratory diseases, including bronchopulmonary dysplasia (BPD), and recent studies have increased aware- ness that PH worsens the clinical course, morbidity and mortality of BPD. Recent evidence indicates that up to 18% of all extremely low-birth-weight infants will develop some degree of PH during their hospitalization, and the incidence rises to 25–40% of the infants with established BPD. Risk factors are not yet well understood, but new evidence shows that fetal growth restriction is a significant predictor of PH. Echocardiography remains the primary method for evaluation of BPD-associated PH, and the development of standardized screening timelines and techniques for identification of infants with BPD-associated PH remains an important ongoing topic of investigation. The use of pulmonary vasodilator medications, such as nitric oxide, sildenafil, and others, in the BPD population is steadily growing, but additional studies are needed regarding their long-term safety and efficacy.
An update on pharmacologic approaches to bronchopulmonary dysplasia

Sailaja Ghanta, Kristen Tropea Leeman, and Helen Christou
Seminars In Perinatology 37 (2013)115–123
http://dx.doi.org/10.1053/j.semperi.2013.01.008

Bronchopulmonary dysplasia (BPD) is the most prevalent long-term morbidity in surviving extremely preterm infants and is linked to increased risk of reactive airways disease, pulmonary hypertension, post-neonatal mortality, and adverse neurodevelopmental outcomes. BPD affects approximately 20% of premature newborns, and up to 60% of premature infants born before completing 26 weeks of gestation. It is characterized by the need for assisted ventilation and/or supplemental oxygen at 36 weeks postmenstrual age. Approaches to prevention and treatment of BPD have evolved with improved understanding of its pathogenesis. This review will focus on recent advancements and detail current research in pharmacotherapy for BPD. The evidence for both current and potential future experimental therapies will be reviewed in detail. As our understanding of the complex and multifactorial pathophysiology of BPD changes, research into these current and future approaches must continue to evolve.

Methylxanthines

Diuretics and bronchodilators

Corticosteroids

Macrolide antibiotics

Recombinant human Clara cell 10-kilodalton protein(rhCC10)

Vitamin A

Surfactant

Leukotriene receptor antagonist

Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

Moustapha Kassem and Paolo Bianco
Cell Jan 15, 2015; 160: 17-19
http://dx.doi.org/10.1016/j.cell.2014.12.034

The nature, biological characteristics, and contribution to organ physiology of skeletal stem cells are not completely determined. Chan et al. and Worthley et al. demonstrate that a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors, can be identified in mice and demonstrate its role in skeletal tissue maintenance and regeneration.

The groundbreaking concept that bone, cartilage, marrow adipocytes, and hematopoiesis-supporting stroma could originate from a common progenitor and putative stem cell was surprising at the time when it was formulated (Owen and Friedenstein, 1988). The putative stem cell, nonhematopoietic in nature, would be found in the postnatal bone marrow stroma, generate tissues previously thought of as foreign to each other, and support the turnover of tissues and organs that self-renew at a much slower rate compared to other tissues associated with stem cells (blood, epithelia). This concept also connected bone and bone marrow as parts of a single-organ system, implying their functional interplay. For many years, the evidence underpinning the concept has been incomplete.

While multipotency of stromal progenitors has been demonstrated by in vivo transplantation experiments, self-renewal, the defining property of a stem cell, has not been easily demonstrated until recently in humans (Sacchetti et al., 2007) and mice (Mendez-Ferrer et al., 2010). Meanwhile, a confusing and plethoric terminology has been introduced into the literature, which diverted and confounded the search for a skeletal stem cell and its physiological significance (Bianco et al., 2013).

Two studies in this issue of Cell (Chan et al., 2015; Worthley et al., 2015), using a combination of rigorous single-cell analyses and lineage tracing technologies, mark significant steps toward rectifying the course of skeletal stem cell discovery by making several important points, within and beyond skeletal physiology.

First, a stem cell for skeletal tissues, and a system of more restricted, downstream progenitors can in fact be identified and linked to defined phenotype(s) in the mouse. The system is framed conceptually, and approached experimentally, similar to the hematopoietic system.

Second, based on its assayable functions and potential, the stem cell at the top of the hierarchy is defined as a skeletal stem cell (SSC). As noted earlier (Sacchetti et al., 2007) (Bianco et al., 2013), this term clarifies, well beyond semantics, that the range of tissues that the self-renewing stromal progenitor (originally referred to as an ‘‘osteogenic’’ or ‘‘stromal’’ stem cell) (Owen and Friedenstein, 1988) can actually generate in vivo, overlaps with the range of tissues that make up the skeleton.

Third, these cells are spatially restricted, local residents of the bone/bone marrow organ. The systemic circulation is not a sizable contributor to their recruitment to locally deployed functions.

Fourth, a native skeletogenic potential is inherent to the system of progenitor/ stem cells found in the skeleton, and internally regulated by bone morphogenetic protein (BMP) signaling. This is reflected in the expression of regulators and antagonists of BMP signaling within the system, highlighting potential feedback mechanisms modulating expansion or quiescence of specific cell compartments.

Fifth, in cells isolated from other tissues, an assayable skeletogenic potential is not inherent: it can only be induced de novo by BMP reprogramming. These two studies (Chan et al., 2015, Worthley et al., 2015) corroborate the classical concept of ‘‘determined’’ and ‘‘inducible’’ skeletal progenitors (Owen and Friedenstein, 1988): the former residing in the skeleton, the latter found in nonskeletal tissues; the former capable of generating skeletal tissues, in vivo and spontaneously, the latter requiring reprogramming signals in order to acquire a skeletogenic capacity; the former operating in physiological bone formation, the latter in unwanted, ectopic bone formation in diseases such as fibrodysplasia ossificans progressiva.

To optimize our ability to obtain specific skeletal tissues for medical application, the study by Chan et al. offers a glimpse of another facet of the biology of SSC lineages and progenitors. Chan et al. show that a homogeneous cell population inherently committed to chondrogenesis can alter its output to generate bone if cotransplanted with multipotent progenitors. Conversely, osteogenic cells can be shifted to a chondrogenic fate by blockade of vascular endothelial growth factor receptor, consistent with the avascular and hypoxic milieu of cartilage. This has two important implications:

commitment is flexible in the system;
the choir is as important as the soloist and can modulate the solo tune.

Reversibility and population behavior thus emerge as two features that may be characteristic, albeit not unique, of the stromal system, resonating with conceptually comparable evidence in the human system.

The two studies by Chan et al. and Worthely et al. emphasize the relevance not only of their new data, but also of a proper concept of a skeletal stem cell per se, for proper clinical use. Confusion arising from improper conceptualization of skeletal stem cells has markedly limited clinical development of skeletal stem cell biology.

Gremlin 1 Identifies a Skeletal Stem Cell with Bone, Cartilage, and Reticular Stromal Potential

Daniel L. Worthley, Michael Churchill, Jocelyn T. Compton, Yagnesh Tailor, et al.
Cell, Jan 15, 2015; 160: 269–284
http://dx.doi.org/10.1016/j.cell.2014.11.042

The stem cells that maintain and repair the postnatal skeleton remain undefined. One model suggests that perisinusoidal mesenchymal stem cells (MSCs) give rise to osteoblasts, chondrocytes, marrow stromal cells, and adipocytes, although the existence of these cells has not been proven through fate-mapping experiments. We demonstrate here that expression of the bone morphogenetic protein (BMP) antagonist gremlin 1 defines a population of osteochondroreticular (OCR) stem cells in the bone marrow. OCR stem cells self-renew and generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes. OCR stem cells are concentrated within the metaphysis of long bones not in the perisinusoidal space and are needed for bone development, bone remodeling, and fracture repair. Grem1 expression also identifies intestinal reticular stem cells (iRSCs) that are cells of origin for the periepithelial intestinal mesenchymal sheath. Grem1 expression identifies distinct connective tissue stem cells in both the bone (OCR stem cells) and the intestine (iRSCs).

Identification and Specification of the Mouse Skeletal Stem Cell

Charles K.F. Chan, Eun Young Seo, James Y. Chen, David Lo, A McArdle, et al.
Cell, Jan 15, 2015; 160: 285–298
http://dx.doi.org/10.1016/j.cell.2014.12.002

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Bone mesenchymal development

The bone-remodeling cycle

Nuclear receptor modulation – Role of coregulators in selective estrogen receptor modulator (SERM) actions

Qin Feng, Bert W. O’Malley
Steroids 90 (2014) 39–43
http://dx.doi.org/10.1016/j.steroids.2014.06.008

Selective estrogen receptor modulators (SERMs) are a class of small-molecule chemical compounds that bind to estrogen receptor (ER) ligand binding domain (LBD) with high affinity and selectively modulate ER transcriptional activity in a cell- and tissue-dependent manner. The prototype of SERMs is tamoxifen, which has agonist activity in bone, but has antagonist activity in breast. Tamoxifen can reduce the risk of breast cancer and, at same time, prevent osteoporosis in postmenopausal women. Tamoxifen is widely prescribed for treatment and prevention of breast cancer. Mechanistically the activity of SERMs is determined by the selective recruitment of coactivators and corepressors in different cell types and tissues. Therefore, understanding the coregulator function is the key to understanding the tissue selective activity of SERMs.

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

Owen J. Tamplin, Ellen M. Durand, Logan A. Carr, Sarah J. Childs, et al.
Cell, Jan 15, 2015; 160: 241–252
http://dx.doi.org/10.1016/j.cell.2014.12.032

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.

Neonatal anemia

Sanjay Aher, Kedar Malwatkar, Sandeep Kadam
Seminars in Fetal & Neonatal Medicine (2008) 13, 239e247
http://dx.doi.org:/10.1016/j.siny.2008.02.009

Neonatal anemia and the need for red blood cell (RBC) transfusions are very common in neonatal intensive care units. Neonatal anemia can be due to blood loss, decreased RBC production, or increased destruction of erythrocytes. Physiologic anemia of the newborn and anemia of prematurity are the two most common causes of anemia in neonates. Phlebotomy losses result in much of the anemia seen in extremely low birthweight infants (ELBW). Accepting a lower threshold level for transfusion in ELBW infants can prevent these infants being exposed to multiple donors.

Management of anemia in the newborn

Naomi L.C. Luban
Early Human Development (2008) 84, 493–498
http://dx.doi.org:/10.1016/j.earlhumdev.2008.06.007

Red blood cell (RBC) transfusions are administered to neonates and premature infants using poorly defined indications that may result in unintentional adverse consequences. Blood products are often manipulated to limit potential adverse events, and meet the unique needs of neonates with specific diagnoses. Selection of RBCs for small volume (5–20 mL/kg) transfusions and for massive transfusion, defined as extracorporeal bypass and exchange transfusions, are of particular concern to neonatologists. Mechanisms and therapeutic treatments to avoid transfusion are another area of significant investigation. RBCs collected in anticoagulant additive solutions and administered in small aliquots to neonates over the shelf life of the product can decrease donor exposure and has supplanted the use of fresh RBCs where each transfusion resulted in a donor exposure. The safety of this practice has been documented and procedures established to aid transfusion services in ensuring that these products are available. Less well established are the indications for transfusion in this population; hemoglobin or hematocrit alone are insufficient indications unless clinical criteria (e.g. oxygen desaturation, apnea and bradycardia, poor weight gain) also augment the justification to transfuse. Comorbidities increase oxygen consumption demands in these infants and include bronchopulmonary dysplasia, rapid growth and cardiac dysfunction. Noninvasive methods or assays have been developed to measure tissue oxygenation; however, a true measure of peripheral oxygen offloading is needed to improve transfusion practice and determine the value of recombinant products that stimulate erythropoiesis. The development of such noninvasive methods is especially important since randomized, controlled clinical trials to support specific practices are often lacking, due at least in part, to the difficulty of performing such studies in tiny infants.
The Effect of Blood Transfusion on the Hemoglobin Oxygen Dissociation Curve of Very Early Preterm Infants During the First Week of Life

Virginie De HaUeux, Anita Truttmann, Carmen Gagnon, and Harry Bard
Seminars in Perinatology, 2002; 26(6): 411-415
http://dx.doi.org:/10.1053/sper.2002.37313

This study was conducted during the first week of life to determine the changes in Ps0 (PO2 required to achieve a saturation of 50% at pH 7.4 and 37~ and the proportions of fetal hemoglobin (I-IbF) and adult hemoglobin (HbA) prior to and after transfusion in very early preterm infants. Eleven infants with a gestational age <–27 weeks have been included in study. The hemoglobin dissociation curve and the Ps0 was determined by Hemox-analyser. Liquid chromatography was also performed to determine the proportions of HbF and HbA. The mean gestational age of the 11 infants was 25.1 weeks (-+1 weeks) and their mean birth weight was 736 g (-+125 g). They received 26.9 mL/kg of packed red cells. The mean Ps0 prior and after transfusion was 18.5 +- 0.8 and 21.0 + 1 mm Hg (P = .0003) while the mean percentage of HbF was 92.9 -+ 1.1 and 42.6 -+ 5.7%, respectively. The data of this study show a decrease of hemoglobin oxygen affinity as a result of blood transfusion in very early preterm infants prone to O 2 toxicity. The shift in HbO 2 curve after transfusion should be taken into consideration when oxygen therapy is being regulated for these infants.

Effect of neonatal hemoglobin concentration on long-term outcome of infants affected by fetomaternal hemorrhage

Mizuho Kadooka, H Katob, A Kato, S Ibara, H Minakami, Yuko Maruyama
Early Human Development 90 (2014) 431–434
http://dx.doi.org/10.1016/j.earlhumdev.2014.05.010

Background: Fetomaternal hemorrhage (FMH) can cause severe morbidity. However, perinatal risk factors for long-term poor outcome due to FMH have not been extensively studied. Aims: To determine which FMH infants are likely to have neurological sequelae.
Study design: A single-center retrospective observational study. Perinatal factors, including demographic characteristics, Kleihauer–Betke test, blood gas analysis, and neonatal blood hemoglobin concentration ([Hb]), were analyzed in association with long-term outcomes.
Subjects: All 18 neonates referred to a Neonatal Intensive Care Unit of Kagoshima City Hospital and diagnosed with FMH during a 15-year study period. All had a neonatal [Hb] b7.5 g/dL and 15 of 17 neonates tested had Kleihauer–Betke test result N4.0%.
Outcome measures: Poor long-term outcome was defined as any of the following determined at 12 month old or more: cerebral palsy, mental retardation, attention deficit/hyperactivity disorder, and epilepsy.
Results: Nine of the 18 neonates exhibited poor outcomes. Among demographic characteristics and blood variables compared between two groups with poor and favorable outcomes, significant differences were observed in [Hb] (3.6 ± 1.4 vs. 5.4 ± 1.1 g/dL, P = 0.01), pH (7.09 ± 0.11 vs. 7.25 ± 0.13, P = 0.02) and base deficits (17.5 ± 5.4 vs. 10.4 ± 6.0 mmol/L, P = 0.02) in neonatal blood, and a number of infants with [Hb] ≤ 4.5 g/dL (78%[7/9] vs. 22%[2/9], P= 0.03), respectively. The base deficit in neonatal arterial blood increased significantly with decreasing neonatal [Hb].
Conclusions: Severe anemia causing severe base deficit is associated with neurological sequelae in FMH infants

Clinical and hematological presentation among Indian patients with common hemoglobin variants

Khushnooma Italia, Dipti Upadhye, Pooja Dabke, Harshada Kangane, et al.
Clinica Chimica Acta 431 (2014) 46–51
http://dx.doi.org/10.1016/j.cca.2014.01.028

Background: Co-inheritance of structural hemoglobin variants like HbS, HbD Punjab and HbE can lead to a variable clinical presentation and only few cases have been described so far in the Indian population.
Methods: We present the varied clinical and hematological presentation of 22 cases (HbSD Punjab disease-15, HbSE disease-4, HbD Punjab E disease-3) referred to us for diagnosis.
Results: Two of the 15 HbSDPunjab disease patients had moderate crisis, one presented with mild hemolytic anemia; however, the other 12 patients had a severe clinical presentation with frequent blood transfusion requirements, vaso occlusive crisis, avascular necrosis of the femur and febrile illness. The 4 HbSE disease patients had a mild to moderate presentation. Two of the 3 HbD Punjab E patients were asymptomatic with one patient’s sibling having a mild presentation. The hemoglobin levels of the HbSD Punjab disease patients ranged from 2.3 to 8.5 g/dl and MCV from 76.3 to 111.6 fl. The hemoglobin levels of the HbD Punjab E and HbSE patients ranged from 10.8 to 11.9 and 9.8 to 10.0 g/dl whereas MCV ranged from 67.1 to 78.2 and 74.5 to 76.0 fl respectively.
Conclusions: HbSD Punjab disease patients should be identified during newborn screening programs and managed in a way similar to sickle cell disease. Couple at risk of having HbSD Punjab disease children may be given the option of prenatal diagnosis in subsequent pregnancies.

Sickle cell anemia is the most common hemoglobinopathy seen across the world. It is caused by a point mutation in the 6th codon of the beta (β) globin gene leading to the substitution of the amino acid glutamic acid to valine. The sickle gene is frequently seen in Africa, some Mediterranean countries, India, Middle East—Saudi Arabia and North America. In India the prevalence of hemoglobin S (HbS) carriers varies from 2 to 40% among different population groups and HbS is mainly seen among the scheduled tribe, scheduled caste and other backward class populations in the western, central and parts of eastern and southern India. Sickle cell anemia has a variable clinical presentation in India with the most severe clinical presentation seen in central India whereas patients in the western region show a mild to moderate clinical presentation.

Hemoglobin D Punjab (HbD Punjab) (also known as HbD Los-Angeles, HbD Portugal, HbD North Carolina, D Oak Ridge and D Chicago) is another hemoglobin variant due to a point mutation in codon 121 of the β globin gene resulting in the substitution of the amino acid glutamic acid to glycine. It is a widely distributed hemoglobin with a relatively low prevalence of 0.86% in the Indo-Pak subcontinent, 1–3% in north-western India, 1–3% in the Black population in the Caribbean and North America and has also been reported among the English. It accounts for 55.6% of all the Hb variants seen in the Xenjiang province of China.

Hemoglobin E (HbE) is the most common abnormal hemoglobin in Southeast Asia. In India, the frequency ranges from 4% to 51% in the north eastern region and 3% to 4% in West Bengal in the east. The HbE mutation (β26 GAG→AAG) creates an alternative splice site and the βE chain is insufficiently synthesized, hence the phenotype of this disorder is that of a mild form of β thalassemia.

Though these 3 structural variants are prevalent in different regions of India, their interaction is increasingly seen in all states of the country due to migration of people to different regions for a better livelihood. There are very few reports on interaction of these commonly seen Hb variants and the phenotypic–genotypic presentation of these cases is important for genetic counseling and management.

HbF of patients with HbSD Punjab disease with variable clinical severity. The HbF values of 4 patients are not included as they were post blood transfusion

The genotypes of the patients were confirmed by restriction enzyme digestion and ARMS (Fig). Patients 1 to 15 were characterized as compound heterozygous for HbS and HbD Punjab whereas patients 16 to 19 were characterized as compound heterozygous for HbS and HbE. Patient nos. 20 to 22 were characterized as compound heterozygous for HbE and HbD Punjab.

Molecular characterization of HbS and HbDPunjab by restriction enzyme digestion and of HbE by ARMS.

The 3 common β globin gene variants of hemoglobin, HbS, HbE and HbD Punjab are commonly seen in India, with HbS having a high prevalence in the central belt and some parts of western, eastern and southern India, HbE in the eastern and north eastern region whereas HbD is mostly seen in the north western part of India. These hemoglobin variants have been reported in different population groups. However, with migration and intermixing of the different populations from different geographic regions, occasional cases of HbSD Punjab and HbSE are being reported. There are several HbD variants like HbD Punjab, HbD Iran, HbD Ibadan. However, of these only HbD Punjab interacts with HbS to form a clinically significant condition as the glutamine residue facilitates polymerization of HbS. HbD Iran and HbD Ibadan are non-interacting and produce benign conditions like the sickle cell trait. The first case of HbSD Punjab disease was a brother and sister considered to have atypical sickle cell disease in 1934. This family was further reinvestigated and reported as the first case of HbD Los Angeles which has the same mutation as the HbD Punjab. Serjeant et al. reported HbD Punjab in an English parent in 6 out of 11 HbSD-Punjab disease cases. This has been suggested to be due to the stationing of nearly 50,000 British troops on the Indian continent for a period of 200 y and the introduction into Britain of their Anglo-Indian children.

HbSD Punjab disease shows a similar pattern to HbS homozygous on alkaline hemoglobin electrophoresis but can be differentiated on acid agar gel electrophoresis and on HPLC. In HbSD Punjab disease cases, the peripheral blood films show anisocytosis, poikilocytosis, target cells and irreversibly sickled cells. Values of HbF and HbA2 are similar to those in sickle homozygous cases. HbSD Punjab disease is characterized by a moderately severe hemolytic anemia.

Twenty-one cases of HbSDPunjab were reported by Serjeant of which 16 were reported by different workers among patients originating from Caucasian, Spanish, Australian, Irish, English, Portuguese, Black, American, Venezuelan, Caribbean, Mexican, Turkish and Jamaican backgrounds. Yavarian et al. 2009 reported a multi centric origin of HbD Punjab which in combination with HbS results in sickle cell disease. Patel et al. 2010 have also reported 12 cases of HbSD Punjab from the Orissa state of eastern India. Majority of these cases were symptomatic, presenting with chronic hemolytic anemia and frequent painful crises.

HbF levels >20% were seen in 4 out of our 11 clinically severe patients of HbSD-Punjab disease with the mean HbF levels of 16.8% in 8 clinically severe patients, while 3 clinically severe patients were post transfused. However, the 3 patients with a mild to moderate clinical presentation showed a mean HbF level of 8.6%. This is in contrast to the relatively milder clinical presentation associated with high HbF seen in patients with sickle cell anemia. This was also reported by Adekile et al. 2010 in 5 cases of HbS-DLos Angeles where high HbF did not ameliorate the severe clinical presentation seen in these patients.

These 15 cases of HbSD^Punjab disease give us an overall idea of the severe clinical presentation of the disease in different regions of India. However the HbD^PunjabE cases were milder or asymptomatic and the HbSE cases were moderately symptomatic. Since most of the cases of HbSD^Punjab disease were clinically severe, it is important to pick up these cases during newborn screening and enroll them into a comprehensive care program with the other sickle cell disease patients with introduction of therapeutic interventions such as penicillin prophylaxis if required and pneumococcal immunization. In fact, 2 of our cases (No. 6 and 7) were identified during newborn screening for sickle cell disorders. The parents can be given information on home care and educated to detect symptoms that may lead to serious medical emergencies. The parents of these patients as well as the couples who are at risk of having a child with HbSD^Punjab disease could also be counseled about the option of prenatal diagnosis in subsequent pregnancies. It is thus important to document the clinical and hematological presentation of compound heterozygotes with these common β globin chain variants.

Common Hematologic Problems in the Newborn Nursery

Jon F. Watchko
Pediatr Clin N Am – (2015) xxx-xxx
http://dx.doi.org/10.1016/j.pcl.2014.11.011

Common RBC disorders include hemolytic disease of the newborn, anemia, and polycythemia. Another clinically relevant hematologic issue in neonates to be covered herein is thrombocytopenia. Disorders of white blood cells will not be reviewed.

KEY POINTS

(1) Early clinical jaundice or rapidly developing hyperbilirubinemia are often signs of hemolysis, the differential diagnosis of which commonly includes immune-mediated disorders, red-cell enzyme deficiencies, and red-cell membrane defects.

(2) Knowledge of the maternal blood type and antibody screen is critical in identifying non-ABO alloantibodies in the maternal serum that may pose a risk for severe hemolytic disease in the newborn.

(3) Moderate to severe thrombocytopenia in an otherwise well-appearing newborn strongly suggests immune-mediated (alloimmune or autoimmune) thrombocytopenia.

Hemolytic conditions in the neonate

1. Immune-mediated (positive direct Coombs test) a. Rhesus blood group: Anti-D, -c, -C, -e, -E, CW, and several others

b. Non-Rhesus blood groups: Kell, Duffy, Kidd, Xg, Lewis, MNS, and others

c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

a. Glucose-6-phosphate dehydrogenase (G6PD) deficiency

b. Pyruvate kinase deficiency

c. Others

3. RBC membrane defects

a. Hereditary spherocytosis

b. Elliptocytosis

c. Stomatocytosis

d. Pyknocytosis

e. Others

4. Hemoglobinopathies

a. alpha-thalassemia

b. gamma-thalassemia

Standard maternal antibody screeningAlloantibody Blood Group

D, C, c, E, e, f, CW, V Rhesus

K, k, Kpa, Jsa Kell

Fya, Fyb Duffy

Jka, Jkb Kidd

Xga Xg

Lea, Leb Lewis

S, s, M, N MNS

P1 P

Lub Lutheran

Non-ABO alloantibodies reported to cause moderate to severe hemolytic disease of the newbornWithin Rh system: Anti-D, -c, -C, -Cw, -Cx, -e, -E, -Ew, -ce, -Ces, -Rh29, -Rh32, -Rh42, -f, -G, -Goa, -Bea, -Evans, -Rh17, -Hro, -Hr, -Tar, -Sec, -JAL, -STEM

Outside Rh system: Anti-LW, -K, -k, -Kpa, -Kpb, -Jka, -Jsa, -Jsb, -Ku, -K11, -K22, -Fya, -M, -N, -S, -s, -U, -PP1 pk, -Dib, -Far, -MUT, -En3, -Hut, -Hil, -Vel, -MAM, -JONES, -HJK, -REIT

Red Blood Cell Enzymopathies

G6PD9 and pyruvate kinase (PK) deficiency are the 2 most common red-cell enzyme disorders associated with marked neonatal hyperbilirubinemia. Of these, G6PD deficiency is the more frequently encountered and it remains an important cause of kernicterus worldwide, including the United States, Canada, and the United Kingdom, the prevalence in Western countries a reflection in part of immigration patterns and intermarriage. The risk of kernicterus in G6PD deficiency also relates to the potential for unexpected rapidly developing extreme hyperbilirubinemia in this disorder associated with acute severe hemolysis.

Red Blood Cell Membrane Defects

Establishing a diagnosis of RBC membrane defects is classically based on the development of Coombs-negative hyperbilirubinemia, a positive family history, and abnormal RBC smear, albeit it is often difficult because newborns normally exhibit a marked variation in red-cell membrane size and shape. Spherocytes, however, are not often seen on RBC smears of hematologically normal newborns and this morphologic abnormality, when prominent, may yield a diagnosis of hereditary spherocytosis (HS) in the immediate neonatal period. Given that approximately 75% of families affected with hereditary spherocytosis manifest an autosomal dominant phenotype, a positive family history can often be elicited and provide further support for this diagnosis. More recently, Christensen and Henry highlighted the use of an elevated mean corpuscular hemoglobin concentration (MCHC) (>36.0 g/dL) and/or elevated ratio of MCHC to mean corpuscular volume, the latter they term the “neonatal HS index” (>0.36, likely >0.40) as screening tools for HS. An index of greater than 0.36 had 97% sensitivity, greater than 99% specificity, and greater than 99% negative predictive value for identifying HS in neonates. Christensen and colleagues also provided a concise update of morphologic RBC features that may be helpful in diagnosing this and other underlying hemolytic conditions in newborns.

The diagnosis of HS can be confirmed using the incubated osmotic fragility test when coupled with fetal red-cell controls or eosin-5-maleimide flow cytometry. One must rule out symptomatic ABO hemolytic disease by performing a direct Coombs test, as infants so affected also may manifest prominent micro-spherocytosis. Moreover, HS and symptomatic ABO hemolytic disease can occur in the same infant and result in severe hyperbilirubinemia and anemia. Of other red-cell membrane defects, only hereditary elliptocytosis, stomato-cytosis, and infantile pyknocytosis have been reported to exhibit significant hemolysis in the newborn period. Hereditary elliptocytosis and stomatocytosis are both rare. Infantile pyknocytosis, a transient red-cell membrane abnormality manifesting itself during the first few months of life, is more common.

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

Albumin less than 3.0 g/dL
Data from Maisels MJ, Bhutani VK, Bogen D, et al. Hyperbilirubinemia in the newborn infant > or 535 weeks’ gestation: an update with clarifications. Pediatrics 2009; 124:1193–8.

Polycythemia

Polycythemia (venous hematocrit 65%) in seen in infants across a range of conditions associated with active erythropoiesis or passive transfusion.76,77 They include, among others, placental insufficiency, the infant of a diabetic mother, recipient in twin-twin transfusion syndrome, and several aneuploidies, including trisomy. The clinical concern related to polycythemia is the risk for microcirculatory complications of hyperviscosity. However, determining which polycythemic infants are hyperviscous and when to intervene is a challenge.

Liver

Metabolic disorders presenting as liver disease

Germaine Pierre, Efstathia Chronopoulou
Paediatrics and Child Health 2013; 23(12): 509-514
The liver is a highly metabolically active organ and many inherited metabolic disorders have hepatic manifestations. The clinical presentation in these patients cannot usually be distinguished from liver disease due to acquired causes like infection, drugs or hematological disorders. Manifestations include acute and chronic liver failure, cholestasis and hepatomegaly. Metabolic causes of acute liver failure in childhood can be as high as 35%. Certain disorders like citrin deficiency and Niemann-Pick C disease may present in infancy with self-limiting cholestasis before presenting in later childhood or adulthood with irreversible disease. This article reviews important details from the history and clinical examination when evaluating the pediatric patient with suspected metabolic disease, the specialist and genetic tests when investigating, and also discusses specific disorders, their clinical course and treatment. The role of liver transplantation is also briefly discussed. Increased awareness of this group of disorders is important as in many cases, early diagnosis leads to early intervention with improved outcome. Diagnosis also allows genetic counselling and future family planning.

Adult liver disorders caused by inborn errors of metabolism: Review and update

Sirisak Chanprasert, Fernando Scaglia
Molecular Genetics and Metabolism 114 (2015) 1–10
http://dx.doi.org/10.1016/j.ymgme.2014.10.011

Inborn errors of metabolism (IEMs) are a group of genetic diseases that have protean clinical manifestations and can involve several organ systems. The age of onset is highly variable but IEMs afflict mostly the pediatric population. However, in the past decades, the advancement in management and new therapeutic approaches have led to the improvement in IEM patient care. As a result, many patients with IEMs are surviving into adulthood and developing their own set of complications. In addition, some IEMs will present in adulthood. It is important for internists to have the knowledge and be familiar with these conditions because it is predicted that more and more adult patients with IEMs will need continuity of care in the near future. The review will focus on Wilson disease, alpha-1 antitrypsin deficiency, citrin deficiency, and HFE-associated hemochromatosis which are typically found in the adult population. Clinical manifestations and pathophysiology, particularly those that relate to hepatic disease as well as diagnosis and management will be discussed in detail.

Inborn errors of metabolism (IEMs) are a group of genetic diseases characterized by abnormal processing of biochemical reactions, resulting in accumulation of toxic substances that could interfere with normal organ functions, and failure to synthesize essential compounds. IEMs are individually rare, but collectively numerous. The clinical presentations cover a broad spectrum and can involve almost any organ system. The age of onset is highly variable but IEMs afflict mostly the pediatric population.

Wilson disease is an autosomal recessive genetic disorder of copper metabolism. It is characterized by an abnormal accumulation of inorganic copper in various tissues, most notably in the liver and the brain, especially in the basal ganglia. The disease was first described in 1912 by Kinnier Wilson, and affects between 1 in 30,000 and 1 in 100,000 individuals. Clinical features are variable and depend on the extent and the severity of copper deposition. Typically, patients tend to develop hepatic disease at a younger age than the neuropsychiatric manifestations. Individuals withWilson disease eventually succumb to complications of end stage liver disease or become debilitated from neurological problems, if they are left untreated.

The clinical presentations of Wilson disease are varied affecting many organ systems. However, the overwhelming majority of cases display hepatic and neurologic symptoms. In general, patients with hepatic disease present between the first and second decades of life although patients as young as 3 years old or over 50 years old have also been reported. The most common modes of presentations are acute self-limited hepatitis and chronic active hepatitis that are indistinguishable from other hepatic disorders although liver aminotransferases are generally much lower than in autoimmune or viral hepatitis. Acute fulminant hepatic failure is less common but is observed in approximately 3% of all cases of acute liver failure. Symptoms of acute liver failure include jaundice, coagulopathy, and hepatic encephalopathy. Cirrhosis can develop over time and may be clinically silent. Hepatocellular carcinoma (HCC) is rarely associated with Wilson disease, but may occur in the setting of cirrhosis and chronic inflammation.

Copper is an essential element, and is required for the proper functioning of various proteins and enzymes. The total body content of copper in a healthy adult individual is approximately 70–100 mg, while the daily requirements are estimated to be between 1 and 5 mg. Absorption occurs in the small intestine. Copper is taken up to the hepatocytes via the copper transporter hTR1. Once inside the cell, copper is bound to various proteins including metallothionein and glutathione, however, it is the metal chaperone, ATOX1 that helps direct copper to the ATP7B protein for intracellular transport and excretion. At the steady state, copper will be bound to ATP7B and is then incorporated to ceruloplasmin and secreted into the systemic circulation. When the cellular copper concentration arises, ATP7B protein will be redistributed from the trans-Golgi network to the prelysosomal vesicles facilitating copper excretion into the bile. The molecular defects in ATP7B lead to a reduction of copper excretion. Excess copper is accumulated in the liver causing tissue injury. The rate of accumulation of copper varies among individuals, and it may depend on other factors such as alcohol consumption, or viral hepatitis infections. If the liver damage is not severe, patients will accumulate copper in various tissues including the brain, the kidney, the eyes, and the musculoskeletal system leading to clinical disease. A failure of copper to incorporate into ceruloplasmin leads to secretion of the unsteady protein that has a shorter half-life, resulting in the reduced concentrations of ceruloplasmin seen in most patients with Wilson disease.

Wilson disease used to be a progressive fatal condition during the first half of the 20th century because there was no effective treatment available at that time. Penicillamine was the first pharmacologic agent introduced in 1956 for treating this condition. Penicillamine is a sulfhydryl-bearing amino acid cysteine doubly substituted with methyl groups. This drug acts as a chelating agent that promotes the urinary excretion of copper. It is rapidly absorbed in the gastrointestinal track, and over 80% of circulating penicillamine is excreted via the kidneys. Although it is very effective, approximately 10%–50% of Wilson disease patients with neuropsychiatric presentations may experience worsening of their symptoms, and often times the worsening symptoms may not be reversible.

Alpha1-antitrypsin deficiency

Alpha1-antitrypsin deficiency (AATD) is one of the most common genetic liver diseases in children and adults, affecting 1 in 2000 to 1 in 3000 live births worldwide. It is transmitted in an autosomal co-dominant fashion with variable expressivity. Alpha1 antitrypsin (A1AT) is a member of the serine protease inhibitor (SERPIN) family. Its function is to counteract the proteolytic effect of neutrophil elastase and other neutrophil proteases. Mutations in the SERPINA1, the gene encoding A1AT, result in changes in the protein structure with the PiZZ phenotype being the most common cause of liver and lung disease-associated AATDs. Although, it classically causes early onset chronic obstructive pulmonary disease (COPD) in adults, liver disease characterized by chronic inflammation, hepatic fibrosis, and cirrhosis is not uncommon in the adult population. Decreased plasma concentration of A1AT predisposes lung tissue to be more susceptible to injury from protease enzymes. However, the underlying mechanism of liver injury is different, and is believed to be caused by accumulation of polymerized mutant A1AT in the hepatocyte endoplasmic reticulum (ER). Currently, there is no specific treatment for liver disease-associated AATD, but A1AT augmentation therapy is available for patients affected with pulmonary involvement.

A1AT is a single-chain, 52-kDa polypeptide of approximately 394 amino acids [56]. It is synthesized in the liver, circulates in the plasma, and functions as an inhibitor of neutrophil elastase and other proteases such as cathepsin G, and proteinase 3. A1AT has a globular shape composed of two central β sheets surrounded by a small β sheet and nine α helices. The pathophysiology underlying liver disease is thought to be a toxic gain-of-function mutation associated with the PiZZ phenotypes. This hypothesis has been supported by the fact that null alleles which produce no detectable plasma A1AT, are not associated with liver disease. In addition, the transgenic mouse model of AATD PiZZ developed periodic acid-Schiff-positive diastase-resistant intrahepatic globule early in life similar to AATD patients. The PiZZ phenotype results in the blockade of the final processing of A1AT in the liver, as only 15% of the A1AT reaches the circulation whereas 85% of non-secreted protein is accumulated in the hepatocytes.

Citrin deficiency

Citrin deficiency is a relatively newly-defined autosomal recessive disease. It encompasses two different sub-groups of patients, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), and adult onset citrullinemia type 2 (CTLN 2).

AGC2 exports aspartate out of the mitochondrial matrix in exchange for glutamate and a proton. Thus, this protein has an important role in ureagenesis and gluconeogenesis. In CTLN2, a defect in this protein is believed to limit the supply of aspartate for the formation of argininosuccinate in the cytosol resulting in impairment of ureagenesis. Interestingly, the mouse model of citrin deficiency (Ctrn−/−) fails to develop symptoms of CTLN2 suggesting that the mitochondrial aspartate is not the only source of ureagenesis. However, it should be noted that the rodent liver expresses higher glycerol-phosphate shuttle activity than the human counterpart. With the intact glycerol-phosphate dehydrogenase, it can compensate for the deficiency of AGC2, as demonstrated by the AGC2 and glycerol-phosphate dehydrogenase double knock-out mice that exhibit similar features to those observed in human CTLN2.

HFE-associated hemochromatosis

HFE-associated hemochromatosis is an inborn error of iron metabolism characterized by excessive iron storage resulting in tissue and organ damage. It is the most common autosomal recessive disorder in the Caucasian population, affecting 0.3%–0.5% of individuals of Northern European descent. The term “hemochromatosis” was coined in 1889 by the German pathologist Friedrich Daniel Von Recklinghausen, who described it as bronze stain of organs caused by a blood borne pigment.

The classic clinical triad of cirrhosis, diabetes, and bronze skin pigmentation is rarely observed nowadays given the early recognition, diagnosis, and treatment of this condition. The most common presenting symptoms are nonspecific including weakness, lethargy, and arthralgia.

The liver is a major site of iron storage in healthy individuals and as such it is the organ that is universally affected in HFE-associated hemochromatosis. Elevation of liver aminotransferases indicative of hepatocyte injury is the most common mode of presentation and it can be indistinguishable from other causes of hepatitis. Approximately 15%–40% of patients with HFE-associated hemochromatosis have other liver conditions, including chronic viral hepatitis B or C infection, nonalcoholic fatty liver disease, and alcoholic liver disease.

The liver in haemochromatosis

Rune J. Ulvik
Journal of Trace Elements in Medicine and Biology xxx (2014) xxx–xxx
http://dx.doi.org/10.1016/j.jtemb.2014.08.005

The review deals with genetic, regulatory and clinical aspects of iron homeostasis and hereditary hemochromatosis. Hemochromatosis was first described in the second half of the 19th century as a clinical entity characterized by excessive iron overload in the liver. Later, increased absorption of iron from the diet was identified as the pathophysiological hallmark. In the 1970s genetic evidence emerged supporting the apparent inheritable feature of the disease. And finally in 1996 a new “hemochromato-sis gene” called HFE was described which was mutated in about 85% of the patients. From the year2000 onward remarkable progress was made in revealing the complex molecular regulation of iron trafficking in the human body and its disturbance in hemochromatosis. The discovery of hepcidin and ferroportin and their interaction in regulating the release of iron from enterocytes and macrophages to plasma were important milestones. The discovery of new, rare variants of non-HFE-hemochromatosis was explained by mutations in the multicomponent signal transduction pathway controlling hepcidin transcription. Inhibited transcription induced by the altered function of mutated gene products, results in low plasma levels of hepcidin which facilitate entry of iron from enterocytes into plasma. In time this leads to progressive accumulation of iron and subsequently development of disease in the liver and other parenchymatous organs. Being the major site of excess iron storage and hepcidin synthesis the liver is a cornerstone in maintaining normal systemic iron homeostasis. Its central pathophysiological role in HFE-hemochromatosis with downgraded hepcidin synthesis, was recently shown by the finding that liver transplantation normalized the hepcidin levels in plasma and there was no sign of iron accumulation in the new liver.

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

Roberto Murgas Torrazza, Nan Li, Josef Neu
Pathophysiology 21 (2014) 21–27
http://dx.doi.org/10.1016/j.pathophys.2013.11.011

Necrotizing enterocolitis (NEC) is an enigmatic disease that affects primarily premature infants. It often occurs suddenly and when it occurs, treatment attempts at treatment often fail and results in death. If the infant survives, there is a significant risk of long term sequelae including neurodevelopmental delays. The pathophysiology of NEC is poorly understood and thus prevention has been difficult. In this review, we will provide an overview of why progress may be slow in our understanding of this disease, provide a brief review diagnosis, treatment and some of the current concepts about the pathophysiology of this disease.

Necrotizing enterocolitis (NEC) has been reported since special care units began to house preterm infants .With the advent of modern neonatal intensive care approximately 40 years ago, the occurrence and recognition of the disease markedly increased. It is currently the most common and deadly gastro-intestinal illness seen in preterm infants. Despite major efforts to better understand, treat and prevent this devastating disease, little if any progress has been made during these 4 decades. Underlying this lack of progress is the fact that what is termed “NEC” is likely more than one disease, or mimicked by other diseases, each with a different etiopathogenesis.

Human gut microbiome

Term or near term infants with “NEC” when compared to matched controls usually have occurrence of their disease in the first week after birth, have a significantly higher frequency of prolonged rupture of membranes, chorio-amnionitis, Apgar score <7 at 1 and 5 min, respiratory problems, congenital heart disease, hypoglycemia, and exchange transfusions. When a “NEC” like illness presents in term or near term infants, it should be noted that these are likely to be distinct in pathogenesis than the most common form of NEC and should be differentiated as such.

The infants who suffer primary ischemic necrosis are term or near term infants (although this can occur in preterms) who have concomitant congenital heart disease, often related to poor left ventricular output or obstruction. Other factors that have been associated with primary ischemia are maternal cocaine use, hyperviscosity caused by polycythemia or a severe antecedent hypoxic–ischemic event. Whether the dis-ease entity that results from this should be termed NEC can be debated on historical grounds, but the etiology is clearly different from the NEC seen in most preterm infants.

The pathogenesis of NEC is uncertain, and the etiology seems to be multifactorial. The “classic” form of NEC is highly associated with prematurity; intestinal barrier immaturity, immature immune response, and an immature regulation of intestinal blood flow (Fig.). Although genetics appears to play a role, the environment, especially a dysbiotic intestinal microbiota acting in concert with host immaturities predisposes the preterm infant to disruption of the intestinal epithelia, increased permeability of tight junctions, and release of inflammatory mediators that leads to intestinal mucosa injury and therefore development of necrotizing enterocolitis.

NEC is a multifactorial disease

What causes NEC? NEC is a multifactorial disease with an interaction of several etiophathologies

It is clear from this review that there are several entities that have been described as NEC. What is also clear is that despite having some overlap in the final parts of the pathophysiologic cascade that lead to necrosis, the disease that is most commonly seen in the preterm infant is likely to have an origin that differs markedly from that seen in term infants with congenital heart disease or severe hypoxic–ischemic injury. Thus, epidemiologic studies will need to differentiate these entities, if the aim is to dissect common features that are most highly associated with development of the disease. At this juncture, we areleft with more of a population based preventative approach, where the use of human milk, evidence based feeding guide-lines, considerations for microbial therapy once these are proved safe and effective and approved as such by regulatory authorities, and perhaps even measures that prevent prematurity will have a major impact on this devastating disease.

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines

Influenced by the microbiota, intestinal epithelial cells (IECs) elaborate cytokines, including thymic stromal lymphoprotein (TSLP), transforming growthfactor (TGF), and interleukin-10 (IL-10), that can influence pro-inflammatory cytokine production by dendritic cells (DC) and macrophages present in the laminapropria (GALT) and Peyer’s patches. Signals from commensal organisms may influence tissue-specific functions, resulting in T-cell expansion and regulation of the numbers of Th-1,
Th-2, and Th-3 cells. Also modulated by the microbiota, other IEC derived factors, including APRIL (a proliferation-inducing ligand),B-cell activating factor (BAFF), secretory leukocyte peptidase inhibitor (SLPI), prostaglandin E2(PGE2), and other metabolites, directly regulate functions ofboth antigen presenting cells and lymphocytes in the intestinal ecosystem. NK: natural killer cell; LN: lymph node; DC: dendritic cells.Modified from R. Sharma, C. Young, M. Mshvildadze, J. Neu, Intestinal microbiota does it play a role in diseases of the neonate? NeoReviews 10 (4) (2009)e166, with permission

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Current Issues in the Management of Necrotizing Enterocolitis

Marion C. W. Henry and R. Lawrence Moss
Seminars in Perinatology, 2004; 28(3): 221-233
http://dx.doi.org:/10.1053/j.semperi.2004.03.010

Necrotizing enterocolitis is almost exclusively a disease of prematurity, with 90% of all cases occurring in premature infants and 90% of those infants weighing less than 2000 g. Prematurity is the only risk factor for necrotizing enterocolitis consistently identified in case control studies and the disease is rare in countries where prematurity is uncommon such as Japan and Sweden. When necrotizing enterocolitis does occur in full-term infants, it appears to by a somewhat different disease, typically associated with some predisposing condition.

NEC occurs in one to three in 1,000 live births and most commonly affects babies born between 30-32 weeks. It is most often diagnosed during the second week of life and occurs more often in previously fed infants. The mortality from NEC has been cited as 10% to 50% of all NEC cases. Surgical mortality has decreased over the last several decades from 70% to between 20 and 50%. The incremental cost per case of acute hospital care is estimated at $74 to 186 thousand compared to age matched controls, not including additional costs of long term care for the infants’ with lifelong morbidity. Survivors may develop short bowel syndrome, recurrent bouts of catheter-related sepsis, malabsorption, malnutrition, and TPN induced liver failure.

Although extensive research concerning the pathophysiology of necrotizing enterocolitis has occurred, a complete understanding has not been fully elucidated. The classic histologic finding is coagulation necrosis; present in over 90% of specimens. This finding suggests the importance of ischemia in the pathogenesis of NEC. Inflammation and bacterial overgrowth also are present. These findings support the assumptions by Kosloske that NEC occurs by the interaction of 3 events:

intestinal ischemia,
colonization by pathogenic bacteria and
excess protein substrate in the intestinal lumen.

Additionally, the immunologic immaturity of the neonatal gut has been implicated in the development of NEC. Reparative tissue changes including epithelial regeneration, formation of granulation tissue and fibrosis, and mixed areas of acute and chronic inflammatory changes suggest that the pathogenesis of NEC may involve a chronic process of injury and repair.

Premature newborns born prior to the 32^nd week of gestational age may have compromised intestinal peristalsis and decreased motility. These motility problems may lead to poor clearance of bacteria, and subsequent bacterial overgrowth. Premature infants also have an immature intestinal tract in terms of immunologic immunity.

There are fewer functional B lymphocytes present and the ability to produce sufficient secretory IgA is reduced. Pepsin, gastric acid and mucus are also not produced as well in prematurity. All of these factors may contribute to the limited proliferation of intestinal flora and the decreased binding of these flora to mucosal cells (Fig).

Role of nitric oxide in the pathogenesis of NEC

Role of nitric oxide in the pathogenesis of NEC.

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis

Characteristics of the immature gut leading to increased risk of necrotizing enterocolitis.

As understanding of the pathophysiology of necrotizing enterocolitis continues to evolve, a unifying concept is emerging. Initially, there is likely a subclinical insult leading to NEC. This may arise from a brief episode of hypoxia or infection. With colonization of the intestines, bacteria bind to the injured mucosa eliciting an inflammatory response which leads to further inflammation.

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

Silvia Arboleya, Borja Sanchez,, Christian Milani, Sabrina Duranti, et al.
Pediatr 2014;-:—). http://dx.doi.org/10.1016/j.jpeds.2014.09.041

Objectives Assess the establishment of the intestinal microbiota in very low birth-weight preterm infants and to evaluate the impact of perinatal factors, such as delivery mode and perinatal antibiotics.
Study design We used 16S ribosomal RNA gene sequence-based microbiota analysis and quantitative polymerase chain reaction to evaluate the establishment of the intestinal microbiota. We also evaluated factors affecting the microbiota, during the first 3 months of life in preterm infants (n = 27) compared with full-term babies (n = 13).
Results Immaturity affects the microbiota as indicated by a reduced percentage of the family Bacteroidaceae during the first months of life and by a higher initial percentage of Lactobacillaceae in preterm infants compared with full term infants. Perinatal antibiotics, including intrapartum antimicrobial prophylaxis, affects the gut microbiota, as indicated by increased Enterobacteriaceae family organisms in the infants.

Human gut microbiome

Conclusions Prematurity and perinatal antibiotic administration strongly affect the initial establishment of microbiota with potential consequences for later health.

Ischemia and necrotizing enterocolitis: where, when, and how

Philip T. Nowicki
Seminars in Pediatric Surgery (2005) 14, 152-158
http://dx.doi.org:/10.1053/j.sempedsurg.2005.05.003

While it is accepted that ischemia contributes to the pathogenesis of necrotizing enterocolitis (NEC), three important questions regarding this role subsist. First, where within the intestinal circulation does the vascular pathophysiology occur? It is most likely that this event begins within the intramural microcirculation, particularly the small arteries that pierce the gut wall and the submucosal arteriolar plexus insofar as these represent the principal sites of resistance regulation in the gut. Mucosal damage might also disrupt the integrity or function of downstream villous arterioles leading to damage thereto; thereafter, noxious stimuli might ascend into the submucosal vessels via downstream venules and lymphatics. Second, when during the course of pathogenesis does ischemia occur? Ischemia is unlikely to the sole initiating factor of NEC; instead, it is more likely that ischemia is triggered by other events, such as inflammation at the mucosal surface. In this context, it is likely that ischemia plays a secondary, albeit critical role in disease extension. Third, how does the ischemia occur? Regulation of vascular resistance within newborn intestine is principally determined by a balance between the endothelial production of the vasoconstrictor peptide endothelin-1 (ET-1) and endothelial production of the vasodilator free radical nitric oxide (NO). Under normal conditions, the balance heavily favors NO-induced vasodilation, leading to a low resting resistance and high rate of flow. However, factors that disrupt endothelial cell function, eg, ischemia-reperfusion, sustained low-flow perfusion, or proinflammatory mediators, alter the ET-1:NO balance in favor of constriction. The unique ET-1–NO interaction thereafter might facilitate rapid extension of this constriction, generating a viscous cascade wherein ischemia rapidly extends into larger portions of the intestine.

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation. Small mesenteric arteries pierce the muscularis layers and terminate in the submucosa where they give rise to 1A (1st order) arterioles. 2A (2nd order) arterioles arise from the 1A. Although not shown here, these 2A arterioles connect merge with several 1A arterioles, thus generating an arteriolar plexus, or manifold that serves to pressurize the terminal downstream microvasculature. 3A (3rd order) arterioles arise from the 2A and proceed to the mucosa, giving off a 4A branch just before descent into the mucosa. This 4A vessel travels to the muscularis layers. Each 3A vessel becomes the single arteriole perfusing each villus.

Collectively, these studies indicate that disruption of endothelial cell function has the potential to disrupt the normal balance between NO and ET-1 within the newborn intestinal circulation, and that such an event can generate significant ischemia. In this context, it is important to note that NO and ET-1 each regulate the expression and activity of the other. An increased [NO] within the microvascular environment reduces ET-1 expression and compromises ligand binding to the ETA receptor (thus decreasing its contractile efficacy), while ET-1 compromises eNOS expression. Thus, factors that upset the balance between NO and ET-1 will have an immediate and direct effect on vascular tone, but also exert an additional indirect effect by extenuating the disruption of balance between these two factors.

It is not difficult to construct a hypothesis that links the perturbations of I/R and sustained low-flow perfusion with an initial inflammatory insult. Initiation of an inflammatory process at the mucosal–luminal interface could have a direct impact on villus and mucosal 3A arterioles, damaging arteriolar integrity and disrupting villus hemodynamics. Ascent of proinflammatory mediators to the submucosal 1A–2A arteriolar plexus could occur via draining venules and lymphatics, generating damage to vascular effector systems therein; these mediators might include cytokines and platelet activating factor, as these elements have been recovered from human infants with NEC. This event, coupled with a generalized loss of 3A flow throughout a large portion of the mucosal surface, could compromise flow rate within the submucosal arteriolar plexus.

Necrotizing enterocolitis: An update

Loren Berman, R. Lawrence Moss
Seminars in Fetal & Neonatal Medicine 16 (2011) 145e150
http://dx.doi.org:/10.1016/j.siny.2011.02.002

Necrotizing enterocolitis (NEC) is a leading cause of death among patients in the neonatal intensive care unit, carrying a mortality rate of 15e30%. Its pathogenesis is multifactorial and involves an over reactive response of the immune system to an insult. This leads to increased intestinal permeability, bacterial translocation, and sepsis. There are many inflammatory mediators involved in this process, but thus far none has been shown to be a suitable target for preventive or therapeutic measures. NEC usually occurs in the second week of life after the initiation of enteral feeds, and the diagnosis is made based on physical examination findings, laboratory studies, and abdominal radiographs. Neonates with NEC are followed with serial abdominal examinations and radiographs, and may require surgery or primary peritoneal drainage for perforation or necrosis. Many survivors are plagued with long term complications including short bowel syndrome, abnormal growth, and neurodevelopmental delay. Several evidence-based strategies exist that may decrease the incidence of NEC including promotion of human breast milk feeding, careful feeding advancement, and prophylactic probiotic administration in at-risk patients. Prevention is likely to have the greatest impact on decreasing mortality and morbidity related to NEC, as little progress has been made with regard to improving outcomes for neonates once the disease process is underway.

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

Nima Parvaneh, Jean-Laurent Casanova, LD Notarangelo, ME Conley
J Allergy Clin Immunol 2013;131:314-23.
http://dx.doi.org/10.1016/j.jaci.2012.11.051

The characterization of primary immunodeficiencies (PIDs) in human subjects is crucial for a better understanding of the biology of the immune response. New achievements in this field have been possible in light of collaborative studies; attention paid to new phenotypes, infectious and otherwise; improved immunologic techniques; and use of exome sequencing technology. The International Union of Immunological Societies Expert Committee on PIDs recently reported on the updated classification of PIDs. However, new PIDs are being discovered at an ever-increasing rate. A series of 19 novel primary defects of immunity that have been discovered after release of the International Union of Immunological Societies report are discussed here. These new findings highlight the molecular pathways that are associated with clinical phenotypes and suggest potential therapies for affected patients.

Combined Immunodeficiencies

T-cell receptor a gene mutation: T-cell receptor ab1 T-cell depletion

T cells comprise 2 distinct lineages that express either ab or gd T-cell receptor (TCR) complexes that perform different tasks in immune responses. During T-cell maturation, the precise order and efficacy of TCR gene rearrangements determine the fate of the cells. Productive β-chain gene rearrangement produces a pre-TCR on the cell surface in association with pre-Tα invariant peptide (β-selection). Pre-TCR signals promote α-chain recombination and transition to a double-positive stage (CD41CD81). This is the prerequisite for central tolerance achieved through positive and negative selection of thymocytes.

Ras homolog gene family member H deficiency: Loss of naive T cells and persistent human papilloma virus infections
MST1 deficiency: Loss of naive T cells

New insight into the role of MST1 as a critical regulator of T-cell homing and function was provided by the characterization of 8 patients from 4 unrelated families who had homozygous nonsense mutations in STK4, the gene encoding MST1. MST1 was originally identified as an ubiquitously expressed kinase with structural homology to yeast Ste. MST1 is the mammalian homolog of the Drosophila Hippo protein, controlling cell growth, apoptosis, and tumorigenesis. It has both proapoptotic and antiapoptotic functions.

Lymphocyte-specific protein tyrosine kinase deficiency: T-cell deficiency with CD41 lymphopenia

Defects in pre-TCR– and TCR-mediated signaling lead to aberrant T-cell development and function (Fig). One of the earliest biochemical events occurring after engagement of the (pre)-TCR is the activation of lymphocyte-specific protein tyrosine kinase (LCK), a member of the SRC family of protein tyrosine kinases. This kinase then phosphorylates immunoreceptor tyrosine-based activation motifs of intracellular domains of CD3 subunits. Phosphorylated immunoreceptor tyrosine-based activation motifs recruit z-chain associated protein kinase of 70 kDa, which, after being phosphorylated by LCK, is responsible for activation of critical downstream events. Major consequences include activation of the membrane-associated enzyme phospholipase Cg1, activation of the mitogen-activated protein kinase, nuclear translocation of nuclear factor kB (NFkB), and Ca21/Mg21 mobilization. Through these pathways, LCK controls T-cell development and activation. In mice lacking LCK, T-cell development in the thymus is profoundly blocked at an early double-negative stage.

TCR signaling

TCR signaling. Multiple signal transduction pathways are stimulated through the TCR. These pathways collectively activate transcription factors that organize T-cell survival, proliferation, differentiation, homeostasis, and migration. Mutant molecules in patients with TCR-related defects are indicated in red.

Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

Idiopathic CD41 lymphopenia (ICL) is a very heterogeneous clinical entity that is defined, by default, by persistent CD41 T-cell lymphopenia (<300 cells/mL or <20% of total T cells) in the absence of HIV infection or any other known cause of immunodeficiency.

Well-Defined Syndromes with Immunodeficiency

Wiskott-Aldrich syndrome protein–interacting protein deficiency: Wiskott-Aldrich syndrome-like phenotype

In hematopoietic cells Wiskott-Aldrich syndrome protein (WASP) is stabilized through forming a complex with WASP interacting protein (WIP).

Phospholipase Cg2 gain-of-function mutations: Cold urticaria, immunodeficiency, and autoimmunity/autoinflammatory

This is a unique phenotype, sharing features of antibody deficiency, autoinflammatory diseases, and immune dysregulatory disorders, making its classification difficult. Two recent studies validated the pleiotropy of genetic alterations in the same gene.

Predominantly Antibody Defects

Defect in the p85a subunit of phosphoinositide 3-kinase: Agammaglobulinemia and absent B cells
CD21 deficiency: Hypogammaglobulinemia
LPS-responsive beige-like anchor deficiency:
Hypogammaglobulinemia with autoimmunity and

early colitis

Defects Of Immune Dysregulation

Pallidin deficiency: Hermansky-Pudlak syndrome type 9
CD27 deficiency: Immune dysregulation and
persistent EBV infection

Congenital Defects Of Phagocyte Number, Function, Or Both

Interferon-stimulated gene 15 deficiency: Mendelian susceptibility to mycobacterial diseases

Defects In Innate Immunity

NKX2-5 deficiency: Isolated congenital asplenia
Toll/IL-1 receptor domain–containing adaptor inducing IFN-b and TANK-binding kinase 1 deficiencies: Herpes simplex encephalitis
Minichromosome maintenance complex component 4 deficiency: NK cell deficiency associated with growth retardation and adrenal insufficiency

Autoinflammatory Disorders

A disintegrin and metalloproteinase 17 deficiency: Inflammatory skin and bowel disease

Cross-talk between monocyte.macrophage cells and T.NK lymphocytes

Cross-talk between monocyte/macrophage cells and T/NK lymphocytes. Genes in the IL-12/IFN-g pathway are particularly important for protection against mycobacterial disease. IRF8 is an IFN-g–inducible transcription factor required for the induction of various target genes, including IL-12. The NF-kB essential modulator (NEMO) mutations in the LZ domain impair CD40-NEMO–dependent pathways. Some gp91phox mutations specifically abolish the respiratory burst in monocyte-derived macrophages. ISG15 is secreted by neutrophils and potentiates IFN-g production by NK/T cells. Genetic defects that preclude monocyte development (eg, GATA2) can also predispose to mycobacterial infections (not shown). Mutant molecules in patients with unusual susceptibility to infection are indicated in red.

The field of PIDs is advancing at full speed in 2 directions. New genetic causes of known PIDs are being discovered (eg, CD21 and TRIF). Moreover, new phenotypes qualify as PIDs with the identification of a first genetic cause (eg, generalized pustular psoriasis). Recent findings contribute fundamental knowledge about immune system biology and its perturbation in disease. They are also of considerable clinical benefit for the patients and their families. A priority is to further translate these new discoveries into improved diagnostic methods and more effective therapeutic strategies, promoting the well-being of patients with PIDs.

Primary immunodeficiencies

Luigi D. Notarangelo
J Allergy Clin Immunol 2010; 125(2): S182-194
http://dx.doi.org:/10.1016/j.jaci.2009.07.053

In the last years, advances in molecular genetics and immunology have resulted in the identification of a growing number of genes causing primary immunodeficiencies (PIDs) in human subjects and a better understanding of the pathophysiology of these disorders. Characterization of the molecular mechanisms of PIDs has also facilitated the development of novel diagnostic assays based on analysis of the expression of the protein encoded by the PID-specific gene. Pilot newborn screening programs for the identification of infants with severe combined immunodeficiency have been initiated. Finally, significant advances have been made in the treatment of PIDs based on the use of subcutaneous immunoglobulins, hematopoietic cell transplantation from unrelated donors and cord blood, and gene therapy. In this review we will discuss the pathogenesis, diagnosis, and treatment of PIDs, with special attention to recent advances in the field.

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Endocrine Action on Midbrain

Posted in Biological Networks, Calcium Signaling, Cell Biology, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Curation, Cytoskeleton, Developmental biology, Diabetes Mellitus, Disease Biology, Embryology, Endocrine Diseases, Genomic Endocrinology, Innovations in Neurophysiology & Neuropsychology, Lipid metabolism, Metabolism, Metabolomics, Neurodegenerative Diseases, Neurological Diseases, Nutrition, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Preimplantation Genetic Diagnosis and Reproductive Genomics, Proteomics, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, Signaling, Signaling & Cell Circuits, Small Molecules in Development of Therapeutic Drugs, tagged brain, cannabinoids, Cognition, glutamate, gonadotrophs, GPCRs, midbrain, neurodegeneration, neuroendocrinology, neuronal plasticity, sexual dimorphism, sterois hormones on February 12, 2015| Leave a Comment »

Endocrine Action on Midbrain

Writer and Curator: Larry H. Bernstein, MD, FCAP

Brain’s Role in Browning White Fat
Insulin and leptin act on specialized neurons in the mouse hypothalamus to promote conversion of white to beige fat.

By Anna Azvolinsky | January 15, 2015

JUSTIN HEWLETT, MNHS MULTIMEDIA, MONASH UNIVERSITY

Ever since energy-storing white fat has been shown to convert to metabolically active beige fat, through a process called browning, scientists have been trying to understand how this switch occurs. The immune system has been shown to contribute to activation of brown fat cells. Now, researchers from Monash University in Australia and their colleagues have shown that insulin and leptin—two hormones that regulate glucose metabolism and satiety and hunger cues—activate “satiety” neurons in the mouse hypothalamus to promote the conversion of white fat to beige. The results are published today (January 15) in Cell.

Hypothalamic appetite-suppressing proopiomelanocortin (POMC) neurons are known to relay the satiety signals in the bloodstream to other parts of the brain and other tissues to promote energy balance. “What is new here is that one way that these neurons promote calorie-burning is to stimulate the browning of white fat,” said Xiaoyong Yang, who studies the molecular mechanisms of metabolism at the Yale University School of Medicine, but was not involved in the work. “The study identifies how the brain communicates to fat tissue to promote energy dissipation.”

“The authors show that [insulin and leptin] directly interact in the brain to produce nervous-system signaling both to white and brown adipose tissue,” said Jan Nedergaard, a professor of physiology at Stockholm University who also was not involved in the study. “This is a nice demonstration of how the acute and chronic energy status talks to the thermogenic tissues.”

Although the differences between beige and brown fat are still being defined, the former is currently considered a metabolically active fat—which converts the energy of triglycerides into heat—nestled within white fat tissue. Because of their energy-burning properties, brown and beige fat are considered superior to white fat, so understanding how white fat can be browned is a key research question. Exposure to cold can promote the browning of white fat, but the ability of insulin and leptin to act in synergy to signal to the brain to promote browning was not known before this study, according the author Tony Tiganis, a biochemist at Monash.

White fat cells steadily produce leptin, while insulin is produced by cells of the pancreas in response to a surge of glucose into the blood. Both hormones are known to signal to the brain to regulate satiety and body weight. To explore the connection between this energy expenditure control system and fat tissue, Garron Dodd, a postdoctoral fellow in Tiganis’s laboratory, and his colleagues deleted one or both of two phosphatase enzymes in murine POMC neurons. These phosphatase enzymes were previously known to act in the hypothalamus to regulate both glucose metabolism and body weight, each regulating either leptin or insulin signaling. When both phosphatases were deleted, mice had less white fat tissue and increased insulin and leptin signaling.

“These [phosphatase enzymes] work in POMC neurons by acting as ‘dimmer switches,’ controlling the sensitivity of leptin and insulin receptors to their endogenous ligands,” Dodd told The Scientist in an e-mail. The double knockout mice also had an increase in beige fat and more active heat-generating brown fat. When fed a high-fat diet, unlike either the single knockout or wild-type mice, the double knockout mice did not gain weight, suggesting that leptin and insulin signaling to POMC neurons is important for controlling body weight and fat metabolism.

The researchers also infused leptin and insulin directly into the hypothalami of wild-type mice, which promoted the browning of white fat. But when these hormones were infused but the neuronal connections between the white fat and the brain were physically severed, browning was prevented. Moreover, hormone infusion and cutting the neuronal connection to only a single fat pad resulted in browning only in the fat pad that maintained signaling ties to the brain. “This really told us that direct innervation from the brain is necessary and that these hormones are acting together to regulate energy expenditure,” said Tiganis.

These results are “really exciting as, perhaps, resistance to the actions of leptin and insulin in POMC neurons is a key feature underlying obesity in people,” said Dodd.

Another set of neurons in the hypothalamus, the agouti-related protein expressing (AgRP) or “hunger” neurons, are activated by hunger signals and promote energy storage. Along with Tamas Horvath, Yale’s Yang recently showed that fasting activates AgRP neurons that then suppress the browning of white fat. “These two stories are complimentary, providing a bigger picture: that the hunger and satiety neurons control browning of fat depending on the body’s energy state,” said Yang. Activation of POMC neurons during caloric intake protects against diet-induced obesity while activation of AgRP neurons tells the body to store energy during fasting.

Whether these results hold up in humans has yet to be explored. Expression of the two phosphatases in the hypothalamus is known to be higher in obese people, but it is not clear whether this suppresses the browning of white fat.

“One of the next big questions is whether this increased expression and prevention of insulin plus leptin signaling, and conversion of white to brown fat perturbs energy balance and promotes obesity,” said Tiganis. Another, said Dodd, is whether other parts of the brain are involved in signaling to and from adipose tissue.

Dodd et al., “Leptin and insulin act on POMC neurons to promote the browning of white fat,”

Cell, 2015. http://dx.doi.org:/10.1016/j.cell.2014.12.022 http://medicine.yale.edu/lab/horvath/index.aspx

Our main interest is the neuroendocrine regulation of homeostasis with particular emphasis on metabolic disorders, such as obesity and diabetes, and the effect of metabolic signals on higher brain functions and neurodegeneration. We have active research programs to pursue the role of synaptic plasticity in the mediation of peripheral hormones’ effects on the central nervous system.

We also study the role of mitochondrial membrane potential in normal and pathological brain functions with particular emphasis on the acute effect of mitochondria in neuronal transmission and neuroprotection. We combine classical neurobiological approaches, including electrophysiology and neuroanatomy, with endocrine and genetic techniques to better understand biological events at the level of the organism.

Leptin and Insulin Act on POMC Neurons to Promote the Browning of White Fat

Garron T. Dodd, Stephanie Decherf, Kim Loh, Stephanie E. Simonds, Florian Wiede, Eglantine Balland, Troy L. Merry, et al.

http://dx.doi.org/10.1016/j.cell.2014.12.022

Highlights

Insulin and leptin act synergistically on POMC neurons to promote WAT browning
Increased POMC-mediated WAT browning prevents diet-induced obesity
PTP1B and TCPTP attenuate leptin and insulin signaling in POMC neurons
Combined PTP1B and TCPTP deficiency in POMC neurons promotes white fat browning

The primary task of white adipose tissue (WAT) is the storage of lipids. However, “beige” adipocytes also exist in WAT. Beige adipocytes burn fat and dissipate the energy as heat, but their abundance is diminished in obesity. Stimulating beige adipocyte development, or WAT browning, increases energy expenditure and holds potential for combating metabolic disease and obesity. Here, we report that insulin and leptin act together on hypothalamic neurons to promote WAT browning and weight loss. Deletion of the phosphatases PTP1B and TCPTP enhanced insulin and leptin signaling in proopiomelanocortin neurons and prevented diet-induced obesity by increasing WAT browning and energy expenditure. The coinfusion of insulin plus leptin into the CNS or the activation of proopiomelanocortin neurons also increased WAT browning and decreased adiposity. Our findings identify a homeostatic mechanism for coordinating the status of energy stores, as relayed by insulin and leptin, with the central control of WAT browning. http://www.cell.com/cms/attachment/2023992410/2043906325/fx1.jpg

Light on the Brain

Researchers find that photoreceptors expressed in zebrafish hypothalamus contribute to light-dependent behavior.

By Sabrina Richards | September 20, 2012

A 21 day old zebrafish. Their optical clarity and relatively easy maintenance make them a favorite for geneticists and developmental biologists. In this fish, the muscles can be seen as chevron shapes in the tail, the swim bladder as a “bubble” just behind the head, and the food that the fish has been eating as a brown patch just below the swim bladder.

Juvenile zebrafish. Shawn Burgess, NHGRI

Zebrafish larvae without eyes or pineal glands can still respond to light using photopigments located deep within their brains. Published today (September 20) in Current Biology, the findings are the first to link opsins, photoreceptors in the hypothalamus and other brain areas, to increased swimming in response to darkness, a behavior researchers hypothesize may help the fish move toward better-lit environments.

“[It’s a] strong demonstration that opsin-dependent photoreceptors in deep brain areas affect behaviors,” said Samer Hattar, who studies light reception in mammals at Johns Hopkins University but did not participate in the research.

Photoreceptors in eyes enable vision, and photoreceptors in the pineal gland, a small endocrine gland located in the center of the vertebrate brain, regulate circadian rhythms. But photoreceptors are also found in other brain areas of both invertebrates and vertebrate lineages. The function of these extraocular photoreceptors has been best studied in birds, where they regulate seasonal reproduction, explained Harold Burgess, a behavioral neurogeneticist at the Eunice Kennedy Shriver National Institute for Child Health and Human Development. Many opsins have been reported in the brains of tiny and transparent larval zebrafish, raising the possibility that light could be stimulating the photoreceptors even deep in the brain. To test for behaviors that may be regulated by deep brain photoreceptors, Burgess and his colleagues in Wolfgang Driever’s lab at the University of Freiburg removed the eyes of zebrafish larvae, and compared their behavior to larvae that retained their eyes. Although most light-dependent behavior required eyes, the eyeless larvae did respond when the lights were turned off, increasing their activity for a several minutes, though to a somewhat lesser extent than control larvae. But the fact that they responded at all suggests that non-retinal photoreceptors contributed to the behavior.

To confirm the role of the deep brain photoreceptors, the researchers also tested eyeless larvae that had been genetically modified to block expression of photoreceptors in the pineal gland. This fish still showed this jump in activity for several minutes after entering darkness.

Two different types of opsins—melanopsin and multiple tissue opsin—are expressed in the same type of neuron in zebrafish hypothalamus. Burgess and his colleagues looked at zebrafish missing the transcription factor Orthopedia, which is unique to these neurons, and found that the darkness-induced activity boost is nearly absent in these fish. To further narrow the search for the responsible photoreceptors, the researchers overexpressed melanopsin in hypothalamus neurons that co-express Orthopedia and melanopsin, and found that it increased the sensitivity of eyeless zebrafish to reductions in light. The results point to both melanopsin and Orthopedia as key players in modulating this behavior and pinpoint the location to neurons that coexpress these factors in the zebrafish hypothalamus.

Interestingly, the hypothalamus is one of the oldest parts of the vertebrate brain, said Detlev Arendt, a developmental biologist at the European Molecular Biology Laboratory in Heidelberg. “It’s very possible that this is one of the oldest functions”—one that evolved in “non-visual organisms” that had no eyes but still needed to sense light.

Although not as directed and efficient as eye-dependent behaviors that help fish swim toward light, Burgess speculates that deep brain opsins can still benefit zebrafish larvae. “You could imagine situation where it can’t see light, if a leaf falls on it and it doesn’t know where to swim. I think this behavior puts it in a hyperactive state where it swims wildly for several minutes until it reaches enough light for eyes to take over,” he explained, noting that such behavior is common in invertebrates.

It remains to be seen whether these deep brain opsins regulate other behaviors, perhaps in similar fashion to seasonal hormonal regulation in birds, but Hattar believes it is likely. “It’s beyond reasonable doubt there are many functions for these deep brain photoreceptors.”

Fernandes et al., “Deep brain photoreceptors control light-seeking behavior in zebrafish larvae,” Current Biology, 22:1-6, 2012.

Neuroendocrine basis of sexuality, mood, anxiety, social consciousness

Physiology, signaling, and pharmacology of galanin peptides and receptors: Three decades of emerging diversity

Lang, R., Gundlach, A.L., Holmes, F.E., (…), Hökfelt, T., Kofler, B.
Pharmacological Reviews 2015: 67 (1), pp. 118-175
http://dx.doi.org:/10.1124/pr.112.006536

Galanin was first identified 30 years ago as a “classic neuropeptide,” with actions primarily as a modulator of neurotransmission in the brain and peripheral nervous system. Other structurally-related peptides—galanin-like peptide and alarin—with diverse biologic actions in brain and other tissues have since been identified, although, unlike galanin, their cognate receptors are currently unknown. Over the last two decades, in addition to many neuronal actions, a number of nonneuronal actions of galanin and other galanin family peptides have been described. These include actions associated with neural stem cells, nonneuronal cells in the brain such as glia, endocrine functions, effects on metabolism, energy homeostasis, and paracrine effects in bone. Substantial new data also indicate an emerging role for galanin in innate immunity, inflammation, and cancer. Galanin has been shown to regulate its numerous physiologic and pathophysiological processes through interactions with three G protein–coupled receptors, GAL1, GAL2, and GAL3, and signaling via multiple transduction pathways, including inhibition of cAMP/PKA (GAL1, GAL3) and stimulation of phospholipase C (GAL2). In this review, we emphasize the importance of novel galanin receptor–specific agonists and antagonists. Also, other approaches, including new transgenic mouse lines (such as a recently characterized GAL3 knockout mouse) represent, in combination with viral-based techniques, critical tools required to better evaluate galanin system physiology. These in turn will help identify potential targets of the galanin/galanin-receptor systems in a diverse range of human diseases, including pain, mood disorders, epilepsy, neurodegenerative conditions, diabetes, and cancer.

Estradiol regulates responsiveness of the dorsal premammillary nucleus of the hypothalamus and affects fear- and anxiety-like behaviors in female rats

Litvin, Y., Cataldo, G., Pfaff, D.W., Kow, L.-M.
European Journal of Neuroscience 2014; 40 (2), pp. 2344-2351
10.1111/ejn.12608

Research suggests a causal link between estrogens and mood. Here, we began by examining the effects of estradiol (E2) on rat innate and conditioned defensive behaviors in response to cat odor. Second, we utilized whole-cell patch clamp electrophysiological techniques to assess noradrenergic effects on neurons within the dorsal premammillary nucleus of the hypothalamus (PMd), a nucleus implicated in fear reactivity, and their regulation by E2. Our results show that E2 increased general arousal and modified innate defensive reactivity to cat odor. When ovariectomized females treated with E2 as opposed to oil were exposed to cat odor, they showed elevations in risk assessment and reductions in freezing, indicating a shift from passive to active coping. In addition, animals previously exposed to cat odor showed clear cue + context conditioning 24 h later. However, although E2 persisted in its effects on general arousal in the conditioning task, its effects on fear disappeared. In the patch clamp experiments noradrenergic compounds that typically induce fear clearly excited PMd neurons, producing depolarizations and action potentials. E2 treatment shifted some excitatory effects of noradrenergic agonists to inhibitory, possibly by differentially affecting α- and β-adrenoreceptors. In summary, our results implicate E2 in general arousal and fear reactivity, and suggest these may be governed by changes in noradrenergic responsivity in the PMd. These effects of E2 may have ethological relevance, serving to promote mate seeking even in contexts of ambiguous threat and shed light on the involvement of estrogen in mood and its associated disorders.

Endogenous opiates and behavior: 2013

Richard J. Bodnar
Peptides 62 (2014) 67–136
http://dx.doi.org/10.1016/j.peptides.2014.09.013

This paper is the thirty-sixth consecutive installment of the annual review of research concerning the endogenous opioid system. It summarizes papers published during 2013 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular-biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior, and the roles of these opioid peptides and receptors in pain and analgesia; stress and social status; tolerance and dependence; learning and memory; eating and drinking; alcohol and drugs of abuse; sexual activity and hormones, pregnancy, development and endocrinology; mental illness and mood; seizures and neurologic disorders; electrical-related activity and neurophysiology; general activity and locomotion; gastrointestinal, renal and hepatic functions; cardiovascular responses; respiration and thermoregulation; and immunological responses.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata)

Timothy S Breton, Matthew A DiMaggio, Stacia A Sowe, David L Berlinsky, et al.
Comparative Biochemistry and Physiology, Part A 181 (2015) 45–53
http://dx.doi.org/10.1016/j.cbpa.2014.11.020

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/
midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Evaluation for roles of neurosteroids in modulating forebrain mechanisms controlling vasopressin secretion and related phenomena in conscious rats

Ken’ichi Yamaguchi
Neuroscience Research xxx (2015) xxx–xxx
http://dx.doi.org/10.1016/j.neures.2015.01.002

Anteroventral third ventricular region (AV3V) regulates autonomic functions through a GABAergic mechanism that possesses neuroactive steroid (NS)-synthesizing ability. Although NS can exert effects by acting on a certain type of GABAA-receptor (R), it is not clear whether NS may operate to modulateAV3V GABAergic activity for controlling autonomic functions. This study aimed to investigate the issue.AV3V infusion with a GABAA antagonist bicuculline increased plasma vasopressin (AVP), glucose, blood pressure (BP), and heart rate in rats. These events were abolished by preinjecting its agonist muscimol, whereas the infusion with allopregnanolone, a NS capable of potentiating GABAA-R function, affectednone of the variables in the absence or presence of such bicuculline actions. Similarly, AV3V infusion with pregnanolone sulfate, a NS capable of antagonizing GABAA-R, produced no effect on those variables.AV3V infusion with muscimol was effective in inhibiting the responses of plasma AVP or glucose, orBP to an osmotic loading or bleeding. However, AV3V infusion with aminoglutethimide, a NS synthesis inhibitor, did not affect any of the variables in the absence or presence of those stimuli. These results suggest that NS may not cause acute effects on the AV3V GABAergic mechanism involved in regulating AVP release and other autonomic function.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce, and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106)
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain, independent of secretion from peripheral endocrine glands, such as the gonads and adrenals. A focus in our laboratory for over 25 years has been how production of the pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review. First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, Nmethyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Long-term dysregulation of brain corticotrophin and glucocorticoid receptors and stress reactivity by single early-life pain experience in male and female rats

Nicole C. Victoria, Kiyoshi Inoue, Larry J. Young, Anne Z. Murphy
Psychoneuroendocrinology (2013) 38, 3015—3028
http://dx.doi.org/10.1016/j.psyneuen.2013.08.013

Inflammatory pain experienced on the day of birth (postnatal day 0: PD0) significantly dampens behavioral responses to stress- and anxiety-provoking stimuli in adult rats. However, to date, the mechanisms by which early life pain permanently alters adult stress responses remain unknown. The present studies examined the impact of inflammatory pain, experienced on the day of birth, on adult expression of receptors or proteins implicated in the activation and termination of the stress response, including corticotrophin releasing factor receptors (CRFR1 and CRFR2) and glucocorticoid receptor (GR). Using competitive receptor autoradiography, we show that Sprague Dawley male and female rat pups administered 1% carrageenan into the intraplantar surface of the hindpaw on the day of birth have significantly decreased CRFR1 binding in the basolateral amygdala and midbrain periaqueductal gray in adulthood. In contrast, CRFR2 binding, which is associated with stress termination, was significantly increased in the lateral septum and cortical amygdala. GR expression, measured with in situ hybridization and immunohistochemistry, was significantly increased in the paraventricular nucleus of the hypothalamus and significantly decreased in the hippocampus of neonatally injured adults. In parallel, acute stress-induced corticosterone release was significantly attenuated and returned to baseline more rapidly in adults injured on PD0 in comparison to controls. Collectively, these data show that early life pain alters neural circuits that regulate responses to and neuroendocrine recovery from stress, and suggest that pain experienced by infants in the Neonatal Intensive Care Unit may permanently alter future responses to anxiety- and stress provoking stimuli.

Dysruption of Corticotropin Releasing Factor in hypocampal region

Stress and trauma: BDNF control of dendritic-spine formation and regression

M.R. Bennett, J. Lagopoulos
Progress in Neurobiology 112 (2014) 80–99
http://dx.doi.org/10.1016/j.pneurobio.2013.10.005

Chronic restraint stress leads to increases in brain derived neurotrophic factor (BDNF) mRNA and protein in some regions of the brain, e.g. the basal lateral amygdala (BLA) but decreases in other regions such as the CA3 region of the hippocampus and dendritic spine density increases or decreases in line with these changes in BDNF. Given the powerful influence that BDNF has on dendritic spine growth, these observations suggest that the fundamental reason for the direction and extent of changes in dendritic spine density in a particular region of the brain under stress is due to the changes in BDNF there.
The most likely cause of these changes is provided by the stress initiated release of steroids, which readily enter neurons and alter gene expression, for example that of BDNF. Of particular interest is how glucocorticoids and mineralocorticoids tend to have opposite effects on BDNF gene expression offering the possibility that differences in the distribution of their receptors and of their downstream effects might provide a basis for the differential transcription of the BDNF genes. Alternatively, differences in the extent of methylation and acetylation in the epigenetic control of BDNF transcription are possible in different parts of the brain following stress.
Although present evidence points to changes in BDNF transcription being the major causal agent for the changes in spine density in different parts of the brain following stress, steroids have significant effects on downstream pathways from the TrkB receptor once it is acted upon by BDNF, including those that modulate the density of dendritic spines.
Finally, although glucocorticoids play a canonical role in determining BDNF modulation of dendritic spines, recent studies have shown a role for corticotrophin releasing factor (CRF) in this regard. There is considerable improvement in the extent of changes in spine size and density in rodents with forebrain specific knockout of CRF receptor 1 (CRFR1) even when the glucocorticoid pathways are left intact. It seems then that CRF does have a role to play in determining BDNF control of dendritic spines.

Central CRF system perturbation in an Alzheimer’s disease knockin mouse model

Qinxi Guo, Hui Zheng, Nicholas John Justice
Neurobiology of Aging 33 (2012) 2678–2691
http://dx.doi.org:/10.1016/j.neurobiolaging.2012.01.002

Alzheimer’s disease (AD) is often accompanied by changes in mood as well as increases in circulating cortisol levels, suggesting that regulation of the stress responsive hypothalamic-pituitary-adrenal (HPA) axis is disturbed. Here, we show that amyloid precursor protein (APP) is endogenously expressed in important limbic, hypothalamic, and midbrain nuclei that regulate hypothalamic-pituitary-adrenal axis activity. Furthermore, in a knockin mouse model of AD that expresses familial AD (FAD) mutations of both APP with humanized amyloid beta (hA), and presenilin 1 (PS1), in their endogenous patterns (APP/hA/PS1 animals), corticotropin releasing factor (CRF) levels are increased in key stress-related nuclei, resting corticosteroid levels are elevated, and animals display increased anxiety-related behavior. Endocrine and behavioral phenotypes can be normalized by loss of 1 copy of CRF receptor type-1 (Crfr1), consistent with a perturbation of central CRF signaling in APP/hA/PS1 animals. However, reductions in anxiety and corticosteroid levels conferred by heterozygosity of CRF receptor type-1 do not improve a deficit in working memory observed in APP/hA/PS1 mice, suggesting that perturbations of the CRF system are not the primary cause of decreased cognitive performance.

Alzheimer’s disease-like neuropathology of gene-targeted APP-SLxPS1mut mice expressing the amyloid precursor protein at endogenous levels

Christoph Kohler, Ulrich Ebert, Karlheinz Baumann, and Hannsjorg Schroeder
Neurobiology of Disease 20 (2005) 528 – 540
http://dx.doi.org:/10.1016/j.nbd.2005.04.009

Most transgenic mice used for preclinical evaluation of potential disease-modifying treatments of Alzheimer’s disease develop major histopathological features of this disease by several-fold overexpression of the human amyloid precursor protein. We studied the phenotype of three different strains of gene-targeted mice which express the amyloid precursor protein at endogenous levels. Only further crossing with transgenic mice overexpressing mutant human presenilin1 led to the deposition of extracellular amyloid, accompanied by the deposition of apolipoprotein E, an astrocyte and microglia reaction, and the occurrence of dilated cholinergic terminals in the cortex. Features of neurodegeneration, however, were absent. The pattern of plaque development and deposition in these mice was similar to that of amyloid precursor protein overproducing strains if crossed to presenilin1-transgenics. However, plaque development started much later and developed slowly until the age of 18 months but then increased more rapidly.

Central Cholinergic Functions In Human Amyloid Precursor Protein Knock-In/Presenilin-1 Transgenic Mice

Hartmann, C. Erb, U. Ebert, K. H. Baumann, A. Popp, G. Koenig, J. Klein
Neuroscience 125 (2004) 1009–1017
http://dx.doi.org:/10.1016/j.neuroscience.2004.02.038

Alzheimer’s disease is characterized by amyloid peptide formation and deposition, neurofibrillary tangles, central cholinergic dysfunction, and dementia; however, the relationship between these parameters is not well understood. We studied the effect of amyloid peptide formation and deposition on central cholinergic function in knock-in mice carrying the human amyloid precursor protein (APP) gene with the Swedish/London double mutation (APP-SL mice) which were crossbred with transgenic mice overexpressing normal (PS1wt) or mutated (M146L; PS1mut) human presenilin-1. APP-SLxPS1mut mice had increased levels of Aβ peptides at 10 months of age and amyloid plaques at 14 months of age while APP-SLPS1wt mice did not have increased peptide levels and did not develop amyloid plaques. We used microdialysis in 15–27 months old mice to compare hippocampal acetylcholine (ACh) levels in the two mouse lines and found that extracellular ACh levels were slightly but significantly reduced in the APP-SLPS1mut mice (-26%; P=0.044). Exploratory activity in the open field increased hippocampal ACh release by two-fold in both mouse lines; total and relative increases were not significantly different for the two strains under study. Similarly, infusion of scopolamine (1 µM) increased hippocampal ACh release to a similar extent (3–5-fold) in both groups. High-affinity choline uptake, a measure of the ACh turnover rate, was identical in both mouse lines. Neurons expressing choline acetyltransferase were increased in the septum of APP-SLPS1mut mice (26%; P =0.046). We conclude that amyloid peptide production causes a small decrease of extracellular ACh levels. The deposition of amyloid plaques, however, does not impair stimulated ACh release and proceeds without major changes of central cholinergic function.

Glutamate Neurotoxicity

Glutamate Neurotoxicity and Diseases of the Nervous System

Dennis W. Choi
Neuron. Oct, 1988; 1: 623-634

A growing number of studies now suggest that the cellular mechanisms which normally participate in signaling in the central nervous system (CNS) can be transformed by disease into instruments of neuronal cell destruction. Excitatory synaptic transmission in the mammalian CNS is principally mediated by L-glutamate. In fact, glutamate excites virtually all central neurons and is present in nerve terminals at millimolar levels (Curtis and Johnston, 1974). Normally, the extracellular levels of glutamate rise to high levels only in the brief and spatially localized fashion appropriate to synaptic transmission. This is fortunate, because as Lucas and Newhouse first showed in 1957, sustained exposure to glutamate can destroy retinal neurons. In a subsequent set of pioneering experiments, Olney (Olney and Sharpe, 1969; Olney et al., 1971) established that this toxicity, which he later called excitotoxicity, was not unique to glutamate or to retinal neurons, but was a feature common to the actions of all excitatory amino acids on central neurons. He postulated therefore that glutamate, or related compounds, might be the cause of the neuronal cell loss found in certain neurological diseases. In recent years, this hypothesis has gathered considerable support, fueled by new insights into glutamate receptor function and the development of effective glutamate antagonist drugs. The evidence is most convincing in diseases involving an acute insult to the brain, as occurs in a stroke, with abrupt deprivation of blood supply. But neurotoxicity due to excitatory amino acids may also be involved in slowly progressive degenerative diseases such as Huntington’s disease. Although the detailed molecular basis of glutamate neurotoxicity is not known, it appears that Ca2+ influx may play a critical role.
Glutamate interacts with at least three classes of membrane receptors, each commonly referred to by preferred pharmacological agonists: N-methyl-o-aspartate (NMDA), quisqualate, and kainate (Watkins and Olverman, 1987) (Figure I). These three classes are linked to membrane cation channels. A second type of quisqualate receptor has been additionally linked to a second messenger system (see below). It has been suggested that all three classes might actually be substates of a single molecular complex, but binding studies and newer physiological studies favor separate structures.

Quisqualate NMDA Kainate

Three Classes of Glutamate Receptors

One type of quisqualate receptor stimulates the formation of inositol 1,4,5-trisphosphate UPS) and diacylglycerol (DAG) from phosphatidylinositol-4,5-biphosphate (PIP,); the other is linked directly to a Na+ ionophore. Activation of the quisqualate receptor-ionophore complex can be potentiated by Zn2+. The NMDA receptor opens a channel permeable to Ca2+ as well as Na+; this receptor-channel complex has several modulatory sites discussed in the text. The kainate receptor opens an ionophore permeable to Na+.

Best defined is the NMDA receptor. This receptor opens a distinctive membrane channel characterized by high conductance (main state about 50 pS), voltage dependent Mgz+ blockade and permeability to both Ca2+ and Na+. The NMDA receptor can be selectively activated by several endogenous compounds, including L-aspartate, homocysteate, and quinolinate. Activation requires the coavailability of glycine in near micromolar concentrations. The action of glutamate at the NMDA receptor can be selectively antagonized: competitively by 2-amino-5-phosphonovalerate (APV) and 2-amino-5-phosphonoheptanoate (APH), or noncompetitively by drugs that bind to the phencyclidine site within the open channel (such as phencyclidine, MK-801, dextrorphan, or ketamine. The NMDA receptor-activated channel can also be blocked noncompetitively by Znz+, most likely at a site different from that which binds Mg2.
Although glutamate has high affinity for all three classes of postsynaptic receptors, it is not easy to demonstrate its neurotoxicity in vivo. Even when directly injected into brain, bypassing the blood-brain barrier, extremely high doses of glutamate are required to create lesions. Mangano & Schwartz found that they could infuse 0.5 crl/hr of a 300 mM glutamate solution into the hippocampus of a rat for 2 weeks without producing neuronal injury. This apparent low in vivo neurotoxic potency of glutamate may represent one reason why Olney’s “glutamate hypothesis” of neurological disease did not initially achieve a more widespread following. However, in fact, glutamate is a potent and rapidly acting neurotoxin; its neurotoxicity in vivo is likely masked by the efficiency of normal cellular uptake mechanisms in removing glutamate from the extracellular space. Glutamate neurotoxicity can be most directly studied in cell culture where bath exposure is not limited by cellular uptake.
The toxic changes produced by glutamate or related excitatory amino acids in vivo are of two sorts:

acute swelling of neuronal dendrites and cell bodies and a
more slowly evolving neuronal degeneration (Olney, 1986).

Axons and glia are relatively spared, although high levels of excitatory amino acids can produce some swelling of glia. A hallmark of excitatory amino acid neurotoxicity is its cellular selectivity, with distinctive patterns of neuronal loss produced by different excitatory amino acids and different routes of administration. For example, Nadler and co-workers (1978) found that intraventricular kainate preferentially destroys hippocampal CA3 neurons but spares dentate granule neurons. Different neuronal subpopulations
may differ in their intrinsic vulnerability to damage.

Possible Mechanisms Involved in Glutamate Neurotoxicity

How Ca*+ may mediate glutamate-induced neuronal degeneration. Glutamate acts on NMDA, non-NMDA, and “metabotropic” receptors (the quisqualate receptor linked to a second messenger system) to produce an increase in cytosolic free Ca*+. This cytosolic Ca *+, in concert with diacylglycerol liberated by the quisqualate-triggered second messenger system, activates protein kinase C, which acts via a number of mechanisms (primarily by altering membrane ion channels) to increase neuronal excitability and further increase cytosolic Ca*+. Elevated cytosolic Ca2+ then activates several enzymes capable of either directly or indirectly (through free radical formation) destroying cellular structure. Glutamate released from synaptic terminals or leaking nonspecifically from ruptured neurons contributes to additional injury propagation.

Glutamate Neurotoxicity in Perspective

The hypothesis that excitatory amino acids may specifically mediate pathological neuronal injury gives new form to this age-old enemy and raises the tantalizing possibility that current molecular and cellular insights into excitatory amino acid transmitter systems might be harnessed to develop an efficacious clinical therapy. Some points of attack are already apparent; others will likely be defined as the biology of excitatory amino acids continues to be unraveled. An intriguing area for investigation is the relationship between excitatory amino acid neurotoxicity and normal neuronal processes such as maturation, neurite outgrowth, and synaptic plasticity.

Glutamate Toxicity in a Neuronal Cell line Involves Inhibition of Cystine Transport Leading to Oxidative Stress

Timothy H. Murphy, M Miyamoto, A Sastre, R Schnaar and JT Coyle
Neuron 1989: 2: 1547-88.

Glutamate binds to both excitatory neurotransmitter binding sites and a W-dependent, quisqualate- and cystine-inhibited transport site on brain neurons. The neuroblastoma-primary retina hybrid cells (NWRE-105) are susceptible to glutamate-induced cytotoxicity. The Cl–dependent transport site to which glutamate and quisqualate (but not kainate or NMDA) bind has a higher affinity for cystine than for glutamate. Towering cystine concentrations in the cell culture medium results in cytotoxicity similar to that induced by glutamate addition in its morphology, kinetics, and CaZ+ dependence. Glutamate-induced cytotoxicity is directly proportional to its ability to inhibit cystine uptake. Exposure to glutamate (or lowered cystine) causes a decrease in glutathione levels and an accumulation of intracellular peroxides. Like NW-RE-105 cells, primary rat hippocampal neurons (but not glia) in culture degenerate in medium with lowered cystine concentration. Thus, glutamate-induced cytotoxicity in N18-RE-105 cells is due to inhibition of cystine uptake, resulting in lowered glutathione levels leading to oxidative stress and cell death.

Mechanism of glutamate-induced neurotoxicity in HT22 mouse hippocampal cells

Masayuki Fukui, Ji-Hoon Song, Jinyoung Choi, Hye Joung Choi, Bao Ting Zhu
European Journal of Pharmacology 617 (2009) 1–11
http://dx.doi.org:/10.1016/j.ejphar.2009.06.059

Glutamate is an endogenous excitatory neurotransmitter. At high concentrations, it is neurotoxic and contributes to the development of certain neurodegenerative diseases. There is considerable controversy in the literature with regard to whether glutamate-induced cell death in cultured HT22 cells (an immortalized mouse hippocampal cell line) is apoptosis, necrosis, or a new form of cell death. The present study focused on investigating the mechanism of glutamate-induced cell death. We found that glutamate induced, in a time dependent manner, both necrosis and apoptosis in HT22 cells. At relatively early time points (8–12 h), glutamate induced mostly necrosis, whereas at late time points (16–24 h), it induced mainly apoptosis. Glutamate-induced mitochondrial oxidative stress and dysfunction were crucial early events required for the induction of apoptosis through the release of the mitochondrial apoptosis-inducing factor (AIF), which catalyzed DNA fragmentation (an ATP-independent process). Glutamate-induced cell death proceeded independently of the Bcl-2 family proteins and caspase activation. The lack of caspase activation likely resulted from the lack of intracellular ATP when the mitochondrial functions were rapidly disrupted by the mitochondrial oxidative stress. In addition, it was observed that activation of JNK, p38, and ERK signaling molecules was also involved in the induction of apoptosis by glutamate. In conclusion, glutamate-induced apoptosis is AIF-dependent but caspase-independent, and is accompanied by DNA ladder formation but not chromatin condensation.

Understanding Low Reliability of Memories for Neutral Information Encoded under Stress: Alterations in Memory-Related Activation in the Hippocampus and Midbrain

Shaozheng Qin, EJ Hermans, HJF van Marle, and G Fernandez, et al.
The Journal of Neuroscience, Mar 21, 2012; 32(12): 4032–4041
http://dx.doi.org:/10.1523/JNEUROSCI.3101-11.2012

Exposure to an acute stressor can lead to unreliable remembrance of intrinsically neutral information, as exemplified by low reliability of eyewitness memories, which stands in contrast with enhanced memory for the stressful incident itself. Stress-sensitive neuromodulators (e.g., catecholamines) are believed to cause this low reliability by altering neurocognitive processes underlying memory formation. Using event-related functional magnetic resonance imaging, we investigated neural activity during memory formation in 44 young, healthy human participants while incidentally encoding emotionally neutral, complex scenes embedded in either a stressful or neutral context.
We recorded event-related pupil dilation responses as an indirect index of phasic noradrenergic activity. Autonomic, endocrine, and psychological measures were acquired to validate stress manipulation. Acute stress during encoding led to a more liberal response bias (more hits and false alarms) when testing memory for the scenes 24 h later. The strength of this bias correlated negatively with pupil dilation responses and positively with stress-induced heart rate increases at encoding. Acute stress, moreover, reduced subsequent memory effects (SMEs; items later remembered vs forgotten) in hippocampus and midbrain, and in pupil dilation responses.
The diminished SMEs indicate reduced selectivity and specificity in mnemonic processing during memory formation. This is in line with a model in which stress-induced catecholaminergic hyperactivation alters phasic neuromodulatory signaling in memory-related circuits, resulting in generalized (gist-based) processing at the cost of specificity. Thus, one may speculate that loss of specificity may yield less discrete memory representations at time of encoding, thereby causing a more liberal response bias when probing these memories.

Neuroendocrinology – Signaling, neuron plasticity and memory

Leptin Signaling Modulates the Activity of Urocortin 1 Neurons in the Mouse Nonpreganglionic Edinger-Westphal Nucleus

Lu Xu, Wim J. J. M. Scheenen, Rebecca L. Leshan, Christa M. Patterson, et al.
Endocrinology 152(3): 979–988, 2011
http://dx.doi.org:/10.1210/en.2010-1143

A recent study systematically characterized the distribution of the long form of the leptin receptor (LepRb) in the mouse brain and showed substantial LepRb mRNA expression in the nonpreganglionic Edinger-Westphal nucleus (npEW) in the rostroventral part of the midbrain. This nucleus hosts the majority of urocortin 1 (Ucn1) neurons in the rodent brain, and because Ucn1 is a potent satiety hormone and electrical lesioning of the npEW strongly decreases food intake, we have hypothesized a role of npEW-Ucn1 neurons in leptin-controlled food intake. Here, we show by immunohistochemistry that npEW-Ucn1 neurons in the mouse contain LepRb and respond to leptin administration with induction of the Janus kinase 2-signal transducer and activator of transcription 3 pathway, both in vivo and in vitro. Furthermore, systemic leptin administration increases the Ucn1 content of then pEW significantly, whereas in mice that lack LepRb (db/db mice), then pEW contains considerably reduced amount of Ucn1. Finally, we reveal by patch clamping of midbrain Ucn1 neurons that leptin administration reduces the electrical firing activity of the Ucn1 neurons. In conclusion, we provide ample evidence for leptin actions that go beyond leptin’s well-known targets in the hypothalamus and propose that leptin can directly influence the activity of the midbrain Ucn1 neurons.

Leptin regulation of hippocampal synaptic function in health and disease

Andrew J. Irving and Jenni Harvey
Trans. R. Soc. B 369: 20130155 http://dx.doi.org/10.1098/rstb.2013.0155

The endocrine hormone leptin plays a key role in regulating food intake and body weight via its actions in the hypothalamus. However, leptin receptors are highly expressed in many extra-hypothalamic brain regions and evidence is growing that leptin influences many central processes including cognition. Indeed, recent studies indicate that leptin is a potential cognitive enhancer as it markedly facilitates the cellular events underlying hippocampal-dependent learning and memory, including effects on glutamate receptor trafficking, neuronal morphology and activity-dependent synaptic plasticity. However, the ability of leptin to regulate hippocampal synaptic function markedly declines with age and aberrant leptin function has been linked to neurodegenerative disorders such as Alzheimer’s disease (AD). Here, we review the evidence supporting a cognitive enhancing role for the hormone leptin and discuss the therapeutic potential of using leptin-based agents to treat AD.

The Y2 receptor agonist PYY3–36 increases the behavioral response to novelty and acute dopaminergic drug challenge in mice

Ulrike Stadlbauer, Elisabeth Weber, Wolfgang Langhans and Urs Meyer
International Journal of Neuropsychopharmacology (2014), 17, 407–419
http://dx.doi.org:/10.1017/S1461145713001223

The gastrointestinal hormone PYY3–36 is a preferential Y2 neuropeptide Y (NPY) receptor agonist. Recent evidence indicates that PYY3–36 acts on central dopaminergic pathways, but its influence on dopamine-dependent behaviors remains largely unknown. We therefore explored the effects of peripheral PYY3–36 treatment on the behavioral responses to novelty and to dopamine-activating drugs in mice. In addition, we examined whether PYY3–36 administration may activate distinct dopamine and γ-aminobutyric acid (GABA) cell populations in the mesoaccumbal and nigrostriatal pathways. We found that i.p. PYY3–36 injection led to a dose-dependent increase in novel object exploration. The effective dose of PYY3–36 (1 μg/100 g body weight) also potentiated the locomotor reaction to the indirect dopamine receptor agonist amphetamine and increased stereotyped climbing/leaning responses following administration of the direct dopamine receptor agonist apomorphine. PYY3–36 administration did not affect activity of midbrain dopaminergic cells as evaluated by double immuno-enzyme staining of the neuronal early gene product c-Fos with tyrosine hydroxylase. PYY3–36 did, however, lead to a marked increase in the number of cells co-expressing c-Fos with glutamic acid decarboxylase in the nucleus accumbens and caudate putamen, indicating activation of GABAergic cells in dorsal and ventral striatal areas. Our results support the hypothesis that acute administration of the preferential Y2 receptor agonist PYY3–36 modulates dopamine-dependent behaviours. These effects do not seem to involve direct activation of midbrain dopamine cells but instead are associated with neuronal activation in the major input areas of the mesoaccumbal and nigrostriatal pathways.

Somatostatin and nociceptin inhibit neurons in the central nucleus of amygdala that project to the periaqueductal grey

Billy Chieng, MacDonald J. Christie
Neuropharmacology 59 (2010) 425e430
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.001

The central nucleus of amygdala (CeA) plays an important role in modulation of the descending antinociceptive pathways. Using whole-cell patch clamp recordings from brain slices, we found that CeA neurons responded to the endogenous ligands somatostatin (SST) and nociceptin/orphanin FQ (OFQ) via an increased K-conductance. Co-application with selective antagonists suggested that SST and OFQ act on SSTR2 and ORL1 receptors, respectively. Taking account of anatomical localisation of recorded neurons, the present study showed that many responsive neurons were located within the medial subdivision of CeA and all CeA projection neurons to the midbrain periaqueductal grey invariably responded to these peptides. Randomly selected agonist-responsive neurons in CeA predominantly classified physiologically as low-threshold spiking neurons. The similarity of SST, OFQ and, as previously reported, opioid responsiveness in a sub-population of CeA neurons suggests converging roles of these peptides to inhibit the activity of projections from CeA to vlPAG, and potentially similar antinociceptive actions in this pathway.

In vitro identification and electrophysiological characterization of dopamine neurons in the ventral tegmental area

Tao A. Zhang, Andon N. Placzek, John A. Dani
Neuropharmacology 59 (2010) 431e436
http://dx.doi.org:/10.1016/j.neuropharm.2010.06.004

Dopamine (DA) neurons in the ventral tegmental area (VTA) have been implicated in brain mechanisms related to motivation, reward, and drug addiction. Successful identification of these neurons in vitro has historically depended upon the expression of a hyperpolarization-activated current (I_h) and immunohistochemical demonstration of the presence of tyrosine hydroxylase (TH), the rate-limiting enzyme for DA synthesis. Recent findings suggest that electrophysiological criteria may be insufficient for distinguishing DA neurons from non-DA neurons in the VTA. In this study, we sought to determine factors that could potentially account for the apparent discrepancies in the literature regarding DA neuron identification in the rodent brain slice preparation. We found that confirmed DA neurons from the lateral VTA generally displayed a larger amplitude I_h relative to DA neurons located in the medial VTA. Measurement of a large amplitude I_h (>100 pA) consistently indicated a dopaminergic phenotype, but non-dopamine neurons also can have I_h current. The data also showed that immunohistochemical TH labeling of DA neurons can render false negative results after relatively long duration (>15 min) wholecell patch clamp recordings. We conclude that whole-cell patch clamp recording in combination with immunohistochemical detection of TH expression can guarantee positive but not negative DA identification in the VTA.

Dopamine Enables In Vivo Synaptic Plasticity Associated with the Addictive Drug Nicotine

Jianrong Tang and John A. Dani
Neuron, Sept 10, 2009; 63, 673–682
http://dx.doi.org:/10.1016/j.neuron.2009.07.025

Addictive drugs induce a dopamine signal that contributes to the initiation of addiction, and the dopamine signal influences drug-associated memories that perpetuate drug use. The addiction process shares many commonalities with the synaptic plasticity mechanisms normally attributed to learning and memory. Environmental stimuli repeatedly linked to addictive drugs become learned associations, and those stimuli come to elicit memories or sensations that motivate continued drug use. Applying in vivo recording techniques to freely moving mice, we show that physiologically relevant concentrations of the addictive drug nicotine directly cause in vivo hippocampal synaptic potentiation of the kind that underlies learning and memory. The drug-induced long-term synaptic plasticity required a local hippocampal dopamine signal. Disrupting general dopamine signaling prevented the nicotine-induced synaptic plasticity and conditioned place preference. These results suggest that dopaminergic signaling serves as a functional label of salient events by enabling and scaling synaptic plasticity that underlies drug-induced associative memory.

NCS-1 in the Dentate Gyrus Promotes Exploration, Synaptic Plasticity, and Rapid Acquisition of Spatial Memory

Bechara J. Saab, John Georgiou, Arup Nath, Frank J.S. Lee, et al.
Neuron, Sept 10, 2009; 63, 643–656
http://dx.doi.org:/10.1016/j.neuron.2009.08.014

The molecular underpinnings of exploration and its link to learning and memory remain poorly understood. Here we show that inducible, modest overexpression of neuronal calcium sensor 1 (Ncs1) selectively in the adult murine dentate gyrus (DG) promotes a specific form of exploratory behavior. The mice also display a selective facilitation of longterm potentiation (LTP) in the medial perforant path and a selective enhancement in rapid-acquisition spatial memory, phenotypes that are reversed by direct application of a cell-permeant peptide (DNIP) designed to interfere with NCS-1 binding to the dopamine type-2 receptor (D2R). Moreover, the DNIP and the D2R-selective antagonist L-741,626 attenuated exploratory behavior, DG LTP, and spatial memory in control mice. These data demonstrate a role for NCS-1 and D2R in DG plasticity and provide insight for understanding how the DG contributes to the origin of exploration and spatial memory acquisition.

Neuroligin 2 Drives Postsynaptic Assembly at Perisomatic Inhibitory Synapses through Gephyrin and Collybistin

Alexandros Poulopoulos, Gayane Aramuni, Guido Meyer, Tolga Soykan, et al.
Neuron 63, 628–642, Sept 10, 2009
http://dx.doi.org:/10.1016/j.neuron.2009.08.023

In the mammalian CNS, each neuron typically receives thousands of synaptic inputs from diverse classes of neurons. Synaptic transmission to the postsynaptic neuron relies on localized and transmitter-specific differentiation of the plasma membrane with postsynaptic receptor, scaffolding, and adhesion proteins accumulating in precise apposition to presynaptic sites of transmitter release. We identified protein interactions of the synaptic adhesion molecule neuroligin 2 that drive postsynaptic differentiation at inhibitory synapses. Neuroligin 2 binds the scaffolding protein gephyrin through a conserved cytoplasmic motif and functions as a specific activator of collybistin, thus guiding membrane tethering of the inhibitory postsynaptic scaffold. Complexes of neuroligin 2, gephyrin and collybistin are sufficient for cell-autonomous clustering of inhibitory neurotransmitter receptors. Deletion of neuroligin 2 in mice perturbs GABAergic and glycinergic synaptic transmission and leads to a loss of postsynaptic specializations specifically at perisomatic inhibitory synapses.

A Subset of Ventral Tegmental Area Neurons is Inhibited by Dopamine, 5-Hydroxytryptamine and Opioids

L. Cameron, M. W. Wessendorf and J. T. Williams
Neuroscience 1997; 77(1), pp. 155–166 PII: S0306-4522(96)00444-7

Neurons originating in the ventral tegmental area are thought to play a key role in the formation of addictive behaviors, particularly in response to drugs such as cocaine and opioids. In this study we identified different populations of ventral tegmental area neurons by the pharmacology of their evoked synaptic potentials and their response to dopamine, 5-hydroxytryptamine and opioids. Intracellular recordings were made from ventral tegmental area neurons in horizontal slices of guinea-pig brain and electrical stimulation was used to evoke synaptic potentials. The majority of cells (61.3%) hyperpolarized in response to dopamine, depolarized to 5-hydroxytryptamine, failed to respond to [Met]5enkephalin and exhibited a slow GABAB-mediated inhibitory postsynaptic potential. A smaller proportion of cells (11.3%) hyperpolarized in response to [Met]5enkephalin, depolarized to 5-hydroxytryptamine, failed to respond to dopamine and did not exhibit a slow inhibitory postsynaptic potential. These two groups of cells corresponded to previously described ‘‘principal’’ and ‘‘secondary’’ cells, respectively. A further group of cells (27.4%) was identified that, like the principal cells, hyperpolarized to dopamine.

However, these ‘‘tertiary cells’’ also hyperpolarized to both 5-hydroxytryptamine and [Met]5enkephalin and exhibited a slow, cocaine-sensitive 5-hydroxytryptamine1A-mediated inhibitory postsynaptic potential. When principal and tertiary cells were investigated immuno-histochemically, 82% of the principal cells were positive for tyrosine hydroxylase compared
with only 29% of the tertiary cells. The 5-hydroxytryptamine innervation of both these cell types was investigated and a similar density of putative contacts was observed near the somata and dendrites in both groups. This latter finding suggests that the existence of a 5-hydroxytryptamine-mediated inhibitory postsynaptic potential in the tertiary cells may be determined by the selective expression of 5-hydroxytryptamine receptors, rather than the distribution or density of the 5-hydroxytryptamine innervation.
We conclude that tertiary cells are a distinct subset of ventral tegmental area neurons where cocaine and μ-opioids both mediate inhibition.

Dopamine reward circuitry: Two projection systems from the ventral midbrain to the nucleus accumbens–olfactory tubercle complex

Satoshi Ikemoto
Brain Research Reviews 56 (2007) 27–78
http://:dx.doi.org:/10.1016/j.brainresrev.2007.05.004

Anatomical and functional refinements of the meso-limbic dopamine system
of the rat are discussed. Present experiments suggest that dopaminergic neurons localized in the posteromedial ventral tegmental area (VTA) and central linear nucleus raphe selectively project to the ventromedial striatum (medial olfactory tubercle and medial nucleus accumbens shell), whereas
the anteromedial VTA has few if any projections to the ventral striatum,
and the lateral VTA largely projects to the ventrolateral striatum (accumbens
core, lateral shell and lateral tubercle). These findings complement the recent behavioral findings that cocaine and amphetamine are more rewarding when administered into the ventromedial striatum than into the ventrolateral striatum. Drugs such as nicotine and opiates are more rewarding when administered into the posterior VTA or the central linear nucleus than into
the anterior VTA. A review of the literature suggests that
(1) the midbrain has corresponding zones for the accumbens core and medial shell;
(2) the striatal portion of the olfactory tubercle is a ventral extension of the nucleus accumbens shell; and
(3) a model of two dopamine projection systems from the ventral midbrain to the ventral striatum is useful for understanding reward function.
The medial projection system is important in the regulation of arousal characterized by affect and drive and plays a different role in goal directed learning than the lateral projection system, as described in the variation–selection hypothesis of striatal functional organization.

Metabolic hormones, dopamine circuits, and feeding

Nandakumar S. Narayanan, Douglas J. Guarnieri, Ralph J. DiLeone
Frontiers in Neuroendocrinology 31 (2010) 104–112
http://dx.doi.org:/10.1016/j.yfrne.2009.10.004

Recent evidence has emerged demonstrating that metabolic hormones such as ghrelin and leptin can act on ventral tegmental area (VTA) midbrain dopamine neurons to influence feeding. The VTA is the origin of mesolimbic dopamine neurons that project to the nucleus accumbens (NAc) to influence behavior. While blockade of dopamine via systemic antagonists or targeted gene delete can impair food intake, local NAc dopamine manipulations have little effect on food intake. Notably, non-dopaminergic manipulations in the VTA and NAc produce more consistent effects on feeding and food choice. More recent genetic evidence supports a role for the substantia nigra-striatal dopamine pathways in food intake, while the VTA–NAc circuit is more likely involved in higher-order aspects of food acquisition, such as motivation and cue associations. This rich and complex literature should be considered in models of how peripheral hormones influence feeding behavior via action on the midbrain circuits.

Control of brain development and homeostasis by local and systemic insulin signaling

Liu, P. Speder & A. H. Brand
Diabetes, Obesity and Metabolism 16 (Suppl. 1): 16–20, 2014

Insulin and insulin-like growth factors (IGFs) are important regulators of growth and metabolism. In both vertebrates and invertebrates, insulin/IGFs are made available to various organs, including the brain, through two routes: the circulating systemic insulin/IGFs act on distant organs via endocrine signaling, whereas insulin/IGF ligands released by local tissues act in a paracrine or autocrine fashion. Although the mechanisms governing the secretion and action of systemic insulin/IGF have been the focus of extensive investigation, the significance of locally derived insulin/IGF has only more recently come to the fore. Local insulin/IGF signaling is particularly important for the development and homeostasis of the central nervous system, which is insulated from the systemic environment by the blood–brain barrier. Local insulin/IGF signaling from glial cells, the blood–brain barrier and the cerebrospinal fluid has emerged as a potent regulator of neurogenesis. This review will address the main sources of local insulin/IGF and how they affect neurogenesis during development. In addition, we describe how local insulin/IGF signaling couples neural stem cell proliferation with systemic energy state in Drosophila and in mammals.

Pharmacology, Physiology, and Mechanisms of Action of Dipeptidyl Peptidase-4 Inhibitors

Erin E. Mulvihill and Daniel J. Drucker
Endocrine Reviews 35: 992–1019, 2014
http://dx.doi.org/10.1210/er.2014-1035

Dipeptidyl peptidase-4 (DPP4) is a widely expressed enzyme transducing actions through an anchored transmembrane molecule and a soluble circulating protein. Both membrane-associated and soluble DPP4 exert
catalytic activity, cleaving proteins containing a position 2 alanine or proline. DPP4-mediated enzymatic cleavage alternatively inactivates peptides or generates new bioactive moieties that may exert competing or novel activities. The widespread use of selective DPP4 inhibitors for the treatment of type 2 diabetes has heightened interest in the molecular mechanisms through which DPP4 inhibitors exert their pleiotropic actions. Here we review the biology ofDPP4with a focus on:
1) identification of pharmacological vs physiological DPP4 substrates; and
2) elucidation of mechanisms of actions of DPP4 in studies employing genetic elimination or chemical reduction of DPP4 activity.
We review data identifying the roles of key DPP4 substrates in transducing the glucoregulatory, anti-inflammatory, and cardiometabolic actions of DPP4 inhibitors in both preclinical and clinical studies. Finally, we highlight experimental pitfalls and technical challenges encountered in studies designed to understand the mechanisms of action and downstream targets activated by inhibition of DPP4.
Dipeptidyl peptidase-4 (DPP4) is a multifunctional protein that exerts biological activity through pleiotropic actions including:

protease activity (1),
association with adenosine deaminase (ADA) (2),
interaction with the extracellular matrix (3),
cell surface coreceptor activity mediating viral entry (4), and
regulation of intracellular signal transduction coupled to control of cell migration and proliferation (5).

The complexity of DPP4 action is amplified by the panoply of bioactive DPP4 substrates, which in turn act as elegant biochemical messengers in multiple tissues, including the immune and neuroendocrine systems.

DPP4 transmits signals across cell membranes and interacts with other membrane proteins (Figure). Remarkably, most of the protein is extracellular, including the C-terminal catalytic domain, a cysteine-rich area, and a large glycosylated region linked by a flexible stalk to the transmembrane segment. Only six N-terminal amino acids are predicted to extend into the cytoplasm. The active site, Ser 630, is flanked by the classic serine peptidase motif Gly-Trp-Ser630-Tyr-Gly-Gly-Tyr-Val.

Membrane-bound DPP4

Membrane-bound DPP4 contains residues 1–766, whereas sDPP4 contains residues 39–766. sDPP4 is lacking the cytoplasmic domain [residues 1–6], transmembrane domain [residues 7–28], and the flexible stalk [residues 29–39]. Both membrane-bound and circulating sDPP4 share many domains including the glycosylated region [residues 101–535, specific residues 85,92, 150], ADA binding domain [340–343], fibronectin binding domain [468–479], cysteine-rich domain [351–506, disulfide bonds are formed from 385–394, 444–472, and 649–762], and the catalytic domain [507–766 including residues composing the catalytic active site 630, 708, and 740].

DPP4 activity is subject to regulation at many levels, including control of gene and protein expression, interaction with binding partners, and modulation of enzyme activity. The DPP4 gene does not contain conventional TATAA or CCAAT promoter sequences but is characterized by a cytosine/guanine-rich promoter region.
DPP4 contains eight to 11 potential N-glycosylation sites, which can contribute to its folding and stability. Although glycosylation may contribute approximately 18–25% of the total molecular weight, mutational analysis has determined that the glycosylation sites are not required for dimerization, catalytic activity, or ADA binding. However, N-terminal sialylation facilitates trafficking of DPP4 to the apical membrane. Interestingly, molecular analysis of DPP4 isoforms isolated from the rat kidney brush border membrane reveals extensive heterogeneity in the oligosaccharides of DPP4.DPP4 was first investigated for its role in hydrolysis of dietary prolyl peptides (58); subsequent studies using DPP4 isolated using immunoaffinity chromatography and ADA binding identified DPP4 as the primary enzyme responsible for the generation of Gly-Prop-nitroanilide substrates in human serum. It is now known that DPP4 can cleave dozens of peptides, including chemokines, neuropeptides, and regulatory peptides, most containing a proline or alanine residue at position 2 of the amino-terminal region. Despite the preference for a position 2 proline, alternate residues (hydroxyproline, dehydroproline > alanine >, glycine, threonine, valine, or leucine) at the penultimate position are also cleaved by DPP4, suggesting a required stereochemistry. The DPP4 cleavage at postproline peptide bonds inactivates peptides and/or generates new bioactive peptides (see Figure), thereby regulating diverse biological processes.

DPP4 cleavage regulates substrate-receptor interactions

DPP4 cleavage regulates substrate/receptor interactions. A, DPP4 cleaves NPY [1–36] and PYY [1–36]. The intact forms of these peptides signal through Y1R-Y5R. After DPP4 cleavage, NPY [3–36] and PYY [3–36] are generated and preferentially signal through the Y2R and Y5R. B, DPP4 cleaves SP [1–11], which signals through the NK1R receptor to generate SP [5–11], which can signal through (NK1R, -2R, -3R).

GHRH and IGF-1

GHRH [1–44] and [1–40] are produced in the arcuate nucleus of the hypothalamus and bind its receptor on the anterior pituitary to stimulate the release of GH, and in turn, GH stimulates hepatic IGF-1 release. GHRH was among the first peptides to be identified as a DPP4 substrate; it is rapidly degraded in rodent and human plasma to GHRH [3–44]/GHRH [3–40], and this cleavage was blocked upon incubation of human plasma with the DPP4 inhibitor, diprotin A (99).GHRH[1–44] or [1–40] exhibits a very short half-life (6 min) andDPP4 cleavage was initially perceived to be a critical regulator of GHRH bioactivity and, in turn, the GH-IGF-1 axis. IGF-1, the downstream effector of GHRH and GH, is a 105-amino acid protein produced mainly by the liver.
IGF-1 was identified as a pharmacological DPP4 substrate by matrix-assisted laser desorption/ionization-time of flight analysis of molecular forms of IGF-1 generated after incubation with DPP4 purified from baculovirus-infected insect cells. However, studies in pigs treated with sitagliptin at doses inhibiting 90% of DPP4 activity failed to demonstrate an increase in active intact IGF-1.
Clinically, treatment of healthy human male subjects with sitagliptin (25–600 mg) for 10 days did not produce increased concentrations of serum IGF-1 or IGF-binding protein 3 as measured by ELISA. Furthermore, Dpp4/ mice or rats do not exhibit increased organ growth or body size. Hence, the available data suggest that although DPP4 cleaves and inactivates both GHRH and IGF-1, enzymatic inactivation by DPP4 is not the major mechanism regulating the bioactivity of the GHRH-IGF-1 axis.

The role of acute cortisol and DHEAS in predicting acute and chronic PTSD symptoms

Joanne Mouthaan, Marit Sijbrandij, Jan S.K. Luitse
Psychoneuroendocrinology (2014) 45, 179—186
http://dx.doi.org/10.1016/j.psyneuen.2014.04.001

Background: Decreased activation of the hypothalamus—pituitary—adrenal (HPA) axis in response to stress is suspected to be a vulnerability factor for posttraumatic stress disorder (PTSD). Previous studies showed inconsistent findings regarding the role of cortisol in predicting PTSD. In addition, no prospective studies have examined the role of dehydroepiandrosterone (DHEA), or its sulfate form DHEAS, and the cortisol-to-DHEA(S) ratio in predicting PTSD. In this study, we tested whether acute plasma cortisol, DHEAS and the cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks and 6 months post-trauma. Methods: Blood samples of 397 adult level-1 trauma center patients, taken at the trauma resuscitation room within hours after the injury, were analyzed for cortisol and DHEAS levels. PTSD symptoms were assessed at 6 weeks and 6 months post-trauma with the Clinician Administered PTSD Scale. Results: Multivariate linear regression analyses showed that lower cortisol predicted PTSD symptoms at both 6 weeks and 6 months, controlling for age, gender, time of blood sampling, injury, trauma history, and admission to intensive care. Higher DHEAS and a smaller cortisol-to-DHEAS ratio predicted PTSD symptoms at 6 weeks, but not after controlling for the same variables, and not at 6 months. Conclusions: Our study provides important new evidence on the crucial role of the HPA-axis in response to trauma by showing that acute cortisol and DHEAS levels predict PTSD symptoms in survivors of recent trauma.
Neurobiology of DHEA and effects on sexuality, mood and cognition

Pluchino, P.Drakopoulos, F.Bianchi-Demicheli, J.M.Wenger
J Steroid Biochem & Molec Biol 145 (2015) 273–280
http://dx.doi.org/10.1016/j.jsbmb.2014.04.012

Dehydroepiandrosterone (DHEA) and its sulfate ester, DHEAS, are the most abundant steroid hormones in the humans. However, their physiological significance, their mechanisms of action and their possible roles as treatment are not fully clarified. Biological actions of DHEA(S) in the brain involve neuroprotection, neurite growth, neurogenesis and neuronal survival, apoptosis, catecholamine synthesis and secretion, as well as anti-oxidant, anti- inflammatory and antiglucocorticoid effects. In addition, DHEA affects neurosteroidogen is and endorphin synthesis/release. We also demonstrated in a model of ovariectomized rats that DHEA therapy increases proceptive behaviors, already after 1 week of treatment, affecting central function of sexual drive. In women, the analyses of clinical outcomes are far from being conclusive and many issues should still be addressed. Although DHEA preparations have been available in the market since the 1990s, there are very few definitive reports on the biological functions of this steroid. We demonstrate that 1 year DHEA administration at the dose of 10mg provided a significant improvement in comparison with vitamin D in sexual function
and in frequency of sexual intercourse in early postmenopausal women. Among symptomatic women, the spectrum of symptoms responding to DHEA requires further investigation, to define the type of sexual symptoms (e.g. decreased sexual function or hypoactive sexual desire disorder) and the degree of mood/cognitive symptoms that could be responsive to hormonal treatment.
In this regard, our findings are promising, although they need further exploration with a larger and more representative sample size.
Although adrenal cortex is considered to be the primary source of DHEAS in the brain, it was reported that DHEAS did not dis- appear or decrease in the brain 15 days neither after orchiectomy, adrenalectomy, or both, nor after the inhibition of adrenal secretion by dexamethasone. DHEA and DHEAS were among the first neurosteroids identified in rat brains. Cytochrome P450c17 was found in a subset of neurons of embryonic rodent brains. While P450c17 protein was readily detected in the brain, the abundance of P450c17 mRNA transcripts in the embryonic mouse brain or hippocampus of adult male rats was low, and was approximated to be 1/200^th of the expression in testis.
DHEAS may be synthesized in the brain from DHEA. Sulfation of DHEA has been observed in the brains of rhesus monkeys in vivo and in human fetal brain slices in vitro. DHEA sulfotransferase (HSTor SULT2A1) is an enzyme that sulfonates DHEA (in addition to pregnenolone).Western blotting and immune-histochemistry showed protein expression of an HST in adult Wistar rat brain. In addition SULT2A1 mRNA expression has been shown in rat brains. DHEAS is predominately transported out of the brain across the blood–brain barrier and DHEAS found in the brain is most likely due to local synthesis . DHEA(S) may mediate some of its actions through conversion into more potent sex steroids and activation of androgen or estrogen receptors in tissue.
According to existing assumption of the biology of depression, DHEA(S) ability to modulate many neurobiological actions could underlie relationships between endogenous and/or exogenously- supplemented DHEA(S) concentrations and depression. There is evidence that DHEAS concentrations are negatively correlated with ratings of depressed mood. However, the remaining literature examining plasma and serum DHEA(S) concentrations in depression is still inconsistent and other plasma indexes were studied in order to more accurately discriminate depressed from nondepressed individuals. Hypothalamic–pituitary–adrenal axis (HPA) hyperactivity has
been demonstrated in chronic diseases affecting nervous system disorders like depression. The end products of HPA axis, glucocorticoids (GCs), regulate many physiological functions and play an important role in affective regulation and dysregulation. Despite DHEAS levels which markedly decrease throughout adulthood, an increase in circulating cortisol with advanced age has been observed in human and nonhuman primates.
The most relevant aspect meriting attention is certainly the controversial finding among the studies that investigate the correlation of the endogenous DHEA sulfate (DHEAS) level, the aging process or organ illness with the results coming from studies focusing on the effects of exogenous DHEAS administration on brain function, sexuality, cardiovascular health and metabolic syndrome. Indeed, the marked age-related decline in serum DHEA and DHEAS has suggested that a deficiency of these steroids may be causally related to the development of a series of diseases that are generally associ- ated with aging. The postulated consequences of low DHEA levels include insulin resistance, obesity, cardiovascular disease, cancer, reduction of the immune defense system as well as psychosocial problems such as depression and a general deterioration in the sensation of well-being and cognitive function, DHEA replacement may seem an attractive treatment opportunity. Nevertheless, the analyses of clinical outcomes are far from being conclusive.

Dehydroepiandrosterone, its metabolites and ion channels

Hill, M. Dusková, L. Stárka
J Steroid Biochem & Molec Biology 145(2015)293–314
http://dx.doi.org/10.1016/j.jsbmb.2014.05.006

This review is focused on the physiological and pathophysiological relevance of steroids influencing the activities of the central and peripheral nervous systems with regard to their concentrations in body fluids and tissues in various stages of human life like the fetal development or pregnancy. The data summarized in this review shows that DHEA and its unconjugated and sulfated metabolites are physiologically and pathophysiologically relevant in modulating numerous ion channels and participate in vital functions of the human organism. DHEA and its unconjugated and sulfated metabolites including 5 _/ _-reduced androstane steroids participate in various physiological and pathophysiological processes like the management of GnRH cyclic release, regulation of glandular and neurotransmitter secretions, maintenance of glucose homeostasis on one hand and insulin insensitivity on the other hand, control of skeletalmuscle and smooth muscle activities including vasoregulation, promotion of tolerance to ischemia and other neuroprotective effects. In respect of prevalence of steroid sulfates over unconjugated steroids in the periphery and the opposite situation in the CNS, the sulfated androgens and androgen metabolites reach relevance in peripheral organs. The unconjugated androgens and estrogens are relevant in periphery and so much the more in the CNS due to higher concentrations of most unconjugated steroids in the CNS tissues than in circulation and peripheral organs.

Neurotrophins are proteins found within a broad range of cell types in the brain and periphery that facilitate neuronal growth, survival, and plasticity. The neurotrophin ‘‘superfamily’’ includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), neurotrophin-4/5 (NT4/5), and neurotrophin-6. Target tissues are hypothesized to regulate neuron survival by making neurotrophins available in limited amounts, resulting in selection of neurons with the best connectivity to the target tissue. NGF, in particular, is released by the target tissue and taken up in responsive neurons by receptor-mediated endocytosis. It is then transported retrogradedly into the cell where it exerts trophic effects. Lu et al. proposed a ‘‘Yin and Yang model,’’ whereby neurotrophic action is mediated by two principal classes of transmembrane receptor systems: the tyrosine kinase (Trk) receptors (including TrkA [selective for NGF], TrkB [selective for BDNF and NT4/5], and TrkC [selective for NT3]) and the neurotrophin receptor p75NTR. Each receptor type binds mature neurotrophins and/or neurotrophin precursors (proneurotrophins), creating a complex ‘‘balance’’ that then causes neuronal survival or death.
DHEA has been shown to evoke NGF mRNA expression in target cells. In a study of pregnant women, Schulte-Herbrüggen et al. showed no relationships between serum DHEAS and NGF. In contrast, we showed that DHEAS independently associated with salivary NGF (sNGF) in military men under baseline conditions, while DHEA did not. We now know that both DHEA(S) and NGF respond affirmatively to stressful insult, yet the association between these analytes during stress exposure is not understood. Characterization of this relationship has implications for prevention and treatment of traumatic stress and injury, degenerative disease management, and nerve repair. In this report, we extended our prior study of neuroprotective properties of DHEAS in men under baseline conditions to a prospective paradigm involving intense stress exposure in both men and women. We hypothesized that

(a) robust associations would prevail between total output of DHEAS and sNGF across the stress trajectory and at each time point,
(b) changes in DHEAS would predict corresponding changes in sNGF, and
(c) baseline DHEAS would positively predict total sNGF output across the stress trajectory.
We also explored the roles of testosterone and cortisol. In light of less definitive prior literature, directional hypotheses were not stated regarding these analytes.

In the first regression model, total hormone output (AUCG) of the independent variables (DHEAS, testosterone, and cortisol) combined to explain 63.7% of variance in sNGF output (F = 65.4, p < 0.001). Standardized beta coefficients revealed that testosterone exerted an independent effect (b = 0.80, p < 0.001), while the other predictors were not significant. In light of this unexpected finding, we then used regression-based causal steps modeling to evaluate whether testosterone mediated a hypothesized direct effect of DHEAS on sNGF. Following this approach, DHEAS predicted sNGF in an initial regression model (b = 0.45, p < 0.001). When testosterone was added, the direct effect of DHEAS (path c0) on sNGF was nearly eradicated and no longer significant (b = .04, p = .57), thus suggesting a mediated effect. An alternate statistical test (Sobel Test; 34) evaluating the hypothesized difference between the total effect (path c) and the direct effect (path c0) of DHEAS on sNGF produced a similar result (test statistic = 4.0, p < 0.001). Fig. 1 depicts positive association of DHEAS to sNGF, while Fig. 2 depicts Positive association of testosterone to sNGF.

Positive association of DHEAS total output and sNGF total output

Positive association of testosterone total output and sNGF total output

Positive association of testosterone total output and sNGF total output.
The models were then decomposed at each time point. At baseline, the independent variables (DHEAS, testosterone, and cortisol) combined to account for 10.2% of variance in sNGF (F = 5.3, p < 0.01). Standardized beta coefficients showed that DHEAS exerted an independent effect on sNGF (b = 0.39, p < 0.001), while the other predictors were not significant. During stress exposure, the independent variables combined to account for 28.0% of variance in NGF (F = 15.8, p < 0.001). Again, DHEAS exerted an independent effect (b = 0.56, p < 0.001) while the other predictors were not significant. During recovery, the predictor set accounted for 18.0% of variance in sNGF (F = 9.2, p < 0.001), and DHEAS exerted an independent effect (b = 0.47, p < 0.001) while the other predictors did not.
The models were then decomposed relative to each change index. In terms of reactivity, the independent variables (DHEAS, testosterone, and cortisol reactivity) and covariate (sex) combined to account for 20.3% of variance in sNGF reactivity (F = 8.2, p < 0.001). Standardized beta coefficients revealed that DHEAS reactivity exerted an independent effect (b = 0.39, p < 0.001), while the other predictors were not significant. In terms of recovery, the predictors combined to account for 28.2% of variance in sNGF recovery (F = 15.5, p < 0.001); DHEAS recovery exerted an independent effect (b = 0.52, p < 0.001), as did testosterone recovery (b = [1]0.27, p < 0.01). In terms of residual elevation/depression, the independent variables explained 12.4% of variance in sNGF residual elevation (F = 6.2, p < 0.001). DHEAS residual elevation exerted an independent effect (b = 0.35, p < 0.001), while the other predictors did not.

Endocrine-Disrupting Chemicals and Human Growth and Maturation: A Focus on Early Critical Windows of Exposure

Julie Fudvoye, Jean-Pierre Bourguignon, Anne-Simone Parent
Vitamins and Hormones, 2014; 94: Chapt 1. 1-25.
http://dx.doi.org/10.1016/B978-0-12-800095-3.00001-8

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with hormone synthesis, metabolism, or action. In addition, some of them could cause epigenetic alterations of DNA that can be transmitted to the following generations. Because the developing organism is highly dependent on sex steroids and thyroid hormones for its maturation, the fetus and the child are very sensitive to any alteration of their hormonal environment. An additional concern about that early period of life comes from the shaping of the homeostatic mechanisms that takes place also at that time with involvement of epigenetic mechanisms along with the concept of fetal origin of health and disease. In this chapter, we will review the studies reporting effects of EDCs on human development. Using a translational approach, we will review animal studies that can shed light on some mechanisms of action of EDCs on the developing organism. We will focus on the major hormone-dependent stages of development: fetal growth, sexual differentiation, puberty, brain development, and energy balance. We will also discuss the possible epigenetic effects of EDCs on human development.

Several studies have reported that prenatal or early postnatal exposure to some EDCs is associated with alterations of cognitive or motor functions in children. Knowing the fundamental role played by thyroid hormones and sex steroids in cortex development, one can hypothesize that disruption of those hormones could cause alteration of the development of the cerebral cortex and of its functions later in life. We will review here the human data suggesting a causal effect for endocrine disrupters on impairment of cortical functions and approach some EDC mechanisms of action using animal models.

Thyroid hormones are known to be essential for brain development. They regulate progenitor proliferation and differentiation, neuron migration, and dendrite outgrowth (Parent, Naveau, Gerard, Bourguignon, & Westbrook, 2011). Even mild thyroid hormone insufficiency in humans can produce measurable deficits in cognitive functions (Zoeller & Rovet, 2004). Thyroid hormone action is mediated by two classes of nuclear receptors (Forrest & Vennstro¨m, 2000) that exhibit differential spatial and temporal expressions in the brain, suggesting that thyroid hormones have variable functions during brain development. This differential expression of thyroid hormone receptors explains the critical period of thyroid hormone action on brain development as suggested by models of maternal hypothyroidism or congenital hypothyroidism.

Depending on the timing of onset of hypothyroidism, the offspring will display problems of visual attention, gross or fine motor skills, or language and memory skills. Similarly, one can hypothesize that disruption of thyroid function by EDCs will have different effects based on the timing of exposure. However, few studies focused on that aspect. Polychlorinated biphenyls (PCBs) form a group of widespread environmental contaminants composed of 209 different congeners used in a wide variety of applications. Their production was banned in the 1970s but PCBs are still present in the environment due to their high stability. PCBs were among the first EDCs identified as responsible for alterations of cognitive functions. Indeed, impaired memory and altered learning abilities have been associated with prenatal exposure to EDCs in humans and In animal models, perinatal exposure to PCBs has been consistently associated with a decrease of thyroid hormone concentration in maternal serum as well as pup serum. Some but not all epidemiological studies in human have found an association between PCB body burden and thyroid hormone levels. This disruption of thyroid function could explain some of the effects of PCBs on the developing brain. Indeed, animal models have shown that the ototoxic effects of PCBs could be partially ameliorated by thyroxin replacement and PCBs seem to alter some of the developmental processes in the cortex and the cerebellum that are dependent on thyroid hormones. However, recent publications raise important issues.

As it is the case for other EDCs, some windows of susceptibility have been identified during pre- and postnatal brain development. Recent studies have shown that exposure to PBDEs causes alteration of thyroid hormone levels in pregnant women and infants as it is the case in rodents. Only very few studies, however, have focused on the molecular or cellular effects of perinatal exposure to PBDEs in vivo. Viberg et al. have reported a decrease of cholinergic nicotinic receptors in the hippocampus after exposure to BDE-99 and BDE-153. However, the link between such a decrease and the behavioral effects of PBDEs is still unclear. Other teams have reported that exposure to PBDEs reduced hippocampal long term potentiation and decreased brain-derived neurotrophic factor expression in the brain. While several studies have reported negative effect of PBDEs on brain development and cognitive function in animals, there is relatively little information about adverse health effects of PBDEs in humans. Some very recent studies have identified a correlation between prenatal exposure to PBDEs and alteration of cognitive functions.

Endocrine-Disrupting Chemicals: Elucidating Our Understanding of Their Role in Sex and Gender-Relevant End Points

Cheryl A. Frye
Vitamins and Hormones, 2014; 94: 41-98
http://dx.doi.org/10.1016/B978-0-12-800095-3.00003-1

Endocrine-disrupting chemicals (EDCs) are diverse and pervasive and may have significant consequence for health, including reproductive development and expression of sex-/gender-sensitive parameters. This review chapter discusses what is known about common EDCs and their effects on reproductively relevant end points. It is proposed that one way that EDCs may exert such effects is by altering steroid levels (androgens or 17-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus. Basic research findings that demonstrate developmentally sensitive end points to androgens and E2 are provided. Furthermore, an approach is suggested to examine differences in EDCs that diverge in their actions at ERs to elucidate their role in sex-/gender-sensitive parameters.

Reproductive dysfunction among adults and emotional, attentional, and behavioral disorders among children are on the rise. Sperm counts and fertility have declined in the last 50 years . Incidence of attention-deficit hyperactivity disorder (ADHD) and autism has increased in the last 30 years. These increases in reproductive dysfunction and developmental disorders may be due to increased exposure to environmental contaminants, although there is controversy about the relationship between exposure and these effects.
Many contaminants in the environment, including polychlorinated biphenyls (PCBs), dioxins, and metals, accumulate in exposed individuals and may have adverse consequences due to effects as endocrine-disrupting chemicals (EDCs). EDCs may have effects by altering steroid levels (androgens or 17β-estradiol, E2) and/or intracellular E2 receptors (ERs) in the hypothalamus and/or hippocampus.
Steroid hormones, during critical periods of development, organize sexual dimorphisms in brain and behavior and give rise to sex differences in later responses to steroid hormones. EDCs can profoundly disrupt reproductive responses following adult exposure and result in pervasive effects that extend throughout the life of their offspring. Many nonreproductive behaviors, such
as spatial performance, activity, and arousal, are also sexually dimorphic and organized and activated by steroid hormones. Thus, EDCs may affect reproductive and the aforementioned nonreproductive parameters by altering E2 levels and/or ER binding in the hypothalamus and/or hippocampus.
Results from the literature and preliminary data will be presented that demonstrate our use of a whole-animal model to begin to investigate effects of exposure (in adulthood and/or development) to EDCs on steroid levels (androgens and E2), actions at ERs (in hypothalamus and hippocampus), and reproductive-sensitive measures (anogenital distance, accessory structure weight, onset of puberty and sexual maturity, and reproductive behavior) and nonreproductive behaviors (spatial performance, play behavior, and arousal) throughout development.

A common feature of many environmental contaminants is their estrogenic effects. Some contaminants can alter production of E2 and/or androgens or act as agonists or antagonists for intracellular or membrane ERs. Thus, the term “endocrine-disrupting chemicals” (EDCs) in this chapter is used to refer to contaminants with these effects. An important question considered here is the extent to which EDCs’ actions to alter E2 levels and/or ER binding in the hypothalamus or hippocampus mitigates effects on reproductive or nonreproductive processes. There are potential pervasive, negative effects of endocrine disrupters on steroid sensitive tissues, which may confer risk to disease states, such as cancer, heart disease, and neurodegenerative disorders. The following discussion provides evidence that exposure to EDCs during development may result in permanent, lifelong differences in sexual function and reproductive ability, as well as cognitive function and/or emotional reactivity/arousal. Gonad development, sex determination, and reproductive success of offspring are highly dependent on sex hormone systems. The developing organism is exquisitely sensitive to alterations in hormone function. In the early embryonic state, the gonads of human males and females are morphologically identical. Sexual differentiation begins under hormonal influence during the fifth and sixth weeks of fetal development, and thus, alterations in hormones during this highly sensitive period can have profound consequences. Disruption of the sex steroid system during fetal stages of life results in profound adverse developmental reproductive effects, as is well known from the effects of DES. The balance of estrogens and androgens is critical for normal development, growth, and functioning of the reproductive system. Although especially important during development, this balance is important throughout life for the preservation of normal feminine or masculine traits, as well as the expression of some sexually dimorphic behaviors (sex, spatial performance, and arousal).

Proposed negative effects of exposure to endocrine disrupters during development in people and in animals. The focus here is on vulnerability to sexually dimorphic processes that are estrogen-sensitive, such as reproductive, cognitive, and emotional development and associated behavioral processes

The existing data clearly indicate that developmental exposure to EDCs can adversely affect sexual development of people and animals; however, there are different effects depending upon the EDCs and when in development exposure occurs. Therefore, we consider the next effects of EDCs exposure at different point in development and the consequences for reproductive development and behavior, as well as E2 levels and hypothalamic ER binding.
Steroid hormones also play a critical role in neurodevelopment that influences not only reproductive but also nonreproductive behaviors that show sex differences. Specific behavioral differences in nonreproductive behaviors between males and females include differences in spatial learning, play, exploration, activity levels, novelty-seeking behavior, and emotional reactivity. These sex dimorphisms are thought to reflect adaptive differences for behavioral strategies in coping as a result of sexual selection. Moreover, these sexually dimorphic behaviors may be relevant for concerns regarding increased developmental, cognitive, or emotional disabilities over the past 30 years. Also, behaviors are particularly sensitive measures of effects of EDCs.
EDCs can alter cognitive development. Some, but not all, studies have shown a predictive relationship between prenatal PCB exposure and cognitive development in infancy through preschool years. EDCs have direct effects on nervous system function. Long-term potentiation (LTP), a form of synaptic plasticity used as a model system for study of cognitive potential, is altered by PCBs and lead. The protein kinase C (PKC)-signaling pathway is involved in the modulation of learning, memory, and motor behavior and may be a target of E2’s actions. PCBs also alter PKC signaling. Although findings provide evidence that EDCs can alter cognitive performance, these measures of cognition are neither sexually dimorphic nor E2- or ER-dependent.
There are sex-specific effects of perinatal PCB and dioxin exposure on spatial learning. Yu-Cheng boys that were prenatally exposed to high levels of PCBs and PCDFs when their mothers were accidentally exposed to these contaminants in rice oil show more disrupted cognitive development, mainly spatial function, than did exposed girls. In animal studies, spatial learning that favors males is mediated by perinatal exposure to androgens. Gestational and lactational exposure to ortho-substituted PCBs produces spatial deficits at adolescence in male mice or adulthood in male rats. The sparse data suggest that developmental exposure to EDCs disrupts spatial memory. Furthermore, Exposure during adulthood to EDCs can also have activational effects on spatial memory. Females exposed to a phytoestrogen-rich diet exhibit “masculinized” spatial performance in a radial arm maze, while males fed with a phytoestrogen-free diet show “feminized” performance.
An important question is what are the mechanisms by which developmental and/or adult exposure to EDCs alters spatial performance? There is evidence for sex differences in spatial performance and activational effects of E2 in adulthood to alter spatial performance of rats. Systemic or intrahippocampal administration of E2 improves spatial performance of female rats. Further, E2’s actions at intracellular ERs in the hippocampus of adults do not seem to be required to mediate these effects on spatial performance.
EDCs may have effects on E2 metabolism in a number of ways. First, some EDCs can alter serum lipid concentrations. Cholesterol is the precursor for the production of E2 and other steroid hormones (see Fig. 3.3). Second, there is also evidence that some EDCs can alter metabolism enzymes that are necessary for converting cholesterol to steroid hormones. Induction of CYP occurs when EDCs, such as TCDD, bind the aromatic hydrocarbon receptor (AhR). There is a firm link between PCBs, enzyme induction, and AhR. The binding of EDCs with AhR can result in antiestrogenic activity through increased metabolism and depletion of endogenous E2. Elevated levels of CYP enzymes, primarily expressed not only in the liver but also in the brain and other tissues, result in increased E2 metabolism and excretion. Alternatively, compounds that are metabolized by P450s may result in a net estrogenic effect if they inhibit endogenous estrogens from being metabolized.
Steroid hormones are lipid molecules with limited solubility in plasma and are accordingly carried through the plasma compartment to target cells by specific plasma transporter proteins. Each transporter protein has a specific ligand-binding domain for its associated hormone. It is generally accepted that the “free” formof the steroid hormone, and not the conjugate of the hormone with its plasma transport protein, enters target cells and binds with the appropriate receptor. Receptors for the steroid hormones are proteins located primarily in the cell nucleus or partitioned between the cytoplasm and the nucleus. The unoccupied steroid receptors may reside in the cell as heterodimeric complexes with the 90 kDa heat-shock protein, which prevents the receptor from binding with the DNA until the receptor has first bound with its steroid hormone. Once the hormone binds to the receptor, the hormone receptor complexes with the heterodimeric heat-shock protein and undergoes a conformational change and is activated. The activated receptor binds with DNA at a specific site, initiating gene transcription.

Traditional effects of steroid hormones at their cognate steroid receptors

Traditional effects of steroid hormones at their cognate steroid receptors, which act as transcription factors. In this example, effects of steroid hormones, such as estradiol, to bind estrogen receptor (ER) subtypes, referred to as ERa and ERb, are shown.

Beyond traditional actions solely through intracellular cognate estrogen receptors (ERs; ERa and ERb), steroids, such as estradiol, and estradiol-mimetics (endocrine disrupters) may have novel actions involving membrane bound ERs, other neurotransmitter systems (e.g., NMDA receptor), and signal transduction cascades (e.g., growth factors, MAPK).

To date, there has been little investigation in a whole-animal model of the effects of EDCs on E2 levels and/or activity at intracellular ERs in the brain. Thus, changes in E2 levels and ER activity in the hypothalamus and hippocampus, concomitant with alterations in endocrine parameters and reproductive behavior and nonreproductive behavior, respectively, are
needed to elucidate tissue specificity of EDCs’ functions and mechanisms.

Low-Dose Effects of Hormones and Endocrine Disruptors

Laura N. Vandenberg
Vitamins and Hormones, 2014; 94: 129-165
http://dx.doi.org/10.1016/B978-0-12-800095-3.00005-5

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules.

Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the “low-dose hypothesis,” which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Since EDCs began to be studied in depth in the 1990s, there has been intense debate over whether the public should be concerned about low level exposures to these chemicals. The low-dose hypothesis, proposed at that time, has steadily accumulated evidence that EDCs have actions at low doses, and these effects are not necessarily predicted from high-dose toxicology testing. In 2002, the NTP expert panel reported evidence for low-dose effects for a small number of EDCs and estradiol. In 2012, an updated approach identified several dozen additional EDCs with evidence for low-dose effects. Further, epidemiology studies continue to find relationships between EDC exposure levels and diseases in the general public, which has raised concerns because the general public is exposed to a large number of environmental chemicals at low doses. For decades, hormones have been known to produce striking changes in tissue morphology, physiology, and behaviors at exceedingly low doses.

A relatively large body of evidence suggests that EDCs, and in particular those environmental chemicals that mimic endogenous hormones, have similar effects at low doses. Although there is still no consensus about the universality of “low-dose effects” in the toxicology community, the Endocrine Society (Diamanti-Kandarakis et al., 2009; Zoeller et al., 2012) believes not only that there is sufficient evidence in support of this phenomenon but also that it is time for public health agencies to make changes to risk assessment paradigms and give greater consideration to studies that specifically identify low-dose effects when considering risks from chemical exposures.

Bisphenol A interferes with synaptic remodeling

Tibor Hajszan, Csaba Leranth
Frontiers in Neuroendocrinology 31 (2010) 519–530
http://dx.doi.org:/10.1016/j.yfrne.2010.06.004

The potential adverse effects of Bisphenol A (BPA), a synthetic xenoestrogen, have long been debated. Although standard toxicology tests have revealed no harmful effects, recent research highlighted what was missed so far: BPA-induced alterations in the nervous system. Since 2004, our laboratory has been investigating one of the central effects of BPA, which is interference with gonadal steroid-induced synaptogenesis and the resulting loss of spine synapses. We have shown in both rats and nonhuman primates that BPA completely negates the ~70–100% increase in the number of hippocampal and prefrontal spine synapses induced by both estrogens and androgens. Synaptic loss of this magnitude may have significant consequences, potentially causing cognitive decline, depression, and schizophrenia, to mention those that our laboratory has shown to be associated with synaptic loss. Finally, we discuss why children may particularly be vulnerable to BPA, which represents future direction of research in our laboratory.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Xiaohong Xu ⁎, Yinping Ye, Tao Li, Lei Chen, Dong Tian, Qingqing Luo, Mei Lu
Toxicology and Applied Pharmacology 249 (2010) 188–196
http://dx.doi.org:/10.1016/j.taap.2010.09.007

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E2. The present results suggest that BPA, like 17β-E2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E2 when it coexists with 17β-E2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

Xiaohong Xu⁎, Tao Li, Qingqing Luo, Xing Hong, Lingdan Xie, Dong Tian
Toxicology and Applied Pharmacology 255 (2011) 221–228
http://dx.doi.org:/10.1016/j.taap.2011.06.022

Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague–Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after foot shock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182, 780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway.

Bisphenol A promotes dendritic morphogenesis of hippocampal neurons through estrogen receptor-mediated ERK1/2 signal pathway

Xiaohong Xu, Yang Lu, Guangxia Zhang, Lei Chen, Dong Tian, et al.
Chemosphere 96 (2014) 129–137
http://dx.doi.org/10.1016/j.chemosphere.2013.09.063

Bisphenol A (BPA), an environmental endocrine disruptor, has attracted increasing attention to its adverse effects on brain developmental process. The previous study indicated that BPA rapidly increased motility and density of dendritic filopodia and enhanced the phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunit NR2B in cultured hippocampal neurons within 30 min. The purpose of the present study was further to investigate the effects of BPA for 24 h on dendritic morphogenesis and the underlying mechanisms. After cultured for 5 d in vitro, the hippocampal neurons from 24 h-old rat were infected by AdV-EGFP to indicate time-lapse imaging of living neurons. The results demonstrated that the exposure of the cultured hippocampal neurons to BPA (10, 100 nM) or 17β-estradiol (17β-E2, 10 nM) for 24 h significantly promoted dendritic development, as evidenced by the increased total length of dendrite and the enhanced motility and density of dendritic filopodia. However, these changes were suppressed by an ERs antagonist, ICI182,780, a non-competitive NMDA receptor antagonist, MK-801, and a mitogen activated ERK1/2-activating kinase (MEK1/2) inhibitor, U0126. Meanwhile, the increased F-actin (filamentous actin) induced by BPA (100 nM) was also completely eliminated by these blockers. Furthermore, the result of western blot analyses showed that, the exposure of the cultures to BPA or 17β-E2 for 24 h promoted the expression of Rac1/Cdc42 but inhibited that of RhoA, suggesting Rac1 (Ras related C3 botulinum toxinsubstrate 1)/Cdc42 (cell divisioncycle 42) and RhoA (Ras homologous A), the Rho family of small GTPases, were involved in BPA- or 17β-E2-induced changes in the dendritic morphogenesis of neurons. These BPA- or 17b-E2-induced effects were completely blocked by ICI182,780, and were partially suppressed by U0126. These results reveal that, similar to 17β-E2, BPA exerts its effects on dendritic morphogenesis by eliciting both nuclear actions and extranuclear-initiated actions that are integrated to influence the development of dendrite in hippocampal neurons.

Tyreoliberin (Trh) – The Regulatory Neuropeptide Of Cns Homeostasis
Danuta Jantas
Advances In Cell Biology 2;(4)/2010 (139–154)
http://dx.doi.org:/10.2478/v10052-010-0008-4

The physiological role of thyreoliberin (TRH) is the preservation of homeostasis within four systems
(i) the hypothalamic-hypophsysiotropic neuroendocrine system,
(ii) the brain stem/midbrain/spinal cord system,
(iii) the limbic/cortical system, and
(iv) the chronobiological system.

Thus TRH, via various cellular mechanisms, regulates a wide range of biological processes (arousal, sleep, learning, locomotive activity, mood) and possesses the potential for unique and widespread applications for treatment of human illnesses. Since the therapeutic potential of TRH is limited by its pharmacological profile (enzymatic instability, short half-life, undesirable effects), several synthetic analogues of TRH were constructed and studied in mono- or adjunct therapy of central nervous system (CNS) disturbances. The present article summarizes the current state of understanding of the physiological role of TRH and describes its putative role in clinical indications in CNS maladies with a focus on the action of TRH analogues.

Breakthrough in neuroendocrinology by discovering novel neuropeptides and neurosteroids: 2. Discovery of neurosteroids and pineal neurosteroids

Kazuyoshi Tsutsui, Shogo Haraguchi
General and Comparative Endocrinology 205 (2014) 11–22
http://dx.doi.org/10.1016/j.ygcen.2014.03.008

Bargmann–Scharrer’s discovery of ‘‘neurosecretion’’ in the first half of the 20th century has since matured into the scientific discipline of neuroendocrinology. Identification of novel neurohormones, such as neuropeptides and neurosteroids, is essential for the progress of neuroendocrinology. Our studies over the past two decades have significantly broadened the horizons of this field of research by identifying novel neuropeptides and neurosteroids in vertebrates that have opened new lines of scientific investigation in neuroendocrinology. We have established de novo synthesis and functions of neurosteroids in the brain of various vertebrates. Recently, we discovered 7α-hydroxypregnenolone (7α-OH PREG), a novel bioactive neurosteroid that acts as a key regulator for inducing locomotor behavior by means of the dopaminergic system. We further discovered that the pineal gland, an endocrine organ located close to the brain, is an important site of production of neurosteroids de novo from cholesterol (CHOL). The pineal gland secretes 7α-OH PREG and 3α,5α-tetrahydroprogesterone (3α,5α-THP; allopregnanolone) that are involved in locomotor rhythms and neuronal survival, respectively. Subsequently, we have demonstrated their mode of action and functional significance. This review summarizes the discovery of these novel neurosteroids and its contribution to the progress of neuroendocrinology.

Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones

Paula Duarte-Guterman, Laia Navarro-Martín, Vance L. Trudeau
General and Comparative Endocrinology 203 (2014) 69–85
http://dx.doi.org/10.1016/j.ygcen.2014.03.015

Thyroid hormones (THs) are well-known regulators of development and metabolism in vertebrates. There is increasing evidence that THs are also involved in gonadal differentiation and reproductive function. Changes in TH status affect sex ratios in developing fish and frogs and reproduction (e.g., fertility), hormone levels, and gonad morphology in adults of species of different vertebrates. In this review, we have summarized and compared the evidence for cross-talk between the steroid hormone and thyroid axes and present a comparative model. We gave special attention to TH regulation of sex steroid synthesis and action in both the brain and gonad, since these are important for gonad development and brain sexual differentiation and have been studied in many species. We also reviewed research showing that there is a TH system, including receptors and enzymes, in the brains and gonads in developing and adult vertebrates. Our analysis shows that THs influences sex steroid hormone synthesis in vertebrates, ranging from fish to pigs. This concept of crosstalk and conserved hormone interaction has implications for our understanding of the role of THs in reproduction, and how these processes may be dysregulated by environmental endocrine disruptors.

Insights into the structure of class B GPCRs

Kaspar Hollenstein, Chris de Graaf, Andrea Bortolato, Ming-Wei Wang, et al.
Trends in Pharmacological Sciences, Jan 2014; 35(1)
http://dx.doi.org/10.1016/j.tips.2013.11.001

The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine, anxiety, and depression). The recently solved crystal structures of the transmembrane domains of the human glucagon receptor and human corticotropin-releasing factor receptor 1 have opened up new opportunities to study the structure and function of class B GPCRs. The current review shows how these structures offer more detailed explanations to previous biochemical and pharmacological studies of class B GPCRs, and provides new insights into their interactions with ligands.

Class B G protein-coupled receptors (GPCRs), also referred to as the secretin family of GPCRs, include receptors for 15 peptide hormones, which can be grouped into five subfamilies based on their physiological role (see Table 1 for an overview) [1]. These receptors are important drug targets in many human diseases, including diabetes, osteoporosis, obesity, cancer, neurodegeneration, cardiovascular disease, headache, and psychiatric disorders. However, the identification of small-molecule oral drugs for this family has proved extremely challenging.

(A,B) Crystal structures of the class B G protein-coupled receptors corticotropin-releasing factor receptor 1 (CRF1) [Protein Data Bank (PDB) identifier: 4K5Y] and glucagon receptor (PDB identifier: 4L6R) are shown in blue and orange ribbons, respectively, in two different views from within the membrane. Transmembrane (TM) helices and helix 8 are labelled. The disulfide bond tethering extracellular loop 2 (ECL2) to the tip of TM3 is shown as purple sticks. In CRF1 the small-molecule antagonist CP-376395 is shown in stick representation with carbon, nitrogen, and oxygen atoms colored magenta, blue, and red, respectively, and as skeletal formula in an inset. (C) Superposition of the two structures, with insets highlighting regions of particular interest. To highlight the structural differences in the extracellular halves of CRF1 and glucagon receptor, the distance of approximately 11 A° between the Ca-atoms of residues 7.33b at the N-terminal end of TM7 is indicated with a red arrow. The small molecule binding pocket is shown as a superposition of the two receptors around CP-376395, illustrating the antagonist binding mode and the substantial structural differences observed for TM6 of the two receptors.

Overview of NMR and crystal structures of class B G protein-coupled

receptor (GPCR) extracellular domains (ECDs; magenta) and their complexes with peptide ligands (different colors). A complete overview of corresponding Protein Data Bank identifiers is presented in Table 1 (not shown). (B) Structure-based sequence alignment of representative peptide ligands of class B GPCR, adopted from Parthier et al. [6]. The residues of the peptide ligands solved in ECD–ligand complex crystal structures are marked using the same colour as in Figure 2A. The residues that are boxed black are found in an α-helical conformation in the complex. Peptide ligand residues that covalently bind receptors in photo-crosslinking or cysteine-trapping studies are colored and boxed green, whereas peptide ligand residues that have been mutated and studied in combination with receptor mutants are colored and boxed red. Note that the first residue of glucagon-like peptide-1 (GLP-1) is His7. A complete overview of all ECD structures and important peptide ligands for all class B GPCRs is presented in Table 1. Putative helix-capping residues [6] are coloured blue and cysteines involved in a disulfide-bridge (calcitonin) are coloured orange. D-phenylalanine (f), and norleucine (m) residues are indicated in stressin and astressin. The last 41 and 99 residues of parathyroid hormone (PTH) and PTH-related protein. (PTHrP), respectively, are not displayed. Abbreviations: CGRP, calcitonin gene-related peptide; CLR, calcitonin receptor-like receptor; CRF, corticotropin-releasing factor; CT, calcitonin; Ext-4, exendin-4; GHRHR, growth hormone releasing hormone receptor; GIP, glucose-dependent insulinotropic peptide; PAC, pituitary adenylate cyclase; PACAP, pituitary adenylate cyclase activating polypeptide; RAMP, receptor-activity modifying proteins; SCTR, secretin receptor; Ucn, urocortin; VPAC, vasoactive pituitary adenylate cyclase.

Figure 3. (not shown) (A) The spatial correspondence between residues in transmembrane (TM) helices of class A and class B G protein-coupled receptors (GPCRs) makes it possible to align the most conserved residues in class A (designated X.50, Ballesteros–Weinstein numbering) and class B (designated X.50b, Wootten numbering) for comparisons between GPCR classes (Box 1). (B) Structural alignment of corticotropin-releasing factor receptor 1 (CRF1; blue) and glucagon receptor (GCGR; orange) to two representative class A GPCRs, histamine H1 receptor (H1R; Protein Data Bank identifier: 3RZE) and CXC-chemokine receptor 4 (CXCR4; Protein Data Bank identifier: 3ODU/3OE0) (in grey). Helices are depicted as cylinders, and the ligands glucagon (for GCGR), CP-376395 (for CRF1), doxepin (for H1R), and IT1t and CVX15 (for CXCR4) are shown as sticks. The

location of the Ca-atoms of the most conserved residues of TM1–3 and TM5 among class A and class B GPCRs (Box 1) are indicated by spheres (TM4 is not depicted for clarity).

The GCGR and CRF1 crystal structures show distinct structural features and different binding pockets compared to class A GPCRs, and give new insights into the molecular details of peptide and small-molecule binding to class B GPCRs. The first two crystal structures of the TM domains of class B GPCRs provide a structural framework that will enable the design of biochemical and biophysical experiments detailing the complex structure of this class of receptors, and facilitate the design of stabilized constructs that might lead to the solution of full-length class B GPCR–ligand complexes. The structures furthermore present more reliable structural templates for the design of specific and potent small molecules for the treatment of type 2 diabetes (GCGR) and depression (CRF1) in particular, and open new avenues for structure-based small-molecule drug discovery for class B GPCRs as a whole.

Novel receptor targets for production and action of allopregnanolone in the central nervous system: a focus on pregnane xenobiotic receptor

Cheryl A. Frye, Carolyn J. Koonce and Alicia A. Walf
Front in Cell Neurosci Apr 2014; 8(106): 1-13.
http://dx.doi.org:/10.3389/fncel.2014.00106

Neurosteroids are cholesterol-based hormones that can be produced in the brain,

independent of secretion from peripheral endocrine glands, such as the gonads and

adrenals. A focus in our laboratory for over 25 years has been how production of the

pregnane neurosteroid, allopregnanolone, is regulated and the novel (i.e., non steroid

receptor) targets for steroid action for behavior. One endpoint of interest has been lordosis, the mating posture of female rodents. Allopregnanolone is necessary and sufficient for lordosis, and the brain circuitry underlying it, such as actions in the midbrain ventral tegmental area (VTA), has been well-characterized. Published and recent findings supporting a dynamic role of allopregnanolone are included in this review.
First, contributions of ovarian and adrenal sources of precursors of allopregnanolone, and the requisite enzymatic actions for de novo production in the central nervous system will be discussed.
Second, how allopregnanolone produced in the brain has actions on behavioral processes that are independent of binding to steroid receptors, but instead involve rapid modulatory actions via neurotransmitter targets (e.g., g-amino butyric acid-GABA, N methyl-D-aspartate- NMDA) will be reviewed.
Third, a recent focus on characterizing the role of a promiscuous nuclear receptor, pregnane xenobiotic receptor (PXR), involved in cholesterol metabolism and expressed in the VTA, as a target for allopregnanolone and how this relates to both actions and production of allopregnanolone will be addressed. For example, allopregnanolone can bind PXR and knocking down expression of PXR in the midbrain VTA attenuates actions of allopregnanolone via NMDA and/or GABAA for lordosis. Our understanding of allopregnanolone’s actions in the VTA for lordosis has been extended to reveal the role of allopregnanolone for broader, clinically-relevant questions, such as neurodevelopmental processes, neuropsychiatric disorders, epilepsy, and aging.

Genetically Encoded Chemical Probes in Cells Reveal the Binding Path of Urocortin-I to CRF Class B GPCR

Irene Coin, Vsevolod Katritch, Tingting Sun, Zheng Xiang, Fai Yiu Siu
Cell Dec 2013; 155, 1258–1269
http://dx.doi.org/10.1016/j.cell.2013.11.008

Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin- I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of
CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights
on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.

Investigation of GPCR-Ligand Interactions under Native Conditions Using Genetically Encoded Chemical Probes GPCRs are integral membrane proteins containing multiple domains and various posttranslational modifications. To understand GPCR-ligand interactions by crystallography, receptors have to be extracted from the cell membrane and modified with a series of expedients such as deglycosylation, therm-stabilizing mutations, fusions with soluble proteins, or complexes with stabilizing nanobodies. We present here a method to investigate GPCR-ligand interactions at the intact fully posttranslationally modified receptor bound to its WT ligand on the membrane of the live cell, which mimics the native conditions for GPCR function. We first genetically incorporated into the receptor the photocrosslinking Uaa Azi, which served as
a proximity probe to provide an overall map of the ligand binding sites on the receptor. We then determined the relative position of the ligand in the binding pocket using a residue-specific chemical crosslinking reaction between Ffact genetically incorporated into the receptor and Cys introduced into the ligand. The derived intermolecular spatial constraints served eventually to build a detailed conformational model for the receptor-ligand complex.

Glutamate Neurons within the Midbrain Dopamine Regions

Morales and D. H. Root
Neuroscience 282 (2014) 60–68
http://dx.doi.org/10.1016/j.neuroscience.2014.05.032

Midbrain dopamine (DA) neurons are hypothesized to play roles in reward-based behavior and addiction, reward prediction and learning by error detection, effort-based decision making, flexible reward-directed behaviors,

incentive salience, stimulus salience (e.g., prediction of rewarding and aversive events), aversion, depression, and fear. The extensive, divergent behavioral roles of midbrain dopamine neurons, predominantly from the ventral tegmental area (VTA), indicate that this system is highly heterogeneous.
This heterogeneity may be reflected in part by the diverse phenotypic characteristics among DAergic neurons and their interactive brain structures.

Midbrain dopamine systems play important roles in Parkinson’s disease, schizophrenia, addiction, and depression. The participation of midbrain dopamine systems in diverse clinical contexts suggests these systems are highly complex. Midbrain dopamine regions contain at least three neuronal phenotypes: dopaminergic, GABAergic, and glutamatergic. Here, we review the locations, subtypes, and functions of glutamatergic neurons within midbrain dopamine regions. Vesicular glutamate transporter 2 (VGluT2) mRNA-expressing neurons are observed within each midbrain dopamine system. Within rat retrorubral field (RRF), large populations of VGluT2 neurons are observed throughout its anteroposterior extent. Within rat substantia nigra pars compacta (SNC), VGluT2 neurons are observed centrally and caudally, and are most dense within the laterodorsal subdivision. RRF and SNC rat VGluT2 neurons lack tyrosine hydroxylase (TH), making them an entirely distinct population of neurons from dopaminergic neurons. The rat ventral tegmental area (VTA) contains the most heterogeneous populations of VGluT2 neurons. VGluT2 neurons are found in each VTA subnucleus but are most dense within the anterior midline subnuclei. Some subpopulations of rat VGluT2 neurons co-express TH or glutamic acid decarboxylase (GAD), but most of the VGluT2 neurons lack TH or GAD. Different subsets of rat VGluT2-TH neurons exist based on the presence or absence of vesicular monoamine transporter 2, dopamine transporter, or D2 dopamine receptor. Thus, the capacity by which VGluT2-TH neurons may release dopamine will differ based on their capacity to accumulate vesicular dopamine, uptake extracellular dopamine, or be autoregulated by dopamine. Rat VTA VGluT2 neurons exhibit intrinsic VTA projections and extrinsic projections to the accumbens and to the prefrontal cortex. Mouse VTA VGluT2 neurons project to accumbens shell, prefrontal cortex, ventral pallidum, amygdala, and lateral habenula. Given their molecular diversity and participation in circuits involved in addiction, we hypothesize that individual VGluT2 subpopulations of neurons play unique roles in addiction and other disorders. This article is part of a Special Issue entitled: Ventral Tegmentum & Dopamine. Published by Elsevier Ltd. On behalf of IBRO.

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Thyroid Function and Disorders

Posted in Amino acids, Calcium Signaling, Clinical Diagnostics, Curation, Developmental biology, Disease Biology, Endocrine Diseases, Genomic Endocrinology, Metabolism, Neurological Diseases, Proteins, Translational Science, tagged blood brain barrier, brain, hyperthyroidism, hypothyroid, MCT8, retinoids, T3, T4, thyroglobulin, Thyroid, thyroid nodule on February 5, 2015| Leave a Comment »

Thyroid Function and Disorders

Writer and Curator: Larry H. Bernstein, MD, FCAP

Normal thyroid function is maintained by endocrine interactions between the hypothalamus, anterior pituitary and thyroid gland [Matfin, 2009]. Iodide is transported across the basement membrane of the thyroid cells by an intrinsic membrane protein called the Na/I symporter (NIS). At the apical border, a second iodide transport protein called pendrin moves iodide into the colloid, where it is involved in hormono-genesis. Once inside the follicle, most of the iodide is oxidized by the enzyme thyroid peroxidase (TPO) in a reaction that facilitates combination with a tyrosine molecule to ultimately form thyroxine (T4) and triiodothyronine (T3). Thyroxine is the major thyroid hormone secreted into the circulation (90%, with T3 composing the other 10%). There is evidence that T3 is the active form of the hormone and that T4 is converted into T3 before it can act physiologically.

All of the major organs in the body are affected by altered levels of thyroid hormone. These actions are mainly mediated by T3. In the cell, T3 binds to a nuclear receptor, resulting in transcription of specific thyroid hormone response genes.

Maternal thyroid hormones are essential for neural development in zebrafish.

Marco A Campinho, João Saraiva, Claudia Florindo, Deborah M Power Molecular endocrinology (Baltimore, Md.) 05/2014;
http://dx.doi.org:/10.1210/me.2014-1032

ABSTRACT Teleost eggs contain an abundant store of maternal thyroid hormones (THs) and early in zebrafish embryonic development all the genes necessary for TH signalling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development an MCT8 knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7 and pax8 genes but downstream of shha and fgf8a signalling. The lack of inhibitory spinal cord interneurons and increased motorneurons in the MCT8 morphants is consistent with their stiff axial body and impaired mobility. MCT8 mutations are associated with X-linked mental retardation in humans and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.
Relationship between thyroid status and renal function in a general population of unselected outpatients

Giuseppe Lippi, Martina Montagnana, Giovanni Targher, Gian Luca Salvagno, Gian Cesare Guidi
Clin Biochem May 2008; 41(7–8): 625-627

When compared with euthyroid subjects, those with TSH < 0.2 mIU/L and > 2.5 mIU/L had increased and decreased estimated glomerular filtration rate (e-GFR), respectively. TSH levels were an independent predictor of e-GFR.

Serum Thyroid-Stimulating Hormone Measurement for Assessment of Thyroid Function and Disease

Douglas S. Ross
Endocr and Metab Clinics of N Am, Jun 2001; 30(2, 1): 245-264

Thyrotropin, or thyroid-stimulating hormone (TSH), is one of a family of glycoprotein hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), and human chorionic gonadotropin (hCG) that share a common α-subunit and a unique β-subunit. Pituitary TSH regulates the secretion of the thyroid hormones T4 (thyroxine) and T3 (triiodothyronine). TSH secretion, in turn, is controlled through negative feedback by thyroid hormone on the pituitary thyrotrope. This relationship is negative log-linear. Small changes in serum free thyroid hormone concentrations result in large changes in serum TSH concentrations, and even subtle changes in thyroid hormone production are best assessed by measurement of serum TSH . Until the late 1980s, the detection limit of TSH assays was within the normal range, and these first-generation TSH assays were useful only for the detection of hypothyroidism. Free T4 measurements were primarily used for assessing thyroid function despite the technical difficulties in free thyroid hormone measurements owing to abnormal binding proteins, changes in binding protein concentrations, and the effects of drugs and illness on thyroid hormone binding. With the use of sensitive second- and third-generation TSH assays, TSH measurement has emerged as the single most useful test of thyroid function. It is widely and appropriately used as a screening test. Unfortunately, the trend has been to rely on TSH measurements alone for the assessment of complicated thyroid disease and patients undergoing treatment for thyroid dysfunction. This article focuses on the potential and real limitations of TSH measurement.
Correlation of creatinine with TSH levels in overt hypothyroidism — A requirement for monitoring of renal function in hypothyroid patients?

Vandana Saini, Amita Yadav, Megha Kataria Arora, Sarika Arora, Ritu Singh, Jayashree Bhattacharjee
Clin Biochem Feb 2012; 45(3): 212-214

Highlights
► Increase serum creatinine levels in both subclinical and overt hypothyroidism. ► Creatinine levels progressively increase with increasing degree of hypothyroidism. ► Increase in creatinine correlated with TSH levels in overt hypothyroid subjects. ► Regular monitoring of renal function is required in hypothyroid patients.

Renal function is influenced by thyroid status. Therefore, this study was done to determine the relationship between renal function and different degrees of thyroid dysfunction.
Design and methods
Thyroid and kidney function tests were analyzed in 47 patients with overt (TSH ≥ 10.0 μIU/L) and 77 patients with subclinical hypothyroidism (TSH 6.0–9.9 μIU/L) in a cross-sectional study. These were compared with 120 age- and sex-matched euthyroid controls.
Results
Overt hypothyroid subjects showed significantly raised serum urea, creatinine and uric acid levels as compared to controls whereas subclinical hypothyroid patients showed significant increased levels of serum urea and creatinine levels. TSH showed significant positive correlation with creatinine and uric acid values and, fT4 had a negative correlation with uric acid in overt hypothyroidism.
Conclusion
Hypothyroid state is associated with significant derangement in biochemical parameters of renal function. Hence the renal function should be regularly monitored in hypothyroid patients.

Ability of Serum Thyroid-Stimulating Hormone Levels to Reflect Peripheral and Central Thyroid Hormone Action Appropriately

Uncertainty Owing to Heterogeneity of T4 Deiodinases
Uncertainty Owing to Heterogeneity of T3 Receptors
Uncertainty Owing to Resetting of the Threshold for Negative Feedback

Clinical Utility of Thyroid-Stimulating Hormone Measurement
Screening for Thyroid Disease and Assessment of Patients Suspected of Having Thyroid Disease

Limitations of Thyroid-Stimulating Hormone Testing in Patients with Known Thyroid Disease Central Hypothyroidism
Thyrotoxicosis Owing to Inappropriate Thyroid-Stimulating Hormone Secretion
Monitoring Thyroid Hormone Therapy
Patients Treated for Hyperthyroidism

The Pituitary-Thyroid Axis in Nonthyroidal Illness

Measurement of Thyroid-Stimulating Hormone
Drugs that Affect Serum Thyroid-Stimulating Hormone Concentrations

Investigations into the etiology of elevated serum T3 levels in protein-malnourished rats

Robert C. Smallridge, Allan R. Glass, Leonard Wartofsky, Keith R. Latham, Kenneth D. Burman
Metabolism, V June 1982; 31(6): 538-542

Thyroid function studies and the peripheral metabolism of thyroid hormone were examined in rats fed a low protein diet (9% casein) for 4–8 wk. Compared to animals fed a normal protein diet ad libitum, both the low protein rats and a pair-fed control group weighed less at the end of the study. However, serum total T3 levels were significantly higher only in the protein deficient rats. The elevated serum T3 was not explainable by enhanced peripheral T4 to T3 conversion, as there was no evidence of any change in hepatic or renal 5′-deiodinase activity when homogenates were examined for conversion of T4 to T3, reverse T3 to 3,3′-diiodothyronine, or 3′,5′-diiodothyronine to 3′-monoiodothyronine. Neither was there an effect on hepatic T3 receptor maximal binding capacity (204 ± 24 versus 168 ± 15 fmol/mg DNA control) or binding affinity (2.07 ± 0.38 versus 2.49 ± 0.24 × 10−10 M control). In two separate experiments the dialyzable fraction of T3 was significantly lower in the low protein group while free T3 concentrations were unchanged or reduced. In contrast, serum total and free T4 were either normal or reduced and dialyzable T4 was unaffected by protein deficiency. We conclude that while serum total T3 is elevated in rats chronically fed a low protein diet, this elevation is not due to enhanced T4 to T3 conversion. Rather, the increased T3 levels can be accounted for by a striking alteration in protein binding to T3. Moreover, the failure to demonstrate similar changes in serum total and dialyzable T4 suggests that in the rat, protein deficiency has different effects on binding to the two major thyroid hormones. Dietary induced changes in serum thyroid hormone binding must be kept in mind in nutrition studies in the rat.

Role of thyrotropin in metabolism of thyroid hormones in nonthyroidal tissues

Udaya M. Kabadi
Metabolism, Jun 2006; 55(6): 748-750

T4 conversion into T3 in peripheral tissues is the major source of circulating T3. However, the exact mechanism of this process is ill defined. Several in vitro studies have demonstrated that thyrotropin facilitates deiodination of T4 into T3 in liver and kidneys. However, there is a paucity of in vitro studies confirming this activity of thyrotropin. Therefore, this study was conducted to examine the influence of thyrotropin on thyroid hormone metabolism in nonthyroidal tissues. We assessed T4, T3, reverse T3 (rT3), and T3 resin uptake (T3RU) responses up to 12 hours at intervals of 4 hours in 6 thyroidectomized female mongrel dogs rendered euthyroid with LT4 replacement therapy before and after subcutaneous (SC) administration of bovine thyrotropin (5 U) on one day and normal saline (0.5 mL) on another in a randomized sequence between 08:00 and 09:00 am. Euthyroid state after LT4 replacement was confirmed before thyrotropin administration. Serum T4, T3, rT3, and T3RU all remained unaltered after SC administration of normal saline. No significant alteration was noted in serum T3RU values on SC administration of thyrotropin. However, serum T3 rose progressively reaching a peak at 12 hours with simultaneous declines being noted in both serum T4 and rT3 concentrations (P < .05 vs prethyrotropin values for all determinations). The changes after SC administration were significantly different (P < .001) in comparison to those noted on SC administration of normal saline. Thyrotropin may promote both the conversion of T4 to T3 and metabolism of rT3 into T2 in nonthyroidal tissues via enhancement of the same monodeionase.

Effects of growth hormone administration on fuel oxidation and thyroid function in normal man

Jens Møller, Jens O.L. Jørgensen, Niels Møller, Jens S. Christiansen, Jørgen Weeke
Metabolism, Jul 1992; 41(7): 728-731

In a randomized, double-blind, placebo-controlled, cross-over study, we examined the effects of 14 days of growth hormone (GH) administration (12 IU/d subcutaneously) on energy expenditure (EE), respiratory exchange ratio (RER), and thyroid function in 14 normal adults of normal weight (eight men and six women). EE (kcal/24 h) was significantly elevated after GH administration (2,073 ± 392, [GH], 1,900 ± 310, [placebo], P = .01). RER was significantly lowered during GH administration (0.73 ± 0.04 v 0.78 ± 0.06, P = .02), reflecting increased oxidation of lipids. Total triiodothyronine (TT3) (nmol/L) and free T3 (FT3) (pmol/L) increased significantly during GH (TT3: 1.73 ± 0.06 [GH], 1.48 ± 0.08 [placebo], P = .01; FT3: 6.19 ± 0.56 [GH], 5.49 ± 0.56 [placebo], P = .01). Concomitantly, an insignificant decrease in reverse T3 (rT3) (nmol/L) was observed (0.07 ± 0.01 [GH], 0.15 ± 0.01 [placebo], P = .08). GH caused a highly significant increase in T3/thyroxine (T4 (×100) ratio (1.84 ± 0.12 [GH], 1.37 ± 0.06 [placebo]). Serum thyrotropin (TSH) was not significantly changed by GH. No changes in total thyroxine (TT4) (nmol/L) (98 ± 6 [GH], 111 ± 8 [placebo], P = .40) and free thyroxine (FT4) (pmol/L) (17.4 ± 1.3 [GH], 18.6 ± 1.1 [placebo], P = .37) after 14 days of GH administration were observed. In conclusion, 2 weeks of GH administration increases EE and lipidoxidation. This finding may partly be mediated by an increase in peripheral T4 to T3 conversion.

Studies on the deiodination of thyroid hormones in Xenopus laevis tadpoles

Helen Robinson, Valerie Anne Galton
Gen Compar Endocr, Sept 1976; 30(1): 83-90

Liver and tail tissues from Xenopus laevis tadpoles possess deiodinating systems capable of degrading both thyroxine (T4) and 3,5,3′-triiodothyronine (T3). Deiodinating activity in liver remains at a constant level throughout late development and metamorphosis with the exception of a transient increase at stage 59, the onset of metamorphosis. Tail activity remains constant during development but rises sharply during metamorphosis when the tail is undergoing regression. In contrast to these findings on spontaneously metamorphosing tadpoles, tail tips induced to regress in vitro do not exhibit any rise in deiodinating activity, even when the tail tips are undergoing extensive autolysis. These results indicate that, while a rise in deiodinating activity may coincide temporarily with hormone action during metamorphosis, the two phenomena may be separated. The deiodinating activity present in tadpole tissues appears to be enzymic and possesses properties characteristic of peroxidase activity. The reaction catalyzed by this mechanism does not appear to involve monodeiodination and hence cannot be considered a mechanism for the peripheral conversion of T4 to T3.

Mechanisms governing the relative proportions of thyroxine and 3,5,3′-triiodothyronine in thyroid secretion

Peter Laurberg
Metabolism, Apr 1984; 33(4): 379-392

In subjects with normal thyroid function only a minor part of circulating 3,5,3′-triiodothyronine (T3) originates directly from the thyroid; the majority is produced in the peripheral tissues by deiodination of thyroxine (T4). However, T3 of thyroidal origin constitutes a relatively high fraction of the total T3 produced in many patients with thyroid hyperfunction or hypofunction. Such a relatively high T3 content in the secretion of the thyroid could be caused by a low T4T3 ratio in thyroglobulin. Severe iodine deficiency is a well-known inducer of a low T4T3 ratio, but a low T4T3 ratio can also be produced independent of the iodine content. This is seen in in vitro studies of thyroglobulin iodination when small amounts of DIT are added to the incubation mixture and in vivo in TSH-treated animals and in patients with Graves’ disease. Another mechanism for high thyroidal secretion of T3 could be an enhanced fractional deiodination of T4 to T3 in the thyroid. In vitro thyroid perfusion studies have shown that the T3 content of thyroid secretions is higher than would be expected from the T4T3 ratio of thyroid hydrolysate and that the major mechanism is deiodination of T4 to T3. Thyroxine deiodinases are also present in the human thyroid, and the amount of T4 deiodinase is enhanced in the thyroids from patients with medically treated Graves’ disease and in the hyperstimulated thyroids of rats. Other factors of possible importance for the mixture of T3 and T4 secreted by the thyroid are a relatively faster liberation of T3 than of T4 from thyroglobulin during partial hydrolysis (this faster release of T3 is probably the mechanism behind the more “rapid” secretion of T3 than of T4), or some kind of thyroid heterogeneity leading to pinocytosis and hydrolysis of thyroglobulin with a lower T4T3 ratio than that of average thyroglobulin.

Starvation-induced alterations of circulating thyroid hormone concentrations in man

Thomas J. Merimee, E.S. Fineberg
Metabolism Jan 1976; 25(1): 79-83

Serum concentrations of triiodothyronine (T3), thyroxine (T4), and TSH were examined in seven men and seven women of normal weight during a 60-hr fast. Similar studies were conducted in two women who received daily for 1 mo before and during a similar fast, 0.4 mg and 0.5 mg of l-thyroxine.
The serum concentrations of T3 decreased in each of the untreated normal subjects (sign test of significance, p < 0.001). The mean control concentration of T3 in women was 152 ± 9 ng100 ml (X ± SEM); after 24 hr of fasting, 131 ± 31 ng100 ml; and at the termination of the fast, 90 ± 15 ng100 ml. The latter value differed from the control value with a p value of < 0.01. Similar changes of T3 concentration occurred in men (mean basal T = 160 ± 11 ng100 ml; mean at termination of fast = 87 ± 16 ng100 ml). The range of decrease for T3 in all subjects varied from 24% to 55%.
The mean T4 concentration at the beginning of the fast was 6.9 ± 0.9, and at the termination of the fast, 7.5 ± 0.6 (p = NS). TSH concentrations remained unchanged (Control, 3.8 ± 0.45 μU/ml; at 60 hr, 4.0 ± 0.26 μU/ml, p = NS).
Studies in two women who received, before and during a fast, T4, indicate that a decreased peripheral conversion of T4 to T3 is the most likely mechanism responsible for this change.

Effect of estrogens on thyroid function. II. Alterations in plasma thyroid hormone levels and their metabolism

Ramesh C. Sawhney, Indra Rastogi, Gopal K. Rastogi
Metabolism Mar 1978; 27(3): 279-288

The circulating levels of total triiodothyronine (TT3), thyroxine (TT4, and T4-bbinding globulin (TBG) and the kinetics of T3 and T4 were studied in five menstruating rhesus monkeys before, during, and after prolonged treatment with estradiol monobenzoate (E2B, 50 μg/kg body weight/day subcutaneously). A significant increase over pretreatment (p < 0.01) plasma TT3, TT4, and TBG was recorded on day 6 of E2B therapy. A further significant stepwise increase in these parameters was noted up to day 19 of E2B, when the levels plateaued for the rest of the period of E2B treatment. Two weeks after discontinuation of E2B, plasma TT3, TT4, and TBG had returned to the pretreatment range and remained so up to 40 days of observation. Although the percent free T3 and percent free T4 were significantly decreased (p < 0.01) during E2B therapy, the absolute concentrations of free T3 and free T4 were not altered. After prolonged E2B treatment the metabolic clearance rate, distribution space, and production rate (PR) of both T3 and T4 were decreased (p < 0.01). The extrathyroidal T4 pool (ETT4P) was significantly increased (p < 0.01), whereas ETT3P did not show any significant alterations (p > 0.05). The decreased PR of T4 might have been due to a direct inhibitory effect of E2B on the thyroid, whereas the decrease in PR of T3 might have been due to either decreased conversion of T4 to T3, to decreased secretion by the thyroid, or both.
Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development

Marjolein Heijlen, Anne M. Houbrechts, Veerle M. Darras
Gen Compar Endocr 1 Jul 2013; 188: 289-296

To unravel the role of thyroid hormones (THs) in vertebrate development it is important to have suitable animal models to study the mechanisms regulating TH availability and activity. Zebrafish (Danio rerio), with its rapidly and externally developing transparent embryo has been a widely used model in developmental biology for some time. To date many of the components of the zebrafish thyroid axis have been identified, including the TH transporters MCT8, MCT10 and OATP1C1, the deiodinases D1, D2 and D3, and the receptors TRα and TRβ. Their structure and function closely resemble those of higher vertebrates. Interestingly, due to a whole genome duplication in the early evolution of ray-finned fishes, zebrafish possess two genes for D3 (dio3 and dio3a) and for TRα (thraa and thrab). Transcripts of all identified genes are present during embryonic development and several of them show dynamic spatio-temporal distribution patterns. Transient morpholino-knockdown of D2, D3 or MCT8 expression clearly disturbs embryonic development, confirming the importance of each of these regulators during early life stages. The recently available tools for targeted stable gene knockout will further increase the value of zebrafish to study the role of peripheral TH metabolism in pre- and post-hatch/post-natal vertebrate development.

The consequences of inappropriate treatment because of failure to recognize the syndrome of pituitary and peripheral tissue resistance to thyroid hormone

Samuel Refetoff, Angel Salazar, Terry J. Smith, Neal H. Scherberg
Metabolism Aug 1983; 32(8); 822-834

Since the description of the syndrome of global (peripheral tissues and pituitary) resistance to thyroid hormone, new cases are being recognized with increasing frequency. The patient described herein had a markedly elevated serum TSH concentration of 260 μU/mL at the time of diagnosis. Studies suggest that elevations of serum TSH levels in this and other patients with the syndrome are most likely iatrogenic in origin. The patient was 312 years old when a goiter and a high serum T4 concentration were detected. Despite subtotal thyroidectomy, antithyroid drugs were required to maintain her T4 level in the normal range. She was referred at age 1112 years because of recurrent goiter. Her parents and five older siblings had normal thyroid function. Off therapy, her serum T4 level was 14.9 μg/dL, FT4I was 17.0, T3 was 362 ng/dL, TSH was 260 μU/mL, and antibodies were negative. There were no signs of thyrotoxicosis, her bone age was 7 years, her growth was stunted (third percentile), her intellectual quotient (IQ) was 67, and there was a 30–50 dB sensorineural hearing loss. The presence of a pituitary adenoma was ruled out. Her TSH had normal bioreactivity and rose to 540 μU/mL in response to TRH. Triiodothyronine was given in incremental doses of 50, 100, 200, and 400 μg/d over 28 days. The log concentrations of serum TSH showed an inverse linear correlation with serum T3. While receiving the highest dose of T3, on which the level of serum T3 ranged from 1400 to 2500 ng/dL, the TSH response to TRH normalized (basal 4.2 and peak 20 μU/mL), as did the high levels of serum cholesterol, carotene, and T4. Her BMR rose from +5 to +22%, her IQ rose to 77, and she gained weight without an increase in caloric intake. Only minimal changes were observed in levels of urinary cAMP, hydroxyproline, magnesium, and nitrogen. All values, with the exception of the weight gain, returned to baseline 2 months after T3 treatment was discontinued. The TSH level was suppressed by l-dopa and by prednisone. Long-term therapy with equivalent doses of T4 (from 300 to 1000 μg/d) produced a growth of 3 cm during the initial 6 weeks, 10.5 cm over the ensuring year (above the 10th percentile), and regression of goiter without thyrotoxicosis. The patient exhibited resistance to thyroid hormone in pituitary and peripheral tissues. The optimal dose of T4 replacement could be predicted by studying tissue responses to incremental doses of T3. The marked elevation in serum TSH concentration, stunted growth, and laboratory evidence of hypothyroidism were due to the limited thyroidal reserve caused by thyroidectomy. All patients with an impaired ability to compensate for the defect as a result of inappropriate treatment should be given thyroid hormone in amounts short of producing catabolic effects. Such a dose is expected to normalize the basal serum TSH concentration and its response to TRH.

Solving the mystery of iodine uptake

Valda Vinson
Science 20 Jun 2014; 344(6190), p. 1355
http://dx.doi.org:/10.1126/science.344.6190.1355-a

The thyroid gland produces iodine-containing hormones that regulate metabolism. The cell membrane protein NIS (sodium/iodine symporter) transports iodine into thyroid cells, but because iodine concentrations outside of the cell are so low, how it does so is a mystery. The key? Moving two sodium ions along with the iodine ion, Nicola et al found. NIS also does not bind sodium very tightly, but the high concentrations of sodium outside the cell allow one sodium ion to bind. This binding increases the affinity of NIS for a second sodium ion and also for iodine. With the three ions bound, NIS changes its conformation so that it opens to the inside of the cell, where the sodium concentration is low enough for NIS to release its sodium ions. When the sodium goes away, so does NIS’s affinity for iodine, leading NIS to release it.

Unliganded Thyroid Hormone Receptor α Regulates Developmental Timing via Gene Repression in Xenopus tropicalis

Jinyoung Choi, Ken-ichi T. Suzuki, Tetsushi Sakuma, Leena Shewade, Takashi Yamamoto, and Daniel R. Buchholz
Endocr Feb 2015; 156(2): 735–744 http://dx.doi.org:/10.1210/en.2014-1554

Thyroid hormone (TH) receptor (TR) expression begins early in development in all vertebrates when circulating TH levels are absent or minimal, yet few developmental roles for unliganded TRs have been established. Unliganded TRs are expected to repress TH-response genes, increase tissue responsivity to TH, and regulate the timing of developmental events. Here we examined the role of unliganded TRα in gene repression and development in Xenopus tropicalis. We used transcription activator-like effector nuclease gene disruption technology to generate founder animals with mutations in the TRα gene and bred them to produce F1 offspring with a normal phenotype and a mutant phenotype, characterized by precocious hind limb development. Offspring with a normal phenotype had zero or one disrupted TRα alleles , and tadpoles with the mutant hind limb phenotype had two truncated TRα alleles with frame shift mutations between the two zinc fingers followed by 40–50 mutant amino acids and then an out-of-frame stop codon. We examined TH-response gene expression and early larval development with and without exogenous TH in F1 offspring. As hypothesized, mutant phenotype tadpoles had increased expression of TH-response genes in the absence of TH and impaired induction of these same genes after exogenous TH treatment, compared with normal phenotype animals. Also, mutant hind limb phenotype animals had reduced hind limb and gill responsivity to exogenous TH. Similar results in methimazole-treated tadpoles showed that increased TH-response gene expression and precocious development were not due to early production of TH. These results indicate that unliganded TRα delays developmental progression by repressing TH-response genes.
The discovery of thyroid replacement therapy. Part 2: The critical 19th century
Conceptualizing the link between the thyroid and myxoedema

Stefan Slater
R Soc Med 2011; 104: 59–63. http://dx.doi.org:/10.1258/jrsm.2010.10k051

Sir William Withey Gull (1816–1890)

Frederik Ruysch, anatomist in Leyden around 1690, adopted, according to Albrecht von Haller in 1766, the opinion that a peculiar fluid was elaborated in the gland and poured into the veins’. The 19th century thus began with thyroidology at best in embryo; but during that century endocrinology was born and the thyroid was its standard bearer. In 1836, Thomas Wilkinson King of Guys Hospital, regarded by some as the ‘Father of Endocrinology’, anticipated on the basis of observation and experiment the internal secretion of the thyroid. In a meticulous paper on its anatomy: he wrote of the thyroid gland that ‘its absorbent vessels carry its peculiar secretion to the great veins of the body’. This language is almost identical to that of Ruysch and Haller more than a century earlier. The idea was prompted by the thyroid’s disproportionately large vascular supply in the absence of any evident mechanical or other local function and also at what he described as its ‘peculiar’ fluid. King notes that his view ‘has been indirectly surmised by Morgagni [probably in 1761] and others’.
In 1850, at a meeting of the Royal Medical and Chirurgical Society of London, chaired by Thomas Addison, Thomas Blizzard Curling, surgeon at the London Hospital, provided a clear clinicopathological correlate in a paper entitled ‘Two cases of absence of the thyroid body and symmetrical swellings of fat tissue at the sides of the neck, connected with defective cerebral development’. Postmortem examination in each revealed no trace of thyroid tissue and that the swellings consisted only of fat. Curling’s important observation was not pursued until 1871 when, at another meeting of the Society, Curling himself then in the chair, Charles Hilton Fagge, a physician at Guy’s Hospital, presented a paper on sporadic cretinism. He described four living cases and noted that none of them had a goiter and that one had been well up to the age of eight and, although now physically cretinous at age 16, she remained very intelligent. He referred to Curling’s paper and reached the same conclusion that the ‘healthy thyroid body is capable of exerting a counteracting influence [on cretinism]’.
Two years later, in 1873, Fagge’s senior colleague at Guy’s, Sir William Withey Gull, presented before the Clinical Society of London two of the five cases he had seen of what he called ‘A Cretinoid State supervening in Adult Life in Women’. He described their cretin-like appearance, drawing particular attention to the broad and thick tongue and the guttural voice and its pronunciation ‘as if the tongue were too large for the mouth’. He acknowledged his remarks were tentative, hence, he said, his use of the word ‘cretinoid’, but he had no doubt this was a ‘substantive’ condition and not one of cardiac or renal origin.
Gull was an interesting personality with apparently a remarkable presence, resembling Napoleon in face, form and manner (Figure). In the 1970s, 80 years after his death in 1890, he was the subject of a theory, quickly discredited, that he had been ‘Jack the Ripper’, the killer in the still unsolved murders and mutilations of at least five Whitechapel prostitutes in 1888. He figured in the 1988 TV film series, Jack the Ripper, starring Michael Caine as the detective. Gull is credited with the first description of hypothyroidism in adults and his paper was important in defining a recognizable clinical syndrome.
Then, in 1877, William Miller Ord, read his paper before the Royal Medical and Chirurgical Society of London and proposed the term ‘myxoedema’ for the adult condition. He described the non-pitting, ‘mucous edema’. He also presented an engaging theory to explain the lethargy, inertia and slow responses associated with the disease. He suggested that these might result from the sheathing and insulation of the body in a ‘jelly-like’, mucin-laden integument that interfered with sensory perceptions and stimulation. Six years later, he chaired the committee set up by the Clinical Society of London to investigate the whole matter. He also later undertook some of the earliest metabolic studies of the effects of treating myxoedema with thyroid extract, showing the rapid weight loss and rise in temperature and in urinary volume and nitrogen excretion that occurred.
The key papers, which advanced these English authors observations, were those of the Swiss surgeons, Jaques-Louis Reverdin in Geneva and Emil Theodor Kocher in Bern, Kocher later receiving the Nobel Prize for his work on the thyroid. How fitting it is that it should be two Swiss doctors whose practices unlocked an understanding of the importance of the thyroid. For they each identified the late effects of total ablation (extirpation) of goiters. they

noted the great similarity of Gull’s and Ord’s myxoedema cases with their affected postoperative patients, referring to the comparison as a ‘rapprochement complet’, clearly making the connection. They acknowledged Gull’s primacy in describing the clinical manifestations and Ord’s ‘christening’ the condition ‘myxoedema’, and proposed that surgical cases be known as ‘myxoedème opératoire’. In light of his findings in 1882, Reverdin thereafter sought to conserve a part of the gland during thyroidectomy for goiter, speculating that its complete removal may have been responsible for these late effects. He had noticed that no such problems followed a just unilateral lobectomy. Kocher called the disease picture in his affected cases ‘cachexia strumipriva’ – literally, a bad condition due to the removal of a struma (goiter) without reference to the earlier work of Reverdin. Halsted noted in his monumental review of goiter surgery: ‘It is interesting to follow the argumentation of a mind so exceptionally keen and sane as Kocher’s in its futile efforts to explain insufficiently illuminated phenomena’. In reading Kocher’s 1909 Nobel Prize Lecture (in English translation), one gets the impression that Kocher was aware in 1883 of Gull’s and Ord’s reports, despite not referring to them, and he dismisses Reverdin’s contribution.
There ensued a competition over the contribution to the thyroid discovery. When post-thyroidectomy myxedema wsas brought to the attention of Kocher, he agreed it was analogous to his cases of cachexia strumipriva. It is also obvious that Kocher, like many surgeons of the time, cannot have engaged in routine postoperative outpatient follow-up, for otherwise the ensuing problems in his goiter-operated patients would have been detected years earlier. In respect of this key moment in the history of the thyroid, Reverdin could be said to hold the intellectual property. The thought has been expressed that perhaps he should have shared the 1909 Nobel Prize with Kocher.
The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Adv in Endocr 2014, Article ID 189194, 7 pp http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The Surgical Management of Thyroid Cancer

Sara A. Morrison, Hyunsuk Suh, and Richard A. Hodin
Rambam Maimonides Med J 2014; 5(2):e0008. http://dx.doi.org:/10.5041/RMMJ.10142

There are approximately 63,000 reported cases of thyroid carcinoma annually in the United States, representing roughly 4% of all documented malignancies.1 Diagnosis typically stems from work-up of a thyroid nodule. Data from the Framingham study suggests that palpable thyroid nodules are present in 4% of the US population,2 but non-palpable nodules may exist in up to 67% of the population. Such nodules are often found incidentally secondary to the rising use of imaging modalities in medical settings. The large majority of thyroid nodules are benign, with an overall reported risk of malignancy from 5% to 15%.
Thyroid cancer has been increasing in incidence, with the number of reported cases in the US rising by 25% over the last 3 years. With growing technological advances in the field and improved contributions of diagnostics, surgical decision-making and operative planning have taken on new challenges. Herein, we review the current clinical practice recommendations and active areas of surgical controversy, reflective of the most recently published professional consensus guidelines and a systematic review of the literature.
The use of FNA in current clinical practice has resulted in post-surgical pathology findings of malignancy in over 50% of specimens.7 The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) was developed in order to allow pathologists among varying institutions to communicate results to clinical care-takers with widely under-stood descriptors. Results of FNA biopsies are broken down into the following categories with the corresponding risks of malignancy: non-diagnostic or unsatisfactory (1%–4%), benign (0%–3%), atypia of undetermined significance or follicular lesion of undetermined significance (AUS/FLUS; 5%–15%), follicular neoplasm or suspicious for a follicular neoplasm (FN/sFN; 15%–30%), suspicious for malignancy (60%–75%), and malignant (97%–99%).
Mutational Panels.
AsuragenmiR Inform (Austin, TX, USA) mutation analysis assay and Thyroid Cancer Mutation Panel by Quest Diagnostics (Madison, NJ, USA) are the two main commercially available mutational tests which test for known genetic alterations such as BRAF, RAS, RET/PTC, and PAX8/PPAR. These mutational panels are highly specific for malignancy; however, due to the low overall frequency of these mutations in thyroid cancers, negative results do not rule out cancer. Therefore, mutational panel tests are considered a “rule-in” test. If a preoperative mutational test is positive, the nodule should be considered malignant, and total thyroidectomy should be recommended.
Gene Expression Profiling.
The most widely known gene expression profiling test is Afirma Gene Expression Classifier (Veracyte, San Francisco, CA, USA), and, with its recent clinical validation by Alexander et al., Afirma is already being utilized in many clinical settings. The Afirma Gene Expression Classifier (GEC) is an RNA-based assay that utilizes FNA samples to evaluate 167 molecular genes associated with benign nodules based on their proprietary algorithm. Unlike the mutational panel testing, Afirma testing is considered a “rule-out” test since the test has a high negative predictive value in distinguishing benign nodules. However, a positive result reported as “suspicious” carries only 38% risk of malignancy.
In all, these molecular tests should be utilized judiciously and should be considered as a complementary diagnostic tool in the management of thyroid nodules. In the future, molecular testing could become more cost-effective and accurate as a diagnostic tool while providing prognostic and therapeutic information.
Papillary Thyroid Cancer.
Total thyroidectomy is the gold standard for patients with a preoperative diagnosis of papillary thyroid cancer when the nodule is greater than 1 cm in size. Completion thyroidectomy is indicated in patients who have undergone prior lobectomy and are found on final pathology to have papillary thyroid cancer that is larger than 1 cm. The completion thyroidectomy should generally be performed within 6 months of the original procedure in order to minimize the risk of lymph node metastasis.
Involvement of cervical lymph nodes in papillary thyroid cancer is frequent, reported to occur in up to 50% of patients. The role of neck dissection at the time of total thyroidectomy is somewhat controversial, however, since most of the nodal involvement is microscopic and does not affect overall survival. It is generally agreed upon that a therapeutic neck dissection should be pursued in the setting of well-differentiated thyroid cancer patients with clinically positive lymph nodes, whether in the central or lateral neck compartments. Prophylactic neck dissection is not done for follicular thyroid cancer, as the rates of lymph node metastasis are typically less than 10%.
Medullary thyroid cancer (MTC) comprises 4% of all thyroid malignancies. The majority of cases are sporadic in nature; approximately 20%–25% represent familiar/hereditary syndromes. Diagnosis is commonly made by FNA biopsy with specific staining for the presence of calcitonin in the tissue specimen. All patients with a diagnosis of medullary thyroid cancer must be evaluated for multiple endocrine neoplasia (MEN) 2 and be ruled out for the synchronous presence of pheochromocytoma prior to scheduling thyroid surgery.
Effects of Dose Level of Anti-thyroid Drug Carbimazole on Thermoregulation and Blood Constituents in Male Rabbits (Oryctolagus cuniculus)

Intisar H. Saeed, Abdalla M. Abdelatif and Mohamed E. Elnageeb
Adv in Research 2014; 2(3): 129-144. Article no. AIR.2014.002

Carbimazole (CBZ) is an anti-thyroid drug commonly used in the treatment of hyperthyroidism. The objective of this study was to evaluate the effects of dose level of CBZ on thermoregulation and blood constituents in mature male rabbits. Twenty animals were assigned to 4 groups (A, B, C, D) of 5 each. Group A served as control and treated animals in groups B,C,D, received daily orally CBZ doses of 10, 15 and 20 mg/animal for 3 weeks, respectively.
The values of rectal temperature (Tr,), respiration rate (RR) and heart rate (HR) decreased in treated rabbits and the mean values of HR decreased with increase in the dose level of CBZ. The packed cell volume (PCV), Hb concentration and total leukocyte count (TLC) were lower in CBZ treated rabbits. Serum levels of total protein and globulins increased and serum albumin level decreased in treated groups of rabbits. Serum urea level was lower in CBZ treated groups and there was an increase in serum urea level with increase in CBZ dose level. Serum cholesterol level was higher in treated groups and there was an increase in serum cholesterol level with increase in CBZ dose level. Plasma glucose level decreased significantly in CBZ treated groups compared with the control and the mean values decreased with increase in the dose level of CBZ. The results indicate that the responses of basic physiological parameters were almost dose dependent in the range adopted in this study.
Phosphatase Inhibitor Calyculin A Activates TRPC2 Channels in Thyroid FRTL-5 Cells

Pramod Sukumaran, MY Asghar, C Löf, T Viitanen, and Kid Törnquist
Calcium Signaling Jun 2014; 1(2) http://www.researchpub.org/journal/cs/cs.html

We have previously shown that rat thyroid FRTL-5 cells express a calcium entry pathway regulated by a phosphatase. The nature of the calcium entry pathway is presently unknown. We have also shown that FRTL-5 cells express only the TRPC2 channel of the TRPC family of cation channels. In the present investigation we show, using pharmacological inhibitors, the measurement of sodium and calcium entry, stable TRPC2 knock-down cells, and transfection with a non-conducting form of TRPC2, that the calcium entry pathway regulated by a phosphatase is, in fact, the TRPC2 channel. Our data thus point to a novel mechanism by which the TRPC2 channels can be regulated.

Thyroxine Uptake by Perfused Rat Liver
No Evidence for Facilitation by Five Different Thyroxine-binding Proteins

Carl M. Mendel and Richard A. Weisiger
J. Clin. Invest. 1990; 86: 1840-1847

For each of the five protein-hormone complexes studied, the rate of hepatic uptake of T4 (measured under conditions expected to result in dissociation-limited uptake) closely approximated the rate of spontaneous dissociation of the protein-hormone complex within the hepatic sinusoids. These findings indicate an absence of special cellular mechanisms that facilitate the hepatic uptake of T4 from its plasma binding proteins, and support the view that uptake occurs from the free T4 pool after spontaneous dissociation of T4 from its binding proteins.
Thyroxine Transport and Distribution in Nagase Analbuminemic Rats

Carl M. Mendel, RR Cavalieri, LA Gavin, T Pettersson, and M Inoue
J. Clin. Invest. 1989; 83: 143-148

The postulate that thyroxine (T4) in plasma enters tissues by protein-mediated transport or enhanced dissociation from plasma-binding proteins leads to the conclusion that almost all T4 uptake by tissues in the rat occurs via the pool of albumin bound T4 (Pardridge, W. M., B. N. Premachandra, and G. Fierer. 1985. Am. J. Physiol. 248:G545-G550).
To directly test this postulate, and to test more generally whether albumin might play a special role in T4 transport in the rat, we performed in vivo kinetics studies in six Nagase analbuminemic rats and in six control rats, all of whom had similar serum T4 concentrations and percent free T4 values.
Evaluation of the plasma disappearance curves of simultaneously injected ¹²⁵I-T4 and ^I31I-albumin indicated that the flux of T4 from the extracellular compartment into the rapidly exchangeable intracellular compartment was similar in the analbuminemic rats (51±21 ng/min, mean±SD) and in the control rats (54±15 ng/min), as was the size of the rapidly exchangeable intracellular pool of T4 (1.13±0.53 vs. 1.22±036 Mg). This latter finding was confirmed by direct analysis of tissue samples (liver, kidney, and brain). We also performed in vitro kinetics studies using the isolated perfused rat liver. The single-pass fractional extraction by normal rat liver of T4 in pooled analbuminemic rat serum was indistinguishable from that of T4 in pooled control rat serum (10.9±3.3%, n = 3, vs. 11.4±3.4%). When > 98% of the albumin was removed from normal rat serum by chromatography with Affi-Gel blue, the single-pass fractional extraction of T4 (measured by a bolus injection method) did not change (16.3±2.1%, n = 5, vs. 15.2±2.5%). These data provide the first valid experimental test of the enhanced

dissociation hypothesis and indicate that there is no special, substantive role for albumin in T4 transport in the rat.
Influence of thyroid receptors on breast cancer cell proliferation

Conde, R. Paniagua, J. Zamora, M. J. Blanquez, B. Fraile, A. Ruiz & M. I. Arenas
Ann Oncol 2005; http://dx.doi.org:/10.1093/annonc/mdj040

Background: The involvement of thyroid hormones in the development and differentiation of normal breast tissue has been established. However, the association between breast cancer and these hormones is controversial. Therefore, the objective of the present study was to determine the protein expression pattern of thyroid hormone receptors in different human breast pathologies and to evaluate their possible relationship with cellular proliferation.
Patients and methods: The presence of thyroid hormone receptors was evaluated by immunohistochemistry and western blot analysis in 84 breast samples that included 12 cases of benign proliferative diseases, 20 carcinomas in situ and 52 infiltrative carcinomas.
Results: TR-α was detected in the nuclei of epithelial cells from normal breast ducts and acini, while in any pathological type this receptor was located in the cytoplasm. However, TR-b presented a nuclear location in benign proliferative diseases and carcinomas in situ and a cytoplasmatic location in normal breast and infiltrative carcinomas. The highest proliferation index was observed in carcinomas in situ, although in infiltrative carcinomas an inverse correlation between this index and the TR-α expression was encountered.
Conclusions: The results of this study reveal substantial changes in the expression profile of thyroid hormone.
Zebrafish as a model for monocarboxyl transporter 8-deficiency

GD Vatine, D Zada, T Lerer-Goldshtein, A Tovin, G Malkinson, K Yaniv and L Appelbaum
J Biol Chem Nov 2012; Manuscript M112.413831
http://dx.doi.org:/10.1074/jbc.M112.413831

Background: Mutations in the thyroid hormone transporter MCT8 are associated with psychomotor retardation AHDS.
Results: In zebrafish, as in humans, mct8 is expressed primarily in the nervous system. Elimination of MCT8 causes severe neural impairment.
Conclusion: MCT8 is a crucial regulator during zebrafish embryonic development. Significance: Establishment of the first vertebrate model for MCT8-deficiency, which exhibits a neurological phenotype.
Unusual Ratio between Free Thyroxine and Free Triiodothyronine in a Long-Lived Mole-Rat Species with Bimodal Ageing

Yoshiyuki Henning, Christiane Vole, Sabine Begall, Martin Bens, et al.
PlusOne Nov 2014; 9(11),e113698. http://dx.doi.org:/10.1371/journal.pone.0113698

Ansell’s mole-rats (Fukomys anselli) are subterranean, long-lived rodents, which live in eusocial families, where the maximum lifespan of breeders is twice as long as that of non-breeders. Their metabolic rate is significantly lower than expected based on allometry, and their retinae show a high density of S-cone opsins. Both features may indicate naturally low thyroid hormone levels.
In the present study, we sequenced several major components of the thyroid hormone pathways and analyzed free and total thyroxine and triiodothyronine in serum samples of breeding and non-breeding F. anselli to examine whether
a) their thyroid hormone system shows any peculiarities on the genetic level,
b) these animals have lower hormone levels compared to euthyroid rodents (rats and guinea pigs), and
c) reproductive status, lifespan and free hormone levels are correlated.
Genetic analyses confirmed that Ansell’s mole-rats have a conserved thyroid hormone system as known from other mammalian species. Interspecific comparisons revealed that free thyroxine levels of F. anselli were about ten times lower than of guinea pigs and rats, whereas the free triiodothyronine levels, the main biologically active form, did not differ significantly amongst species. The resulting fT4:fT3 ratio is unusual for a mammal and potentially represents a case of natural hypothyroxinemia.
Comparisons with total thyroxine levels suggest that mole-rats seem to possess two distinct mechanisms that work hand in hand to downregulate fT4 levels reliably. We could not find any correlation between free hormone levels and reproductive status, gender or weight. Free thyroxine may slightly increase with age, based on subsignificant evidence. Hence, thyroid hormones do not seem to explain the different ageing rates of breeders and nonbreeders. Further research is required to investigate the regulatory mechanisms responsible for the unusual proportion of free thyroxine and free triiodothyronine.
Transthyretin Regulates Thyroid Hormone Levels in the Choroid Plexus, But Not in the Brain Parenchyma: Study in a Transthyretin-Null Mouse Model

JA Palha, R Fernandes, GM De Escobar, V Episkopou, M Gottesman, and MJ Saraiva
Endocr 2000; 141(9): 3267–3272.

Transthyretin (TTR) is the major T4-binding protein in rodents. Using a TTR-null mouse model we asked the following questions.
1) Do other T4 binding moieties replace TTR in the cerebrospinal fluid (CSF)?
2) Are the low whole brain total T4 levels found in this mouse model associated with hypothyroidism, e.g. increased 59-deiodinase type 2 (D2) activity and RC3-neurogranin messenger RNA levels?
3) Which brain regions account for the decreased total whole brain T4 levels?
4) Are there changes in T3 levels in the brain?
Our results show the following.
1) No other T4-binding protein replaces TTR in the CSF of the TTR-null mice.
2) D2 activity is normal in the cortex, cerebellum, and hippocampus, and total brain RC3-neurogranin messenger RNA levels are not altered.
3) T4 levels measured in the cortex, cerebellum, and hippocampus are normal. However T4 and T3 levels in the choroid plexus are only 14% and 48% of the normal values, respectively.
4) T3 levels are normal in the brain parenchyma.
The data presented here suggest that TTR influences thyroid hormone levels in the choroid plexus, but not in the brain. Interference with the blood-choroid-plexus-CSF-TTR-mediated route of T4 entry into the brain caused by the absence of TTR does not produce measurable features of hypothyroidism. It thus appears that TTR is not required for T4 entry or for maintenance of the euthyroid state in the mouse brain.
Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

E.C.H. Friesema, S Ganguly, A. Abdalla, J.E.M. Fox, AP. Halestrap, and TJ. Visser
J Biol Chem 2003; Manuscript M300909200
http://dx.doi.org/10.1074/jbc.M300909200

Transport of thyroid hormone across the cell membrane is required for its action and

metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family, and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8, and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced a ~10-fold increase in uptake of 10 nM ¹²⁵I-labeled thyroxine (T4), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine. Due to the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp or Tyr. [¹²⁵I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3’,5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 μM for T4, T3 and rT3. Immunohistochemistry showed high expression in liver, kidney, brain and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.
Thyroid hormones,T3 andT4, in the brain
Amy C. Schroeder and Martin L. Privalsky
Front Endocr Mar 2014; 5 article 40. http://dx.doi.org:/10.3389/fendo.2014.00040

Thyroid hormones (THs) are essential for fetal and post-natal nervous system development and also play an important role in the maintenance of adult brain function. Of the two major THs, T4 (3,5,30,50-tetraiodo-l-thyronine) is classically viewed as an pro-hormone that must be converted toT3 (3,5,30-tri-iodo-l-thyronine) via tissue-level deiodinases for biological activity. THs primarily mediate their effects by binding to thyroid hormone receptor (TR) isoforms, predominantly TRα1 and TRβ1, which are expressed in different tissues and exhibit distinctive roles in endocrinology. Notably, the ability to respond toT4 and toT3 differs for the two TR isoforms, with TRα1 generally more responsive to T4 than TRβ1. TRα1 is also the most abundantly expressed TR isoform in the brain, encompassing 70–80% of all TR expression in this tissue. Conversion of T4 into T3 via deiodinase 2 in astrocytes has been classically viewed as critical for generating local T3 for neurons. However, deiodinase-deficient mice do not exhibit obvious defectives in brain development or function. Considering that TRα1 is well-established as the predominant isoform in brain, and that TRα1 responds to both T3 and T4, we suggest T4 may play a more active role in brain physiology than has been previously accepted.
Thyroid hormone action: astrocyte–neuron communication

Beatriz Morte and Juan Bernal
Front Endocr May 2014; 5, Article 82 http://dx.doi.org:/10.3389/fendo.2014.00082

Thyroid hormone (TH) action is exerted mainly through regulation of gene expression by binding of T3 to the nuclear receptors.T4 plays an important role as a source of intracellular T3 in the central nervous system via the action of the type 2 deiodinase (D2), expressed in the astrocytes. A model of T3 availability to neural cells has been proposed and validated. The model contemplates that brain T3 has a double origin: a fraction is available directly from the circulation, and another is produced locally from T4 in the astrocytes by D2. The fetal brain depends almost entirely on theT3 generated locally. The contribution of systemic T3 increases subsequently during development to account for approximately 50% of total brain T3 in the late postnatal and adult stages. In this article, we review the experimental data in support of this model, and how the factors affectingT3 availability in the brain, such as deiodinases and transporters, play a decisive role in modulating local TH action during development.
The Significance of Thyroid Hormone Transporters in the Brain

Juan Bernal
Endocr Apr 2005; 146(4):1698–1700. http://dx.doi.org:/10.1210/en.2005-0134

The MCT family comprises up to 14 members, some of which are involved in the transport of important substrates for the brain such as lactate and pyruvate. MCT8 has been shown to act as a specific transporter for T4 and T3 and displays slightly higher affinity for T3. Heuer et al. have also studied the regional expression of MCT8 mRNA. In addition to high expression levels in the choroid plexus, they found that MCT8 is expressed in neurons of the neocortex, hippocampus, basal ganglia, amygdala, hypothalamus, and the Purkinje cells of the cerebellum, all regions known to be sensitive to thyroid hormones. Expression of MCT8 in neurons suggests that neuronal uptake of the T3 produced in astrocytes is facilitated by this transporter.
The physiological significance ofMCT8 as a transporter for thyroid hormone is supported by the finding of mutations in humans by Dumitrescu et al. and Friesema et al. The syndrome affects children from an early age and consists of severe developmental delay and neurological damage together with an unusually altered pattern of thyroid hormone levels in blood. The patients presented low total and free T4, high total and free T3, and low rT3. TSH was moderately elevated in two of the patients and normal or slightly elevated in the other five. Inactivating mutations of the MCT8 transporter could result in the altered thyroid hormone levels. In vitro uptake of T4 and T3 by fibroblasts isolated from affected males was strongly reduced, and intracellular D2 was increased 6- to 8-fold. It is thus hypothesized that the resulting increase in intracellularly generated T3 accumulates in blood because of its poor reuptake into cells.
The second trimester is also the period when thyroid hormone receptors increase in concentration in the brain. If MCT8 is needed at this stage of development for T3 entry into neurons, mutations of the transporter could interfere with T3-dependent developmental processes. Knowledge of the ontogenetic patterns of MCT8 in the human fetal brain would certainly be helpful. On the other hand, there is also the possibility that MCT8 mutations interfere with transport of other substrates for brain metabolism that could be even more important than T3 in determining the severity and outcome of the syndrome. Other members of the family transport metabolic substrates such as pyruvate and lactate, but MCT8 so far appears to be specific for iodothyronines

Peripheral markers of thyroid function: The effect of T4 monotherapy versus T4/T3 combination therapy in hypothyroid subjects: A randomized cross-over study

Ulla Schmidt, B Nygaard, EW Jensen, J Kvetny, A Jarløv, and Jens Faber
Endocrine Connections Jan 10, 2013 http://dx.doi.org:/10.1530/EC-12-0

Background: A recent randomized controlled trial suggests that hypothyroid subjects may find L-T4 and L-T3 combination therapy to be

superior to L-T4 monotherapy in terms of quality of life, suggesting that the brain registered increased T3 availability during the

combination therapy.

Hypothesis: Peripheral tissue might also be stimulated during T4/T3 combination therapy compared to T4 monotherapy.
Methods: Serum levels of Sex Hormone-Binding Globulin (SHBG), pro-collagen-1-N-terminal peptide (PINP), and N-terminal pro-brain natriuretic peptide (NT-proBNP) (representing hepatocyte, osteoblast, and cardiomyocyte stimulation, respectively) were measured in 26 hypothyroid subjects in a double blind, randomized, cross-over trial, which compared the replacement therapy with T4/T3 in combination (50 Fg T4 was substituted with 20 Fg T3) to T4 alone (once daily regimens). This was performed to obtain unaltered serum thyroid stimulating hormone (TSH) levels during the trial and between the two treatment groups. Blood sampling was performed 24 hours after the last intake of thyroid hormone medication.
Results: TSH remained unaltered between the groups ((median) 0.83 vs. 1.18 mU/l in T4/T3 combination and T4 mono-therapy, respectively; p=0.534). SHBG increased from (median) 75 nmol/l at baseline to 83 nmol/l in the T4/T3 group (p=0.015), but remained unaltered in the T4 group (67 nmol/l); thus, it was higher in the T4/T3 vs. T4 group (p=0.041). PINP levels were higher in the T4/T3 therapy (48 vs. 40 Fg/l (p<0.001)). NT-proBNP did not differ between the groups. Conclusions: T4/T3 combination therapy in hypothyroidism seems to have more metabolic effects than the T4 monotherapy.
Stimulatory effects of thyroid hormone on brain angiogenesis in vivo and in vitro

Liqun Zhang, CM Cooper-Kuhn, U Nannmark, K Blomgren and HG Kuhn
J Cereb Blood Flow & Metab 2010; 30:323–335. http://dx.doi.org:/10.1038/jcbfm.2009.216

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day 21 (P21) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial growth factor A (Vegfa) and basic fibroblast growth factor (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfα upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing factor to the neurologic deficits induced by thyroid hormone deficiency.

Molecules important for thyroid hormone

synthesis and action – known facts and future perspectives

Klaudia Brix, Dagmar Führer, Heike Biebermann
Thyroid Research 2011, 4(Suppl 1):S9 http://www.thyroidresearchjournal.com/content/4/S1/S9

Thyroid hormones are of crucial importance for the functioning of nearly every organ. Remarkably, disturbances of thyroid hormone synthesis and function are among the most common endocrine disorders affecting approximately one third of the working German population. Over the last ten years our understanding of biosynthesis and functioning of these hormones has increased tremendously. This includes the identification of proteins involved in thyroid hormone biosynthesis like Thox2 and Dehal where mutations in these genes are responsible for certain degrees of hypothyroidism. One of the most important findings was the identification of a specific transporter for triiodothyronine (T3), the monocarboxylate transporter 8 (MCT8) responsible for directed transport of T3 into target cells and for export of thyroid hormones out of thyroid epithelial cells. Genetic disturbances of MCT8 in patients result in a biochemical constellation of high T3 levels in combination with low or normal TSH and thyroxine levels leading to a new syndrome of severe X-linked mental retardation. Importantly mice lacking MCT8 presented only with a mild phenotype, indicating that compensatory mechanisms exist in mice. Moreover, it has become clear that not only genomic actions of T3 exist. T3 is also capable to activate adhesion receptors and it signals via activation of PI3K and MAPK pathways. Most recently, thyroid hormone derivatives were identified, the thyronamines which are decarboxylated thyroid hormones initiating physiological actions like lowering body temperature and heart rate, thereby acting in opposite direction to the classical thyroid hormones. So far it is believed that thyronamines function via the activation of a G-protein coupled receptor, TAAR1. The objective of this review is to summarize the recent findings in thyroid hormone synthesis and action and to discuss their implications for diagnosis of thyroid disease and for treatment of patients.

Retinoic Acid Induces Expression of the Thyroid Hormone Transporter, Monocarboxylate Transporter 8 (Mct8)

T Kogai, Yan-Yun Liu, LL Richter, K Mody, H Kagechika, and GA Brent
J Biol Chem Jun 2010. Manuscript M110.123158
http://www.jbc.org/cgi/doi/10.1074/jbc.M110.123158

Retinoic acid (RA) and thyroid hormone are critical for differentiation and organogenesis in the embryo. The monocarboxylate transporter-8 (Mct8), expressed predominantly in brain and placenta, mediates thyroid hormone uptake from the circulation and is required for normal neural development. RA induces differentiation of F9 mouse teratocarcinoma cells towards neurons as well as extraembryonal endoderm. We hypothesized that Mct8 is functionally expressed in F9 cells and induced by RA. All trans RA (tRA), and other RA receptor (RAR) agonists, dramatically (> 300-fold) induced Mct8. tRA treatment significantly increased uptake of triiodothyronine and thyroxine (4.1 fold and 4.3 fold, respectively), which was abolished by a selective Mct8 inhibitor, bromosulfophthalein. Sequence inspection of the Mct8 promoter region and
5′-rapid amplification of cDNA ends (5’-RACE) PCR analysis in F9 cells identified
11 transcription start sites and a proximal Sp1 site, but no TATA-box. tRA significantly enhanced Mct8 promoter activity through a consensus RA responsive element located 6.6 kilobases upstream of the coding region. Chromatin immunoprecipitation assay demonstrated binding of RAR and retinoid-X receptor (RXR) to the RA response element. The promotion of thyroid hormone uptake through the transcriptional up-regulation of Mct8 by RAR is likely to be important for extraembryonic endoderm development and neural differentiation. This finding demonstrates crosstalk between RA signaling and thyroid hormone signaling in early development at the level of the thyroid hormone transporter.
Abnormal thyroid hormone metabolism in mice lacking the monocarboxylate transporter 8

Marija Trajkovic, Theo J. Visser, Jens Mittag, Sigrun Horn, et al.
J. Clin. Invest. 2007; 117:627–635. http://dx.doi.org:/10.1172/JCI28253

In humans, inactivating mutations in the gene of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8; SLC16A2) lead to severe forms of psychomotor retardation combined with imbalanced thyroid hormone serum levels. The MCT8-null mice described here, however, developed without overt deficits but also exhibited distorted 3,5,3′-triiodothyronine (T3) and thyroxine (T4) serum levels, resulting in increased hepatic activity of type 1 deiodinase (D1). In the mutants’ brains, entry of T4 was not affected, but uptake of T3 was diminished. Moreover, the T4 and T3 content in the brain of MCT8-null mice was decreased, the activity of D2 was increased, and D3 activity was decreased, indicating the hypothyroid state of this tissue. In the CNS, analysis of T3 target genes revealed that in the mutants, the neuronal T3 uptake was impaired in an area-specific manner, with strongly elevated thyrotropin-releasing hormone transcript levels in the hypothalamic paraventricular nucleus and slightly decreased RC3 mRNA expression in striatal neurons; however, cerebellar Purkinje cells appeared unaffected, since they did not exhibit dendritic outgrowth defects and responded normally to T3 treatment in vitro.
In conclusion, the circulating thyroid hormone levels of MCT8-null mice closely resemble those of humans with MCT8 mutations, yet in the mice, CNS development is only partially affected.
3-Monoiodothyronamine: the rationale for its action as an endogenous adrenergic-blocking neuromodulator

HS Gompf, JH Greenberg, G Aston-Jones, A Ianculescu, TS Scanlan, and MB Dratman
Brain Res. 2010 Sep 10; 1351: 130–140. http://dx.doi.org:/10.1016/j.brainres.2010.06.067

The investigations reported here were designed to gain insights into the role of
3-monoiodothyronamine (T1AM) in the brain, where the amine was originally identified and characterized.
Extensive deiodinase studies indicated that T1AM was derived from the T4 metabolite, reverse triiodothyronine (revT3), while functional studies provided well-confirmed evidence that T1AM has strong adrenergic blocking effects. Because a state of adrenergic overactivity prevails when triiodothyronine (T3) concentrations becomes excessive, the possibility that T3’s metabolic partner, revT3, might give rise to an antagonist of those T3 actions was thought to be reasonable.
All T1AM studies thus far have required use of pharmacological doses.
Therefore we considered that choosing a physiological site of action was a priority and focused on the locus coeruleus (LC), the major noradrenergic control center in the brain. Site-directed injections of T1AM into the LC elicited a significant, dose-dependent neuronal firing rate change in a subset of adrenergic neurons with an EC50=2.7 μM, a dose well within the physiological range. Further evidence for its physiological actions came from autoradiographic images obtained following intravenous carrier-free 125I-labeled T1AM injection. These showed that the amine bound with high affinity to the LC and to other selected brain nuclei, each of which is both an LC target and a known T3 binding site. This new evidence points to a physiological role for T1AM as an endogenous adrenergic-blocking neuromodulator in the central noradrenergic system.

Thyroid hormones are transported through the blood-brain barrier

Thyroid hormones are transported through the blood-brain barrier (OATP) or the blood-CSF barrier (OATP and MCT8). In the astrocytes and tanycytes T4 is converted to T3 which then enters the neurons through MCT8. In the neurons both T4 and T3 are degraded by D3. T3 from the tanycytes may reach the portal vessels in the median eminence. Other transporters may be present on the astrocyte or tanycyte membranes. In most cases the transport could be bidirectional, although only one direction is shown.
Juan Bernal – Instituto de Investigaciones Biomedicas – 28029 Madrid, Spain

the interactions of maternal, placental and fetal thyroid

Old and new concepts of thyroid hormone action.

A: Old concept of thyroid hormone action. In former times it was assumed that thyroid hormones are able to pass the plasma membrane by passive transport. Once in the cytosol T4 is deiodinated to T3 which exerts genomic effects by binding to the thyroid hormone receptor (TR). After hetero-dimerization with other nuclear receptors like retinoic X receptor (RXR), transcriptional regulation is initiated resulting in activation or inactivation of target genes.
B: New concepts of thyroid hormone action. Thyroid hormones enter a target cell via specific transporters, e.g. T3 uses the monocarboxylate transporter MCT8 while T4 entry is mediated by Lat2 or Oatp14. Moreover, T3 can interact with avb3 integrins to induce ERK1/2 signalling. Cytosolic T3 exerts genomic effects but can additionally also act by non-genomic means after TR binding and activation of down-stream PI-3 kinase. Likewise, the naturally occurring iodothyronine T2 is believed to stimulate metabolic rates via mitochondrial pathways, thereby bypassing genomic regulation. Besides thyroid hormones, derivatives like the thyronamines T1AM or T0AM, modulate the action of T3, e.g. counter-acting its effects in certain target cells. Thyronamines (TAMs) bind to and activate G-protein coupled receptors (GPCRs) of the trace amine associated receptor (TAAR) family. So far, it is only known that TAAR1 is activated by TAMs and signals via adenylylcyclase (AC) activation with subsequent rise of cAMP levels. However other GPCRs are likely targets for thyroid hormone derivatives

Brix et al.: Molecules important for thyroid hormone synthesis and action – known facts and future perspectives. Thyroid Research 2011 4(Suppl 1):S9.
http://dx.doi.org:/10.1186/1756-6614-4-S1-S9

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Proteomics

Writer and Curator: Larry H. Bernstein, MD, FCAP

The previous discussion concerned genomics, metabolomics, and cancer. The discussion that follows is concerned with the expanding filed of proteomics, which has implication for disease discovery, pharmaceutical targeting, and diagnostics.

The human proteome – a scientific opportunity for transforming diagnostics, therapeutics, and healthcare

Marc Vidal, Daniel W Chan, Mark Gerstein, Matthias Mann, Gilbert S Omenn, et al.
Clinical Proteomics 2012, 9:6 http://www.clinicalproteomicsjournal.com/content/9/1/6

A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26–27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

Proteomics Pioneer Award 2013: Professor Amos Bairoch, University of Geneva, Switzerland

Eupa Open Proteomics 2 (2014) 34 http://dx.doi.org/10.1016/j.euprot.2013.12.002

Amos Bairoch has always been fascinated by computer science, genetics and biochemistry. His fi rst project, as a PhD student, was the development of PC/Gene, a MS-DOS based software package for the analysis of protein and nucleotide sequences. While working on this project, he realized that there was no single resource for protein sequences, and started to develop the first annotated protein sequence database, which became Swiss-Prot and was first released in July 1986. In 1988, he created PROSITE, a database of protein families and domains, and a little later ENZYME, an enzyme nomenclature database.

Amos Bairoch led the Swiss-Prot group from its creation in 1988 until 2009. During this period, Swiss-Prot became the primary protein sequence resource in the world and has been a key research instrument for both bioinformaticians and laboratory-based scientists, particularly in the field of proteomics.

Since 2009, Amos Bairoch’s group is developing neXtProt, a knowledgebase
specifically dedicated to human proteins.neXtProt has been chosen as the reference protein database for the HUPO Human Proteome Projects.

For his major contributions in the field of proteomic databases, Amos Bairoch received the Friedrich Miescher Award from the Swiss Society of Biochemistry in 1993, the Helmut Horten Foundation Incentive Award in 1995, the Pehr Edman award and the European Latsis Prize in 2004, the Otto Naegeli prize in 2010, and the HUPO Distinguished Achievement Award in Proteomic Sciences in 2011.

National Heart, Lung, and Blood Institute Clinical Proteomics Working Group Report

CB Granger, JE Van Eyk, SC Mockrin and N. Leigh Anderson
Circulation. 2004;109:1697-1703
http://dx.doi.org:/10.1161/01.CIR.0000121563.47232.2A

The National Heart, Lung, and Blood Institute (NHLBI) Clinical Proteomics Working Group was charged with identifying opportunities and challenges in clinical proteomics and using these as a basis for recommendations aimed at directly improving patient care. The group included representatives of clinical and translational research, proteomic technologies, laboratory medicine, bioinformatics, and 2 of the NHLBI Proteomics Centers, which form part of a program focused on innovative technology development. This report represents the results from a one-and-a-half-day meeting on May 8 and 9, 2003. For the purposes of this report, clinical proteomics is defined as the systematic, comprehensive, large-scale identification of protein patterns (“fingerprints”) of disease and the application of this knowledge to improve patient care and public health through better assessment of disease susceptibility, prevention of disease, selection of therapy for the individual, and monitoring of treatment response.

The -omics era: Proteomics and lipidomics in vascular research

Athanasios Didangelos, Christin Stegemann, Manuel Mayr
Atherosclerosis 221 (2012) 12– 17
http://dx.doi.org:/10.1016/j.atherosclerosis.2011.09.043

The retention of proatherogenic low-density lipoprotein (LDL) particles on the subendothelial extracellular matrix (ECM) is a hallmark of atherosclerosis. Apolipoprotein B (apoB)-containing lipoprotein particles are trapped in the arterial intima by proteoglycans in atherosclerosis-prone areas and eventually become modified, commonly by aggregation and oxidation. The initial accumulation of proatherogenic lipoproteins initiates an inflammatory response, which results in the release of proteolytic enzymes and induces the dedifferentiation of vascular smooth muscle cells (SMCs) resulting in alterations of their matrix producing properties. The precise mechanisms responsible for the accumulation of certain matrix components and subsequent lipoprotein retention on the vessel wall are not fully elucidated. Undoubtedly, ECM remodeling contributes to the formation of atherosclerotic lesions and the lipid composition of apolipoproteins influences their binding properties to the matrix. An unbiased discovery approach, which is not limited to known molecules of presumed importance, will be invaluable for the identification of novel, previously unknown mediators of disease. Although descriptive, the detailed examination of atherosclerotic plaques using advanced proteomics and lipidomics techniques can generate novel insights and form the basis for further mechanistic investigations.

The Revolution in Proteomics Ionization –
CaptiveSpray nanoBooster™
Bruker, LC-MS Source

Bruker CaptiveSpray principle:

Stable and robust nanoflow LC/MS is still a challenge in proteomics analysis. The Bruker CaptiveSpray source is a revolutionary ion source with a patented design that provides provides easy operation just as simple normal flow electrospray.

CaptiveSpray delivers nanospray sensitivity, resists plugging, and provides reproducible uninterrupted flow for even the most complex proteomics samples.

CaptiveSpray nanoBooster brings your MS to the next performance level and provides even higher flexibility.

Boost nanoflow sensitivity
• Push up ID rates
• Enabling Glycoanalysis
• Supercharging capability

CaptiveSpray provides a vortex gas that sweeps around the emitter spray tip to desolvate and to focus the Taylor cone into the MS inlet capillary. The vacuum seal to the MS ion guide draws all of the sample ions into the MS increasing the efficiency of sample transfer from the spray tip into the mass spectrometer. The direct connection to the inlet capillary eliminates the need for any source adjustment making the CaptiveSpray source truly Plug-and-Play.

CaptiveSpray Illustration

Structure elucidation

Tissue Proteomics for the Next Decade? Towards a Molecular Dimension in Histology

R Longuespee, M Fleron, C Pottier, F Quesada-Calvo, Marie-Alice Meuwis, et al.
OMICS A Journal of Integrative Biology 2014; 18(9)
http://dx.doi.org:/10.1089/omi.2014.0033

Currently, sampling methods, biochemical procedures, and MS instrumentations allow scientists to perform ‘‘in depth’’ analysis of the protein content of any type of tissue of interest. This article reviews the salient issues in proteomics analysis of tissues. We first outline technical and analytical considerations for sampling and biochemical processing of tissues and subsequently the instrumental possibilities for proteomics analysis such as shotgun proteomics in an anatomical context. Specific attention concerns formalin fixed and paraffin embedded (FFPE) tissues that are potential ‘‘gold mines’’ for histopathological investigations. In all, the matrix assisted laser desorption/ionization (MALDI) MS imaging, which allows for differential mapping of hundreds of compounds on a tissue section, is currently the most striking evidence of linkage and transition between ‘‘classical’’ and ‘‘molecular’’ histology. Tissue proteomics represents a veritable field of research and investment activity for modern biomarker discovery and development for the next decade.

A transcriptome-proteome integrated network identifies ERp57 as a hub that mediates bone metastasis

N Santana-Codina, R Carretero, R Sanz-Pamplona1, T Cabrera, et al.
The American Society for Biochemistry and Molecular Biology
MCP Apr 26, 2013; Manuscript M112.022772
E-mail: asierra@idibell.cat

Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer.
We used the human breast cancer cell line MDA-MB-231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared to parental cells. We employed a network-modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein–protein interaction network and differential expression. The protein–protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized.
The combination of expression profiles and protein interactions revealed an endoplasmic reticulum-thiol oxidoreductase, ERp57, functioning as a hub which retained 4 downregulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA-A, HLA-B, HLA-E and HLA-F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organ-specific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.

Tandem-repeat protein domains across the tree of life

Kristin K. Jernigan and Seth R. Bordenstein
PeerJ 3:e732; 2015 http://dx.doi.org:/10.7717/peerj.732

Tandem-repeat protein domains, composed of repeated units of conserved stretches of 20–40 amino acids, are required for a wide array of biological functions. Despite their diverse and fundamental functions, there has been no comprehensive assessment of their taxonomic distribution, incidence, and associations with organismal lifestyle and phylogeny.
In this study, we assess for the first time the abundance of armadillo (ARM) and tetratricopeptide (TPR) repeat domains across all three domains in the tree of life and compare the results to our previous analysis on ankyrin (ANK) repeat domains in this journal. All eukaryotes and a majority of the bacterial and archaeal genomes analyzed have a minimum of one TPR and ARM repeat. In eukaryotes, the fraction of ARM-containing proteins is approximately double that of TPR and ANK-containing proteins, whereas bacteria and archaea are enriched in TPR-containing proteins relative to ARM- and ANK-containing proteins.
We show in bacteria that phylogenetic history, rather than lifestyle or pathogenicity, is a predictor of TPR repeat domain abundance, while neither phylogenetic history nor lifestyle predicts ARM repeat domain abundance. Surprisingly, pathogenic bacteria were not enriched in TPR-containing proteins, which have been associated within virulence factors in certain species. Taken together, this comparative analysis provides a newly appreciated view of the prevalence and diversity of multiple types of tandem-repeat protein domains across the tree of life.
A central finding of this analysis is that tandem repeat domain-containing proteins are prevalent not just in eukaryotes, but also in bacterial and archaeal species.

Detection of colorectal adenoma and cancer based on transthyretin and C3a-desArg serum levels

Anne-Kristin Fentz, Monika Sporl, Jorg Spangenberg, Heinz Joachim List, et al.
Proteomics Clin. Appl. 2007, 1, 536–544
http://dx.doi.org:/10.1002/prca.200600664

Colorectal cancer is the second leading cause of cancer death, and it develops from benign colorectal adenomas in over 95% of patients. Early detection of these cancer precursors by screening tests and their removal can potentially eradicate more than 95% of colorectal cancers before they develop.
To discover sensitive and specific biomarkers for improvement of pre-clinical diagnosis of colorectal adenoma and cancer, we analysed in two independent studies (n = 87 and n = 83 patients) serum samples from colorectal cancer (stage III), colorectal adenoma and control patients using SELDI-TOF-MS. Extensive statistical analysis was performed to establish homogeneous patient groups based on their clinical data.
Two biomarkers that were each able to distinguish control patients from either colorectal adenoma or colorectal cancer patients (p,0.001) were identified as transthyretin (pre-albumin) and C3adesArg by MS/MS and were further validated by antibody-based assays (radial immunodiffusion, ELISA). A combination of both proteins clearly indicated the presence of colorectal adenoma or carcinoma. Using a cut-off of >0.225 g/L for transthyretin and >1974 ng/mL for C3a-desArg, we found a sensitivity and specificity for colorectal adenoma of 96% and 70%, respectively.

The essential biology of the endoplasmic reticulum stress response for structural and computational biologists

Sadao Wakabayashi, Hiderou Yoshida
CSBJ Mar 2013; 6(7), e201303010 http://dx.doi.org/10.5936/csbj.201303010

The endoplasmic reticulum (ER) stress response is a cytoprotective mechanism that maintains homeostasis of the ER by upregulating the capacity of the ER in accordance with cellular demands. If the ER stress response cannot function correctly, because of reasons such as aging, genetic mutation or environmental stress, unfolded proteins accumulate in the ER and cause ER stress-induced apoptosis, resulting in the onset of folding diseases, including Alzheimer’s disease and diabetes mellitus. Although the mechanism of the ER stress response has been analyzed extensively by biochemists, cell biologists and molecular biologists, many aspects remain to be elucidated. For example, it is unclear how sensor molecules detect ER stress, or how cells choose the two opposite cell fates (survival or apoptosis) during the ER stress response. To resolve these critical issues, structural and computational approaches will be indispensable, although the mechanism of the ER stress response is complicated and difficult to understand holistically at a glance. Here, we provide a concise introduction to the mammalian ER stress response for structural and computational biologists.

Sequence co-evolution gives 3D contacts and structures of protein complexes

Thomas A Hopf, Charlotta P I Schärfe, João P G L M Rodrigues, et al.
eLife 2014;3:e03430 http://dx.doi.org:/10.7554/eLife.03430

Protein–protein interactions are fundamental to many biological processes. Experimental screens have identified tens of thousands of interactions, and structural biology has provided detailed functional insight for select 3D protein complexes. An alternative rich source of information about protein interactions is the evolutionary sequence record. Building on earlier work, we show that analysis of correlated evolutionary sequence changes across proteins identifies residues that are close in space with sufficient accuracy to determine the three-dimensional structure of the protein complexes. We evaluate prediction performance in blinded tests on 76 complexes of known 3D structure, predict protein–protein contacts in 32 complexes of unknown structure, and demonstrate how evolutionary couplings can be used to distinguish between interacting and non-interacting protein pairs in a large complex. With the current growth of sequences, we expect that the method can be generalized to genome-wide elucidation of protein–protein interaction networks and used for interaction predictions at residue resolution.
S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding

Jorge Alegre-Cebollada, P Kosuri, D Giganti, E Eckels, JA Rivas-Pardo, et al.
Cell, Mar 13, 2014; 156: 1235–1246. http://dx.doi.org/10.1016/j.cell.2014.01.056

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue.
Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes.
We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.
Encounter complexes and dimensionality reduction in protein–protein association

Dima Kozakov, Keyong Li, David R Hall, Dmitri Beglov, Jiefu Zheng, et al.
eLife 2014;3:e01370 http://dx.doi.org:/10.7554/eLife.01370.001

An outstanding challenge has been to understand the mechanism whereby proteins associate. We report here the results of exhaustively sampling the conformational space in protein–protein association using a physics-based energy function. The agreement between experimental intermolecular paramagnetic relaxation enhancement (PRE) data and the PRE profiles calculated from the docked structures shows that the method captures both specific and non-specific encounter complexes. To explore the energy landscape in the vicinity of the native structure, the nonlinear manifold describing the relative orientation of two solid bodies is projected onto a Euclidean space in which the shape of low energy regions is studied by principal component analysis. Results show that the energy surface is canyon-like, with a smooth funnel within a two dimensional subspace capturing over 75% of the total motion. Thus, proteins tend to associate along preferred pathways, similar to sliding of a protein along DNA in the process of protein-DNA recognition.

Cardiovascular Proteomics: Evolution and Potential

Kent Arrell, Irina Neverova and Jennifer E. Van Eyk
Circ Res. 2001;88:763-773 http://dx.doi.org:/doi:/10.1161/hh0801.090193

The development of proteomics is a timely one for cardiovascular research. Analyses at the organ, subcellular, and molecular levels have revealed dynamic, complex, and subtle intracellular processes associated with heart and vascular disease. The power and flexibility of proteomic analyses, which facilitate protein separation, identification, and characterization, should hasten our understanding of these processes at the protein level. Properly applied, proteomics provides researchers with cellular protein “inventories” at specific moments in time, making it ideal for documenting protein modification due to a particular disease, condition, or treatment. This is accomplished through the establishment of species- and tissue-specific protein databases, providing a foundation for subsequent proteomic studies. Evolution of proteomic techniques has permitted more thorough investigation into molecular mechanisms underlying cardiovascular disease, facilitating identification not only of modified proteins but also of the nature of their modification. Continued development should lead to functional proteomic studies, in which identification of protein modification, in conjunction with functional data from established biochemical and physiological methods, has the ability to further our understanding of the interplay between proteome change and cardiovascular disease.

Advances in Proteomic Technologies and Its Contribution to the Field of Cancer

Mehdi Mesri

Advances in Medicine 2014, Article ID 238045, 25 pages http://dx.doi.org/10.1155/2014/238045

Systematic studies of the cancer genome have generated a wealth of knowledge in recent years. These studies have uncovered a number of new cancer genes not previously known to be causal targets in cancer. Genetic markers can be used to determine predisposition to tumor development, but molecularly targeted treatment strategies are not widely available for most cancers. Precision care plans still must be developed by understanding and implementing basic science research into clinical treatment. Proteomics is continuing to make major strides in the discovery of fundamental biological processes as well as more recent transition into an assay platform capable of measuring hundreds of proteins in any biological system. As such, proteomics can translate basic science discoveries into the clinical practice of precision medicine. The proteomic field has progressed at a fast rate over the past five years in technology, breadth and depth of applications in all areas of the bioscience. Some of the previously experimental technical approaches are considered the gold standard today, and the community is now trying to come to terms with the volume and complexity of the data generated. Here I describe contribution of proteomics in general and biological mass spectrometry in particular to cancer research, as well as related major technical and conceptual developments in the field.

Chemoproteomics reveals Toll-like receptor fatty acylation

Nicholas M Chesarino, Jocelyn C Hach, James L Chen, Balyn W Zaro, et al.
BMC Biology 2014, 12:91 http://www.biomedcentral.com/1741-7007/12/91

Background: Palmitoylation is a 16-carbon lipid post-translational modification that increases protein hydrophobicity. This form of protein fatty acylation is emerging as a critical regulatory modification for multiple aspects of cellular interactions and signaling. Despite recent advances in the development of chemical tools for the rapid identification and visualization of palmitoylated proteins, the palmitoyl proteome has not been fully defined. Here we sought to identify and compare the palmitoylated proteins in murine fibroblasts and dendritic cells.
Results: A total of 563 putative palmitoylation substrates were identified, more than 200 of which have not been previously suggested to be palmitoylated in past proteomic studies. Here we validate the palmitoylation of several new proteins including Toll-like receptors (TLRs) 2, 5 and 10, CD80, CD86, and NEDD4. Palmitoylation of TLR2, which was uniquely identified in dendritic cells, was mapped to a transmembrane domain-proximal cysteine. Inhibition of TLR2 S-palmitoylation pharmacologically or by cysteine mutagenesis led to decreased cell surface expression and a decreased inflammatory response to microbial ligands. Conclusions: This work identifies many fatty acylated proteins involved in fundamental cellular processes as well as cell type-specific functions, highlighting the value of examining the palmitoyl proteomes of multiple cell types. Spalmitoylation of TLR2 is a previously unknown immunoregulatory mechanism that represents an entirely novel avenue for modulation of TLR2 inflammatory activity.

Comparative Proteomics and Network Analysis Identify PKC Epsilon Underlying Long-Chain Fatty Acid Signaling

T Yonezawa, R Kurata, A Tajima, X Cui, H Maruta, H Nakaoka, K Nakajima and H Inokio
J Proteomics Bioinform 2014: 7:11 http://dx.doi.org/10.4172/jpb.1000337

Long-chain fatty acid possesses myriad roles in the biological function of the cells, not only as an energy substrate but also as substrates for cell membrane synthesis and as precursors for intracellular signaling molecules. However, little is known about the biological pathways that are stimulated by long-chain fatty acid. In order to identify the pathway of long-chain fatty acid, we performed 2-dimensional gel electrophoresis in the cells treated with or without oleate, and then analyzed 648 protein spots using PDQuest software and narrowed down 22 significant changing spots by statistical criterion. We also tried to determine these spots by MALDI-QIT-TOF-MS and SWISSPROT database query. We identified 11 proteins and predicted the biological network using available data sets from protein-protein interaction database. This prediction indicated that several protein kinase Cs (PKCs) underlie long chain fatty acid signaling. Indeed, oleate stimulated predicted PKC pathways. In expression array, oleate significantly up-regulated only PKC epsilon, but not other PKCs, in transcriptional levels. Collectively, our proteomics and network analysis implicates that PKC epsilon pathway plays an important role in long-chain fatty acid signaling.
Editorial: The art of proteomics translation

Translational Proteomics 2013; 1: 1–2 http://dx.doi.org/10.1016/j.trprot.2013.03.001

Over the years, the difficulties of transferring fundamental proteomics discoveries to clinical applications have caused a lot of frustration to proteomics researchers and clinicians alike, in both academia and industry. One of the reasons for this barrier is the lack of understanding between basic scientists and physicians: they have been trained using opposing concepts. Whilst the former want to control and understand all variables, the latter need rapid actions on patients, rather than absolute certainties. Both disciplines are difficult to con-dense into a single scientist and therefore interdisciplinary associations need to be fostered. Translational research has often been viewed as a two-way street: bedside to bench, and back to bedside. We should perhaps look at it as a roundabout, with the patient and his disease in the center, surrounded by a constant, iterative inter-play between basic, translational and clinical scientists, from both the public and private sectors. Proteomics research needs more than just a translation road bridge from discoveries to cures. Rather, it requires networks of road junctions to fill all the gaps and to allow cross-fertilization and synergies. Translational research and translational proteomics are more than just interesting concepts and hot keywords, they are supposed to improve the quality of people’s lives. With the launch of Translational Proteomics, we want to help the scientific and medical communities overcome the challenges on the long path from discovery to patient care. By focusing on connecting basic proteomics research to its ultimate clinical applications, the Journal will provide a space for publications detailing proteomics experiments, from early discovery to validation and the bedside.

Structural Basis of Diverse Membrane Target Recognitions by Ankyrins

C Wang, Z Wei, K Chen, F Ye, C Yu, V Bennett, and M Zhang
eLife 2014; http:dx.doi.org:/10.7554/eLife.04353

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target protein with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins’ membrane targets, with implications for other proteins containing extended ANK repeat domains.

Fusion of Protein Aggregates Facilitates Asymmetric Damage Segregation

Miguel Coelho, Steven J. Lade, Simon Alberti, Thilo Gross, Iva M. Tolic
PLOS Biology June 2014; 12(6):e1001886
http://dx.doi.org:/10.1371/journal.pbio.1001886

Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.

Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol

Jan-Erik Hoffmann, Y Fermin, R LO Stricker, K Ickstadt, E Zamir
eLife 2014;3:e02257. http://dx.doi.org:/10.7554/eLife.02257.001

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photo-bleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system.

Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress

Jie Wang, Kristeen A Pareja, Chris A Kaiser, Carolyn S Sevier
eLife 2014;3:e03496. http://dx.doi.org:/10.7554/eLife.03496

Oxidative protein folding in the endoplasmic reticulum (ER) has emerged as a potentially significant source of cellular reactive oxygen species (ROS). Recent studies suggest that levels of ROS generated as a byproduct of oxidative folding rival those produced by mitochondrial respiration. Mechanisms that protect cells against oxidant accumulation within the ER have begun to be elucidated yet many questions still remain regarding how cells prevent oxidant-induced damage from ER folding events. Here we report a new role for a central well-characterized player in ER homeostasis as a direct sensor of ER redox imbalance. Specifically we show that a conserved cysteine in the lumenal chaperone BiP is susceptible to oxidation by peroxide, and we demonstrate that oxidation of this conserved cysteine disrupts BiP’s ATPase cycle. We propose that alteration of BiP activity upon oxidation helps cells cope with disruption to oxidative folding within the ER during oxidative stress.

Current perspectives on cadherin-cytoskeleton interactions and dynamics

Xuan Liang, Guillermo A Gomez, Alpha S Yap
Cell Health and Cytoskeleton 2015:7 11–24
http://dx.doi.org/10.2147/CHC.S76107

Cells are linked together dynamically by adhesion molecules, such as the classical cadherins. E-cadherin, which mediates epithelial cell–cell interactions, plays fundamental roles in tissue organization and is often perturbed in diseases such as cancer. It has long been recognized that the biology of E-cadherin arises from cooperation between adhesion and the actin cytoskeleton. A major feature is the generation of contractile forces at junctions, yielding patterns of tension that contribute to tissue integrity and patterning. Here we discuss recent developments in understanding how cadherin junctions integrate signaling and cytoskeletal dynamics to sense and generate force.

N-glycosylation status of E-cadherin controls cytoskeletal dynamics through the organization of distinct β-catenin- and γ-catenin-containing AJs

Basem T Jamal, M Nita-Lazar, Z Gao, B Amin, J Walker, MA Kukuruzinska
Cell Health and Cytoskeleton 2009:1 67–80

N-glycosylation of E-cadherin has been shown to inhibit cell–cell adhesion. Specifically, our recent studies have provided evidence that the reduction of E-cadherin N-glycosylation promoted the recruitment of stabilizing components, vinculin and serine/threonine protein phosphatase 2A (PP2A), to adherens junctions (AJs) and enhanced the association of AJs with the actin cytoskeleton. Here, we examined the details of how N-glycosylation of E-cadherin affected the molecular organization of AJs and their cytoskeletal interactions. Using the hypoglycosylated E-cadherin variant, V13, we show that V13/β-catenin complexes preferentially interacted with PP2A and with the microtubule motor protein dynein. This correlated with dephosphorylation of the microtubule-associated protein tau, suggesting that increased association of PP2A with V13-containing AJs promoted their tethering to microtubules. On the other hand, V13/γ-catenin complexes associated more with vinculin, suggesting that they mediated the interaction of AJs with the actin cytoskeleton. N-glycosylation driven changes in the molecular organization of AJs were physiologically significant because transfection of V13 into A253 cancer cells, lacking both mature AJs and tight junctions (TJs), promoted the formation of stable AJs and enhanced the function of TJs to a greater extent than wild-type E-cadherin. These studies provide the first mechanistic insights into how N-glycosylation of E-cadherin drives changes in AJ composition through the assembly of distinct β-catenin- and γ-catenin-containing scaffolds that impact the interaction with different cytoskeletal components.

Mapping the dynamics of force transduction at cell-cell 4 junctions of epithelial clusters

Mei Rosa Ng, Achim Besser, Joan S. Brugge, Gaudenz Danuser
eLife 2014;10.7554/eLife.03282
http://dx.doi.org/10.7554/eLife.03282

Force transduction at cell-cell adhesions regulates tissue development, maintenance and adaptation. We developed computational and experimental approaches to quantify, with both subcellular and multi-cellular resolution, the dynamics of force transmission in cell clusters. Applying this technology to spontaneously-forming adherent epithelial cell clusters, we found that basal force fluctuations were coupled to E-cadherin localization at the level of individual cell-cell junctions. At the multi-cellular scale, cell-cell force exchange depended on the cell position within a cluster, and was adaptive to reconfigurations due to cell divisions or positional rearrangements. Importantly, force transmission through a cell required coordinated modulation of cell-matrix adhesion and actomyosin contractility in the cell and its neighbors. These data provide insights into mechanisms that could control mechanical stress homeostasis in dynamic epithelial tissues, and highlight our methods as a resource for the study of mechanotransduction in cell-cell adhesions.

G-protein-coupled receptor signaling and polarized actin dynamics drive cell-in-cell invasion

Vladimir Purvanov, Manuel Holst, Jameel Khan, Christian Baarlink, Robert Grosse
eLife 2014;3:e02786. http://dx.doi.org:/10.7554/eLife.02786

Homotypic or entotic cell-in-cell invasion is an integrin-independent process observed in carcinoma cells exposed during conditions of low adhesion such as in exudates of malignant disease. Although active cell-in-cell invasion depends on RhoA and actin, the precise mechanism as well as the underlying actin structures and assembly factors driving the process are unknown. Furthermore, whether specific cell surface receptors trigger entotic invasion in a signal-dependent fashion has not been investigated. In this study, we identify the G-protein-coupled LPA receptor 2 (LPAR2) as a signal transducer specifically required for the actively invading cell during entosis. We find that G12/13 and PDZ-RhoGEF are required for entotic invasion, which is driven by blebbing and a uropod-like actin structure at the rear of the invading cell. Finally, we provide evidence for an involvement of the RhoA-regulated formin Dia1 for entosis downstream of LPAR2. Thus, we delineate a signaling process that regulates actin dynamics during cell-in-cell invasion.

Cytoskeletal Basis of Ion Channel Function in Cardiac Muscle

Matteo Vatta, and Georgine Faulkner
Future Cardiol. 2006 Jul 1; 2(4): 467–476. http://dx.doi.org:/10.2217/14796678.2.4.467

The heart is a force-generating organ that responds to self-generated electrical stimuli from specialized cardiomyocytes. This function is modulated by sympathetic and parasympathetic activity.

In order to contract and accommodate the repetitive morphological changes induced by the cardiac cycle, cardiomyocytes depend on their highly evolved and specialized cytoskeletal apparatus. Defects in components of the cytoskeleton, in the long term, affect the ability of the cell to compensate at both functional and structural levels. In addition to the structural remodeling, the myocardium becomes increasingly susceptible to altered electrical activity leading to arrhythmogenesis. The development of arrhythmias secondary to structural remodeling defects has been noted, although the detailed molecular mechanisms are still elusive. Here I will review the current knowledge of the molecular and functional relationships between the cytoskeleton and ion channels and, I will discuss the future impact of new data on molecular cardiology research and clinical practice.

Structure and transport mechanism of the sodium/proton 2 antiporter MjNhaP1

Cristina Paulino, D Wöhlert , E Kapotova, Ö Yildiz & W Kühlbrandt
eLife 2014; http://dx.doi.org/10.7554/eLife.03583

Sodium/proton antiporters are essential for sodium and pH homeostasis and play a major role in human health and disease. We determined the structures of the archaeal sodium/proton antiporter MjNhaP1 in two complementary states. The inward-open state was obtained by x-ray crystallography in the presence of sodium at pH8, where the transporter is highly active. The outward-open state was obtained by electron crystallography without sodium at pH4, where MjNhaP1 is inactive. Comparison of both structures reveals a 7° tilt of the 6-helix bundle. Na+ uptake measurements indicate non-cooperative transport with an activity maximum at pH7.5. We conclude that binding of a Na+ ion from the outside induces helix movements that close the extracellular cavity, open the cytoplasmic funnel, and result in a ~5 Å vertical relocation of the ion binding site to release the substrate ion into the cytoplasm.

Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5

Michel Becuwe, Sébastien Léon
eLife 2014; http://dx.doi.org/10.7554/eLife.03307

After endocytosis, membrane proteins can recycle to the cell membrane or be degraded in lysosomes. Cargo ubiquitylation favors their lysosomal targeting and can be regulated by external signals, but the mechanism is ill-defined. Here, we studied the post-endocytic trafficking of Jen1, a yeast monocarboxylate transporter, using microfluidics-assisted live cell imaging. We show that the ubiquitin ligase Rsp5 and the glucose-regulated arrestin related (ART) protein Rod1, involved in the glucose-induced internalization of Jen1, are also required for the post-endocytic sorting of Jen1 to the yeast lysosome. This new step takes place at the trans-Golgi network (TGN), where Rod1 localizes dynamically upon triggering endocytosis. Indeed, transporter trafficking to the TGN after internalization is required for their degradation. Glucose removal promotes Rod1 relocalization to the cytosol and Jen1 deubiquitylation, allowing transporter recycling when the signal is only transient. Therefore, nutrient availability regulates transporter fate through the localization of the ART/Rsp5 ubiquitylation complex at the TGN.

McKenney, W Huynh, ME. Tanenbaum, G Bhabha, and RD. Vale
Science Express 19 June 2014 /10.1126/science.1254198
http://www.sciencemag.org/content/early/recent/10.1126/science.1254198

Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, mRNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultra-processive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.

Removal of surface charge–charge interactions from ubiquitin leaves the protein folded and very stable

Vakhtang V. Loladze And George I. Makhatadze
Protein Science (2002), 11:174–177
http://www.proteinscience.org/cgi/doi/10.1101/ps.29902.

The contribution of solvent-exposed charged residues to protein stability was evaluated using ubiquitin as a model protein. We combined site-directed mutagenesis and specific chemical modifications to first replace all Arg residues with Lys, followed by carbomylation of Lys- amino groups. Under the conditions in which all carboxylic groups are protonated (at pH 2), the chemically modified protein is folded and very stable (dG= 18 kJ/mol). These results indicate that surface charge–charge interactions are not an essential fundamental force for protein folding and stability.

Phase Transitions of Multivalent Proteins Can Promote Clustering of Membrane Receptors

Sudeep Banjade and Michael K. Rosen
eLife 2014; http://dx.doi.org/10.7554/eLife.04123

Clustering of proteins into micrometer-sized structures at membranes is observed in many signaling pathways. Most models of clustering are specific to particular systems, and relationships between physical properties of the clusters and their molecular components are not well understood. We report biochemical reconstitution on supported lipid bilayers of protein clusters containing the adhesion receptor Nephrin, and its cytoplasmic partners, Nck and N-WASP. With Nephrin attached to the bilayer, multivalent interactions enable these proteins to polymerize on the membrane surface and undergo two-dimensional phase separation, producing micrometer-sized clusters. Dynamics and thermodynamics of the clusters are modulated by the valencies and affinities of the interacting species. In the presence of the Arp2/3 complex, the clusters assemble actin filaments, suggesting that clustering of regulatory factors could promote local actin assembly at membranes. Interactions between multivalent proteins could be a general mechanism for cytoplasmic adaptor proteins to organize membrane receptors into micrometer-scale signaling zones.

The quantitative architecture of centromeric chromatin

Dani L Bodor, João F Mata, Mikhail Sergeev, Ana Filipa David, et al.
eLife 2014;3:e02137. http://dx.doi.org:/10.7554/eLife.02137

The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion

Jiajie Diao, Patricia Grob, Daniel J Cipriano, Minjoung Kyoung
eLife 2012;1:e00109. http://dx.doi.org:/10.7554/eLife.00109

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.

Cytoskeleton, cytoskeletal interactions, and vascular endothelial function

Jingli Wang, Michael E Widlansky
Cell Health and Cytoskeleton 2012:4 119–127
http://dx.doi.org/10.2147/CHC.S21823

Far from being inert, the vascular endothelium is a critical regulator of vascular function. While the endothelium participates in autocrine, paracrine, and endocrine signaling, it also transduces mechanical signals from the cell surface involving key cell structural elements. In this review, we discuss the structure of the vascular endothelium and its relationship to traditional cardiovascular risk factors and clinical cardiovascular events. Further, we review the emerging evidence that cell structural elements, including the glycocalyx, intercellular junctions, and cytoskeleton elements, help the endothelium to communicate with its environment to regulate vascular function, including vessel permeability and signal transduction via nitric oxide bioavailability. Further work is necessary to better delineate the regulatory relationships between known key regulators of vascular function and endothelial cell structural elements.

Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics

Aurélie Alleaume-Butaux, C Dakowski, M Pietri, S Mouillet-Richard, et al.
Cell Health and Cytoskeleton 2013:5 1–12
http://dx.doi.org/10.2147/CHC.S28081

Neuritogenesis is a dynamic phenomenon associated with neuronal differentiation that allows a rather spherical neuronal stem cell to develop dendrites and axon, a prerequisite for the integration and transmission of signals. The acquisition of neuronal polarity occurs in three steps: (1) neurite sprouting, which consists of the formation of buds emerging from the postmitotic neuronal soma; (2) neurite outgrowth, which represents the conversion of buds into neurites, their elongation and evolution into axon or dendrites; and (3) the stability and plasticity of neuronal polarity. In neuronal stem cells, remodeling and activation of focal adhesions (FAs) associated with deep modifications of the actin cytoskeleton is a prerequisite for neurite sprouting and subsequent neurite outgrowth. A multiple set of growth factors and interactors located in the extracellular matrix and the plasma membrane orchestrate neuritogenesis by acting on intracellular signaling effectors, notably small G proteins such as RhoA, Rac, and Cdc42, which are involved in actin turnover and the dynamics of FAs. The cellular prion protein (PrPC), a glycosylphosphatidylinositol (GPI)-anchored membrane protein mainly known for its role in a group of fatal neurodegenerative diseases, has emerged as a central player in neuritogenesis. Here, we review the contribution of PrPC to neuronal polarization and detail the current knowledge on the signaling pathways fine-tuned by PrPC to promote neurite sprouting, outgrowth, and maintenance. We emphasize that PrPC-dependent neurite sprouting is a process in which PrPC governs the dynamics of FAs and the actin cytoskeleton via β1 integrin signaling. The presence of PrPC is necessary to render neuronal stem cells competent to respond to neuronal inducers and to develop neurites. In differentiating neurons, PrPC exerts a facilitator role towards neurite elongation. This function relies on the interaction of PrPC with a set of diverse partners such as elements of the extracellular matrix, plasma membrane receptors, adhesion molecules, and soluble factors that control actin cytoskeleton turnover through Rho-GTPase signaling. Once neurons have reached their terminal stage of differentiation and acquired their polarized morphology, PrPC also takes part in the maintenance of neurites. By acting on tissue nonspecific alkaline phosphatase, or matrix metalloproteinase type 9, PrPC stabilizes interactions between neurites and the extracellular matrix.

Broader implications: biological and clinical significance of microtubule acetylation

Sharon M Rymut, Thomas J Kelley
Cell Health and Cytoskeleton 2015:7 71–82
http://dx.doi.org/10.2147/CHC.S77040

Microtubule acetylation is a key posttranslational modification that enhances organelle transport, drives cell signaling, and regulates cell cycle regulation. The optimal level of microtubule acetylation is regulated by the acetyltransferase alpha-tubulin-N-acetyltransferase 1and two deacetylases, histone deacetylase 6 and sirtuin-2. Alterations in microtubule acetylation levels have been associated with the pathophysiology of a number of diseases, including various forms of neurodegenerative conditions, cancer, and even cystic fibrosis. In this review, we will highlight the biological and clinical significance of microtubule acetylation and the potential of targeting this pathway for therapeutics.

Inositol-1,4,5-trisphosphate 1 (IP3)-mediated STIM1 oligomerization requires intact mitochondrial Ca2+ uptake

Deak, S. Blass, M. J. Khan, L. N. Groschner, M. Waldeck-Weiermair, et al.
Journal of Cell Science 2014 advanced print

Mitochondria contribute to cell signaling by controlling store-operated Ca2+ entry (SOCE). SOCE is activated by Ca2+ release from the endoplasmic reticulum (ER), whereupon the stromal interacting molecule 1 (STIM1) forms oligomers, redistributes to ER-plasma membrane junctions, and opens plasma membrane Ca2+ channels. Mechanisms by which mitochondria interfere with the complex process of SOCE are insufficiently clarified. In this study we used a shRNA approach to investigate the direct involvement of mitochondrial Ca2+ buffering in SOCE. We demonstrate that knock-down of two proteins that are essential for mitochondrial Ca2+ uptake, either the mitochondrial calcium uniporter (MCU) or uncoupling protein 2 (UCP2), results in decelerated STIM1 oligomerization and impaired SOCE following cell stimulation with an inositol-1,4,5-trisphosphate (IP3)-generating agonist. Upon artificially augmented cytosolic Ca2+-buffering or ER Ca2+ depletion by sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors, STIM1 oligomerization did not rely on intact mitochondrial Ca2+ uptake. However, MCU-dependent mitochondrial sequestration of Ca2+ entering through the SOCE pathway was essential to prevent slow deactivation of SOCE. Our findings show a stimulus specific contribution of mitochondrial Ca2+ uptake to the SOCE machinery likely by shaping cytosolic Ca2+ micro-domains.

Role of forkhead box protein A3 in age-associated metabolic decline

Xinran Ma, Lingyan Xu, Oksana Gavrilov, and Elisabetta Mueller
PNAS | September 30, 2014 | vol. 111 | no. 39 | 14289–14294
www.pnas.org/cgi/doi/10.1073/pnas.1407640111

Aging is associated with increased adiposity and diminished thermogenesis, but the critical transcription factors influencing these metabolic changes late in life are poorly understood. We recently demonstrated that the winged helix factor forkhead box protein A3 (Foxa3) regulates the expansion of visceral adipose tissue in high-fat diet regimens; however, whether Foxa3 also contributes to the increase in adiposity and the decrease in brown fat activity observed during the normal aging process is currently unknown.
Here we report that during aging, levels of Foxa3 are significantlyand selectively up-regulated in brown and inguinal white fat depots, and that midage Foxa3-null mice have increased white fat browning and thermogenic capacity, decreased adipose tissue expansion, improved insulin sensitivity, and increased longevity. Foxa3 gain-of-function and loss-of-function studies in inguinal adipose depots demonstrated a cell-autonomous function for Foxa3 in white fat tissue browning. Furthermore, our analysis revealed that the mechanisms of Foxa3 modulation of brown fat gene programs involve the suppression of peroxisome proliferator activated receptor γ coactivtor 1 α (PGC1α) levels through interference with cAMP responsive element binding protein 1-mediated transcriptional regulation of the PGC1α promoter. Overall, our data demonstrate a role for Foxa3 in energy expenditure and in age-associated metabolic disorders.

Prediction of enzyme function by combining sequence similarity and protein interactions

Jordi Espadaler, Narayanan Eswa, Enrique Querol, Francesc X Avilés, et al.
BMC Bioinformatics 2008, 9:249 http://dx.doi.org:/10.1186/1471-2105-9-249

Background: A number of studies have used protein interaction data alone for protein function prediction. Here, we introduce a computational approach for annotation of enzymes, based on the observation that similar protein sequences are more likely to perform the same function if they share similar interacting partners.
Results: The method has been tested against the PSI-BLAST program using a set of 3,890 protein sequences from which interaction data was available. For protein sequences that align with at least 40% sequence identity to a known enzyme, the specificity of our method in predicting the first three EC digits increased from 80% to 90% at 80% coverage when compared to PSI-BLAST.
Conclusion: Our method can also be used in proteins for which homologous sequences with known interacting partners can be detected. Thus, our method could increase 10% the specificity of genome-wide enzyme predictions based on sequence matching by PSI-BLAST alone.

Plasma Transthyretin Indicates the Direction of both Nitrogen Balance and Retinoid Status in Health and Disease

Ingenbleek Yves and Bienvenu Jacques
The Open Clinical Chemistry Journal, 2008, 1, 1-12

Whatever the nutritional status and the disease condition, the actual transthyretin (TTR) plasma level is determined by opposing influences between anabolic and catabolic alterations. Rising TTR values indicate that synthetic processes prevail over tissue breakdown with a nitrogen balance (NB) turning positive as a result of efficient nutritional support and / or anti-inflammatory therapy. Declining TTR values point to the failure of sustaining NB as an effect of maladjusted dietetic management and / or further worsening of the morbid condition. Serial measurement of TTR thus appears as a dynamic index defining the direction of NB in acute and chronic disorders, serving as a guide to alert the physician on the validity of his therapeutic strategy. The level of TTR production by the liver also works as a limiting factor for the cellular bioavailability of retinol and retinoid derivatives which play major roles in the brain ageing process. Optimal protein nutritional status, as assessed by TTR values within the normal range, prevents the occurrence of vascular and cerebral damages while maintaining the retinoid-mediated memory, cognitive and behavioral activities of elderly persons.

Prof. Dr. Volker Haucke
Institut für Chemie-Biochemie
Takustrasse 6
http://userpage.chemie.fu-berlin.de/biochemie/aghaucke/teaching.html

Eukaryotic cells contain three major types of cytoskeletal filaments

major types of cytoskeletal filaments

Intermediate Filaments support the nuclear membrane and connect cells at cell junctions

microtubules (MTs; green) radiate from MTOCs (yellow) towards the cell periphery

Actin polymerization in vitro reveals a critical dependence of filament assembly on G-actin concentration via a 3-step nucleation mechanism

Binding-proteins and receptors

Motor, visual and emotional deficits in mice after closed-head mild traumatic brain injury are alleviated by the novel CB2 inverse agonist SMM-189
Reiner, A., Heldt, S.A., Presley, C.S., (…), Gurley, S.N., Moore, B.M.
2015 International Journal of Molecular Sciences 16 (1), pp. 758-787

We have developed a focal blast model of closed-head mild traumatic brain injury (TBI) in mice. As true for individuals that have experienced mild TBI, mice subjected to 50-60 psi blast show motor, visual and emotional deficits, diffuse axonal injury and microglial activation, but no overt neuron
loss. Because microglial activation can worsen brain damage after a concussive event and because microglia can be
modulated by their cannabinoid type 2 receptors (CB2), we evaluated the effectiveness of the novel CB2 receptor inverse agonist SMM-189 in altering microglial activation and mitigating deficits after mild TBI. In vitro analysis indicated that SMM-189 converted human microglia from the pro-inflammatory M1 phenotype to the pro-healing M2 phenotype. Studies in mice showed that daily administration of SMM-189 for two weeks beginning shortly after blast greatly reduced the motor, visual, and emotional deficits otherwise evident after 50-60 psi blasts, and prevented brain injury that may contribute to these deficits. Our results suggest that treatment with the CB2 inverse agonist SMM-189 after a mild TBI event can reduce its adverse consequences by beneficially modulating microglial activation. These
findings recommend further evaluation of CB2 inverse agonists as a novel therapeutic approach for treating mild TBI.

The novel small leucine-rich protein chondroadherin-like (CHADL) is expressed in cartilage and modulates chondrocyte differentiation
Tillgren, V., Ho, J.C.S., Önnerfjord, P., Kalamajski, S.
2015 Journal of Biological Chemistry 290 (2), pp. 918-925

The constitution and biophysical properties of extracellular matrices can dramatically influence cellular phenotype during development, homeostasis, or pathogenesis. These effects can be signaled through a differentially regulated assembly of collagen fibrils, orchestrated by a family of collagen-associated small leucine-rich proteins (SLRPs). In this report, we describe the tissue-specific expression and function of a previously uncharacterized SLRP, chondroadherin-like (CHADL). We developed antibodies against CHADL and, by immunohistochemistry, detected CHADL expression mainly in skeletal tissues, particularly in fetal cartilage and in the pericellular space of adult chondrocytes. In situ hybridizations and immunoblots on tissue lysates confirmed this tissue-specific expression pattern. Recombinant CHADL bound collagen in cell culture and inhibited in vitro collagen fibrillogenesis. After Chadl shRNA knockdown, chondrogenic ATDC5 cells increased their differentiation, indicated by increased transcript levels of Sox9, Ihh, Col2a1, and Col10a1. The knockdown increased collagen II and aggrecan deposition in the cell layers.

Microarray analysis of the knockdown samples suggested collagen receptor-related changes, although other upstream effects could not be excluded. Together, our data indicate that the novel SLRP CHADL is expressed in cartilaginous tissues, influences collagen fibrillogenesis, and modulates chondrocyte differentiation. CHADL appears to have a negative regulatory role, possibly ensuring the formation of a stable extracellular matrix.

P53 protein-mediated Up-regulation of MAP kinase phosphatase 3 (MKP-3) contributes to the establishment of the cellular senescent phenotype through dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2)
Zhang, H., Chi, Y., Gao, K., Zhang, X., Yao, J.
2015 Source of the DocumentJournal of Biological Chemistry 290 (2), pp. 1129-1140

Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced ?-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogenactivated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-?. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Dynamics and interaction of Interleukin-4 receptor subunits in living cells
Gandhi, H., Worch, R., Kurgonaite, K., (…), Bökel, C., Weidemann, T.
2015 Biophysical Journal 107 (11), pp. 2515-2527

It has long been established that dimerization of Interleukin-4 receptor (IL-4R) subunits is a pivotal step for JAK/STAT signal transduction. However, ligand-induced complex formation at the surface of living cells has been challenging to observe. Here we report an experimental assay employing trisNTA dyes for orthogonal, external labeling of eGFP-tagged receptor constructs that allows the quantification of receptor heterodimerization by dual-color fluorescence cross-correlation spectroscopy. Fluorescence cross-correlation spectroscopy analysis at the plasma membrane shows that IL-4R subunit dimerization is indeed a strictly ligand-induced process.

Under conditions of saturating cytokine occupancy, we determined intramembrane dissociation constants (Kd,2D) of 180 and 480 receptors per ?m2 for the type-2 complexes IL-4:IL-4R?/IL-13R?1 and IL-13:IL-13R?1/IL-4R?, respectively. For the lower affinity type-1 complex IL-4:IL-4R?/IL-2R?, we estimated a Kd,2D of ?1000 receptors per ?m2. The receptor densities required for effective dimerization thus exceed the typical, average expression levels by several orders of magnitude. In addition, we find that all three receptor subunits accumulate rapidly within a subpopulation of early sorting and recycling endosomes stably anchored just beneath the plasma membrane (cortical endosomes, CEs). The receptors, as well as labeled IL-4 and trisNTA ligands are specifically trafficked into CEs by a constitutive internalization mechanism. This may compensate for the inherent weak affinities that govern ligand-induced receptor dimerization at the plasma membrane. Consistently, activated receptors are also concentrated at the CEs. Our observations thus suggest that receptor trafficking may play an important role for the regulation of IL-4R-mediated JAK/STAT signaling.

Role of mitochondria in nonalcoholic fatty liver disease
Nassir, F., Ibdah, J.A.
2015 International Journal of Molecular Sciences 15 (5), pp. 8713-8742

Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis of NALFD; however the key metabolic aberrations underlying lipid accumulation in hepatocytes and the progression of NAFLD remain to be elucidated. Accumulating and emerging evidence indicate that hepatic mitochondria play a critical role in the development and pathogenesis of steatosis and NAFLD. Here, we review studies that document a link between the pathogenesis of NAFLD and hepatic mitochondrial dysfunction with particular focus on new insights into the role of impaired fatty acid oxidation, the transcription factor peroxisome proliferator-activated receptor-? coactivator-1? (PGC-1?), and sirtuins in development and progression of NAFLD.

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Genomics and Metabolomics Advances in Cancer

Posted in Acute lymphocytic leukemia, Acute myelocytic leukemia, Biological Networks, CANCER BIOLOGY & Innovations in Cancer Therapy, Curation, Cytoskeleton, Disease Biology, Gene Regulation, Genome Biology, Hematology, Human Immune System in Health and in Disease, Metabolism, Metabolomics, Molecular Genetics & Pharmaceutical, Mutant Gene Expression, Proteomics, tagged Cancer - General, genomics, mechanisms of disease, Metabolomics, pathophysiology on January 30, 2015| Leave a Comment »

Genomics and Metabolomics Advances in Cancer

Writer and Curator: Larry H. Bernstein, MD, FCAP

UPDATED 6/01/2019

UPDATED 9/26/2021

Genomics

Unraveling the clonal hierarchy of somatic genomic aberrations

D Prandi, SC Baca, A Romanel, CE Barbieri, Juan-Miguel Mosquera, et al.
Genome Biology 2014, 15:439
http://genomebiology.com/2014/15/8/439

Defining the chronology of molecular alterations may identify milestones in carcinogenesis. To unravel the temporal evolution of aberrations from clinical tumors, we developed CLONET, which upon estimation of tumor admixture and ploidy infers the clonal hierarchy of genomic aberrations. Comparative analysis across 100 sequenced genomes from prostate, melanoma, and lung cancers established diverse evolutionary hierarchies, demonstrating the early disruption of tumor-specific pathways. The analyses highlight the diversity of clonal evolution within and across tumor types that might be informative for risk stratification and patient selection for targeted therapies. CLONET addresses heterogeneous clinical samples seen in the setting of precision medicine.

The Transcription Factor Titration Effect Dictates Level of Gene Expression

RC Brewster, FM Weinert, HG Garcia, D Song, M Rydenfelt, and R Phillips
Cell, Mar 13, 2014;156: 1312–1323
http://dx.doi.org/10.1016/j.cell.2014.02.022

Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number—in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.

Telomere dynamics in human mesenchymal stem cells after exposure to acute oxidative stress

M Harbo, S Koelvraa, N Serakinci, L Bendixa
DNA Repair 2012. http://dx.doi.org/10.1016/j.dnarep.2012.06.003

A gradual shortening of telomeres due to replication can be measured using the standard telomere restriction fragments (TRF) assay and other methods by measuring the mean length of all the telomeres in a cell. In contrast, stress-induced telomere shortening, which is believed to be just as important for causing cellular senescence, cannot be measured properly using these methods. Stress-induced telomere shortening caused by, e.g. oxidative damage happens in a stochastic manner leaving just a few single telomeres critically short. It is now possible to visualize these few ultra-short telomeres due to the advantages of the newly developed Universal single telomere length assay (STELA), and we therefore believe that this method should be considered the method of choice when measuring the length of telomeres after exposure to oxidative stress. In order to test our hypothesis, cultured human mesenchymal stem cells, either primary or hTERT immortalized, were exposed to sub-lethal doses of hydrogen peroxide, and the short term effect on telomere dynamics was monitored by Universal STELA and TRF measurements. Both telomere measures were then correlated with the percentage of senescent cells estimated by senescence-associated β-galactosidase staining. The exposure to acute oxidative stress resulted in an increased number of ultra-short telomeres, which correlated strongly with the percentage of senescent cells, whereas a correlation between mean telomere length and the percentage of senescent cells was absent. Based on the findings in the present study, it seems reasonable to conclude that Universal STELA is superior to TRF in detecting telomere damage caused by exposure to oxidative stress. The choice of method should therefore be considered carefully in studies examining stress-related telomere shortening as well as in the emerging field of lifestyle studies involving telomere length measurements.

tDNA insulators and the emerging role of TFIIIC in genome organization

Kevin Van Bortle and Victor G. Corces
Transcription Dec 12, 2012; 3(6): 1-8. www.landesbioscience.com

Recent findings provide evidence that tDNAs function as chromatin insulators from yeast to humans. TFIIIC, a transcription factor that interacts with the B-box in tDNAs as well as thousands of ETC sites in the genome, is responsible for insulator function. Though tDNAs are capable of enhancer-blocking and barrier activities for which insulators are defined, new insights into the relationship between insulators and chromatin structure suggest that TFIIIC serves a complex role in genome organization. We review the role of tRNA genes and TFIIIC as chromatin insulators, and highlight recent findings that have broadened our understanding of insulators in genome biology.

Structure and organization of insulators in eukaryotes. (A) From yeast to mammals, in organisms in which it has been studied, the TFIIIC protein interacts with the B-box sequence in tRNA genes or sites in the genome named ETC sites.

Synthetic CpG islands reveal DNA sequence determinants of chromatin structure

E Wachter, T Quante, C Merusi, A Arczewska, F Stewart, S Webb, A Bird
eLife 2014;3:e03397. http://dx.doi.org:/10.7554/eLife.03397.001

The mammalian genome is punctuated by CpG islands (CGIs), which differ sharply from the bulk genome by being rich in G + C and the dinucleotide CpG. CGIs often include transcription initiation sites and display ‘active’ histone marks, notably histone H3 lysine 4 methylation. In embryonic stem cells (ESCs) some CGIs adopt a ‘bivalent’ chromatin state bearing simultaneous ‘active’ and ‘inactive’ chromatin marks. To determine whether CGI chromatin is developmentally programmed at specific genes or is imposed by shared features of CGI DNA, we integrated artificial CGI-like DNA sequences into the ESC genome. We found that bivalency is the default chromatin structure for CpG-rich, G + C-rich DNA. A high CpG density alone is not sufficient for this effect, as A + T-rich sequence settings invariably provoke de novo DNA methylation leading to loss of CGI signature chromatin. We conclude that both CpG-richness and G + C-richness are required for induction of signature chromatin structures at CGIs.

Locus-specific mutation databases: pitfalls and good practice based on the p53 experience

Thierry Soussi, Chikashi Ishioka, Mireille Claustres and Christophe Béroud
NATURE REVIEWS | CANCER JAN 2006; 6: 83-90.

Between 50,000 and 60,000 mutations have been described in various genes that are associated with a wide variety of diseases. Reporting, storing and analysing these data is an important challenge as such data provide invaluable information for both clinical medicine and basic science.

The practical value of mutation analysis All studies performed to date show that mutations are, in general, not randomly distributed. Hot-spot regions have been demonstrated, corresponding to a region of DNA that is susceptible to mutations (such as CpG dinucleotides), a codon encoding a key residue in the biological function of the protein, or both (BOX 1). Identification of these hot-spot regions and natural mutants is essential to define crucial regions in an unknown protein.

Locus-specific databases have been developed to exploit this huge volume of data. The p53 mutation database is a paradigm, as it constitutes the largest collection of somatic mutations (22,000). However, there are several biases in this database that can lead to serious erroneous interpretations. We describe several rules for mutation database management that could benefit the entire scientific community.

Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles

A Subramaniana, P Tamayo, VK Mootha, S Mukherjee, BL Ebert, et al.
PNAS Oct 25, 2005; 102(43): 15545–15550
http://pnas.org/cgi/doi/10.1073/pnas.0506580102

Although genomewide RNA expression analysis has become a routine tool in biomedical research, extracting biological insight from such information remains a major challenge. Here, we describe a powerful analytical method called Gene Set Enrichment Analysis (GSEA) for interpreting gene expression data. The method derives its power by focusing on gene sets, that is, groups of genes that share common biological function, chromosomal location, or regulation. We demonstrate how GSEA yields insights into several cancer-related data sets, including leukemia and lung cancer. Notably, where single-gene analysis finds little similarity between two independent studies of patient survival in lung cancer, GSEA reveals many biological pathways in common. The GSEA method is embodied in a freely available software package, together with an initial database of 1,325 biologically defined gene sets.

Mutational landscape and significance across 12 major cancer types

C Kandoth, MD McLellan, F Vandin, Kai Ye, B Niu, C Lu, et al.
NATURE OCT 2013; 502: 333-337. http://dx.doi.org:/10.1038/nature12634

The Cancer Genome Atlas (TCGA) has used the latest sequencing and analysis methods to identify somatic variants across thousands of tumours. Here we present data and analytical results for point mutations and small insertions/deletions from 3,281 tumours across 12 tumour types as part of the TCGA Pan-Cancer effort. We illustrate the distributions of mutation frequencies, types and contexts across tumour types, and establish their links to tissues of origin, environmental/ carcinogen influences, and DNA repair defects. Using the integrated data sets, we identified 127 significantly mutated genes from well-known(for example, mitogen-activated protein kinase, phosphatidylinositol-3-OH kinase,Wnt/b-catenin and receptor tyrosine kinase signalling pathways, and cell cycle control) and emerging (for example, histone, histone modification, splicing, metabolism and proteolysis) cellular processes in cancer. The average number of mutations in these significantly mutated genes varies across tumour types; most tumours have two to six, indicating that the numberof driver mutations required during oncogenesis is relatively small. Mutations in transcriptional factors/regulators show tissue specificity, whereas histone modifiers are often mutated across several cancer types. Clinical association analysis identifies genes having a significant effect on survival, and investigations of mutations with respect to clonal/subclonal architecture delineate their temporal orders during tumorigenesis. Taken together, these results lay the groundwork for developing new diagnostics and individualizing cancer treatment.

Molecular insights into RNA and DNA helicase evolution from the determinants of specificity for a DEAD-box RNA helicase

Anna L. Mallam, David J. Sidote and Alan M. Lambowitz
eLife 2014; http://dx.doi.org:/10.7554/eLife.04630

How different helicase families with a conserved catalytic ‘helicase core’ evolved to function on varied RNA and DNA substrates by diverse mechanisms remains unclear. Here, we used Mss116, a yeast DEAD-box protein that utilizes ATP to locally unwind dsRNA, to investigate helicase specificity and mechanism. Our results define the molecular basis for the substrate specificity of a DEAD-box protein. Additionally, they show that Mss116 has ambiguous substrate-binding properties and interacts with all four NTPs and both RNA and DNA. The efficiency of unwinding correlates with the stability of the ‘closed-state’ helicase core, a complex with nucleotide and nucleic acid that forms as duplexes are unwound. Crystal structures reveal that core stability is modulated by family-specific interactions that favor certain substrates. This suggests how present-day helicases diversified from an ancestral core with broad specificity by retaining core closure as a common catalytic mechanism while optimizing substrate-binding interactions for different cellular functions.

Identification of human TERT elements necessary for telomerase recruitment to telomeres

Jens C Schmidt, Andrew B Dalby, Thomas R Cech
eLife 2014; http://dx.doi.org/10.7554/eLife.03563

Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. Here we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease.

Metabolomics

Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

MN Mindrinos, G Bhanot, SH Dairkee, RW Davis, SS Jeffrey
PLoS ONE 7(5): e33788. http://dx.doi.org:/doi:10.1371/journal.pone.0033788

Background: To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.
Methodology/Principal Findings: We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with nonepithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.
Conclusions/Significance: For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery

Simplifying Disease Complexity part 6 – Bringing Metabolomics into Practice
Dr. Kirk Beebe, Director of Application Science, Metabolon, Inc.

n the previous editions of this 6-part series, we’ve explored numerous example of how metabolomics is bringing success to areas such as cancer, metabolic disease, cardiovascular, and rare disease research. Although we did not devote attention to every area of biology or therapeutic area, the intent of this broad series was not only to convey how metabolomics can be used in a specific area of research (e.g. cancer), but actually, how metabolomics is a central science for interrogating any biological question. So, although it may seem like an oversimplification, to understand whether metabolomics could be used in a research setting one need only ask themselves, “Do I have a biological question that would benefit from a hypothesis-free approach?, am I interested in exploring my system for potential new discoveries? Or do I need a biomarker/better biomarker?

As described in our first part, metabolites have been and continue to be a staple for clinical and in vivo decision making (e.g. cholesterol, glucose, bilirubin, creatinine, thyroid hormone, newborn screening for inborn errors of metabolism (IEMs)). In short, this utility is fundamental to the foundations of biology since metabolism is central to all kingdoms of life and contemporary biology is driven to maintain metabolic homeostasis to maintain the phenotype. An unappreciated point that we leave this series with is that this fundamental nature (the connection of metabolism to the phenotype) confers an important advantage of metabolism for deriving biomarkers and understanding the underlying physiology.

Metabolites are a diagnostic data stream.

Whether a phenotype is driven by a single mutation or a combination of genetic differences, environmental influences or the microbiota, metabolism provides a systems-level diagnostic.

That is, no matter the source of the physiological or phenotypic change (i.e. genes, microbiota, environmental), the change will almost invariably register within metabolism. Thus, modern metabolomic approaches offer the opportunity to more deeply interrogate the “metabolome” to discover more sensitive and specific biomarkers and understand the basis of disease and drug response.

As such, metabolomics has the potential to be able to integrate systems on a number of levels. It is useful through its ability to enrich genomics, transcriptomics and proteomics, thus integrating a number of data streams that provide knowledge and contribute to informed decision-making and patient management1. Using metabolomics, individual tissues can be queried but less invasive sample types (e.g., blood, urine, feces, and/or saliva) can also yield biomarkers and mechanistic insight. The integration of the individual tissues at the level of these more accessible samples can offer an overview of the entire system and inform on important biological pathways. Finally, although the focus of this series was on what metabolomics can bring to biomarker and other related research areas, it should be noted that a combination of metabolomics with other scientific approaches will undoubtedly broaden insight and produce verifiable, validatable biomarkers that track with efficacy and therapy.

As we close this series, we hope that we have conveyed 4 critical points – 1) metabolism is central to biology and hence, key in research and biomarker discovery, 2) the reason for this is due to the fundamental nature of metabolism being central to the development of all life and being the focal point of contemporary biology’s drive to maintain homeostasis, 3) metabolomic is the most powerful way to survey metabolism by offering a simultaneous read-out if hundreds of reactions and pathways, and 4) metabolomics as a practical tool has only recently emerged.

And it is on this last point that we leave the reader with some final considerations. We imagine that, after careful review of the information outlined in this series, many readers will be motivated to explore the use of metabolomics in their research. However, as outlined throughout this series, mature technologies have only recently arisen. Nevertheless, there are many laboratories that perform some version of “metabolomics”. Although the experimental goal often dictates the precise approach, there are 5 critical features that a metabolomic technology must harbor in order for it to achieve a similar purpose as mature omic technologies (e.g. DNA sequencers) in terms of depth of coverage and data quality. These minimally include:

Must be based on an authenticated chemical library
2. Must have procedures for eliminated noise from the data
5. Must have a mechanism to identify novel metabolites
6. Must have robust QC process from sample preparation through statistical analysis
4. Must provide a mechanism to abstract information/interpret the data

References

Eckhart, A.D., Beebe, K. & Milburn, M. Metabolomics as a key integrator for “omic” advancement of personalized medicine and future therapies. Clin Transl Sci 5, 285-288

(2012).

Evans, A., Mitchell, M., Dai, H. & DeHaven, C.D. Categorizing Ion –Features in Liquid Chromatography/Mass Spectrometry Metobolomics Data. Metabolomics 2 (2012).
DeHaven, C.D., Evans, A., Dai, H. & Lawton, K.A. in Metabolomics. (ed. U. Roessner) (InTech, 2012).
Dehaven, C.D., Evans, A.M., Dai, H. & Lawton, K.A. Organization of GC/MS and LC/MS metabolomics data into chemical libraries. J Cheminform 2, 9 (2010).
Evans, A.M., DeHaven, C.D., Barrett, T., Mitchell, M. & Milgram, E. Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Anal Chem 81, 6656-6667 (2009).

Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M Magnusdottir, MM, et al.

Metabolomics Aug 14, 2014; http://dx.doi.org:/10.1007/s11306-014-0721-3
http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

intra- extracellular metabolites

http://link.springer.com/static-content/images/404/
art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework to analyze information-rich omics data sets, and are increasingly being used to investigate metabolic alternations in human diseases. An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data. Herein, we describe a workflow for such an integrative analysis emphasizing on extracellular metabolomics data. We demonstrate, using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM, how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting a more glycolytic phenotype for the CCRF-CEM model and a more oxidative phenotype for the Molt-4 model, which was supported by our experimental data. Gene expression analysis revealed altered expression of gene products at key regulatory steps in those central metabolic pathways, and literature query emphasized the role of these genes in cancer metabolism. Moreover, in silico gene knock-outs identified unique control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model. Thus, our workflow is well suited to the characterization of cellular metabolic traits based on extracellular metabolomic data, and it allows the integration of multiple omics data sets into a cohesive picture based on a defined model context.

Metabolome Informatics Research

Identification of Metabolites in the Normal Ovary and Their Transformation in Primary and Metastatic Ovarian Cancer MOC vs EOC

Genomics and Cancer

Identification of Gene Networks Associated with Acute Myeloid Leukemia by Comparative Molecular Methylation and Expression Profiling

M Dellett, KA O’Hagan, HA Alexandra Colyer and KI Mills
Biomarkers in Cancer 2010:2 43–55 http://www.la-press.com.

Around 80% of acute myeloid leukemia (AML) patients achieve a complete remission, however many will relapse and ultimately die of their disease. The association between karyotype and prognosis has been studied extensively and identified patient cohorts as having favourable [e.g. t(8; 21), inv (16)/t(16; 16), t(15; 17)], intermediate [e.g. cytogenetically normal (NK-AML)] or adverse risk [e.g. complex karyotypes]. Previous studies have shown that gene expression profiling signatures can classify the sub-types of AML, although few reports have shown a similar feature by using methylation markers. The global methylation patterns in 19 diagnostic AML samples were investigated using the Methylated CpG Island Amplification Microarray (MCAM) method and CpG island microarrays containing 12,000 CpG sites. The first analysis, comparing favourable and intermediate cytogenetic risk groups, revealed significantly differentially methylated CpG sites (594 CpG islands) between the two subgroups. Mutations in the NPM1 gene occur at a high frequency (40%) within the NK-AML subgroup and are associated with a more favourable prognosis in these patients. A second analysis comparing the NPM1 mutant and wild-type research study subjects again identified distinct methylation profiles between these two subgroups. Network and pathway analysis revealed possible molecular mechanisms associated with the different risk and/or mutation sub-groups. This may result in a better classification of the risk groups, improved monitoring targets, or the identification of novel molecular therapies.

Molecular Imaging of Proteases in Cancer

Yunan Yang, Hao Hong, Yin Zhang and Weibo Cai
Cancer Growth and Metastasis 2009:2 13–27. http://www.la-press.com

Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction.

Evolutionarily conserved genetic interactions with budding and fission yeast MutS identify orthologous relationships in mismatch repair-deficient cancer cells

E Tosti, JA Katakowski, S Schaetzlein, Hyun-Soo Kim, CJ Ryan, M Shales, et al.
Genome Medicine 2014, 6:68. http://genomemedicine.com/content/6/9/68

Background: The evolutionarily conserved DNA mismatch repair (MMR) system corrects base-substitution and insertion-deletion mutations generated during erroneous replication. The mutation or inactivation of many MMR factors strongly predisposes to cancer, where the resulting tumors often display resistance to standard chemotherapeutics. A new direction to develop targeted therapies is the harnessing of synthetic genetic interactions, where the simultaneous loss of two otherwise non-essential factors leads to reduced cell fitness or death. High-throughput screening in human cells to directly identify such interactors for disease-relevant genes is now widespread, but often requires extensive case-by-case optimization. Here we asked if conserved genetic interactors (CGIs) with MMR genes from two evolutionary distant yeast species (Saccharomyces cerevisiae and Schizosaccharomyzes pombe) can predict orthologous genetic relationships in higher eukaryotes.
Methods: High-throughput screening was used to identify genetic interaction profiles for the MutSα and MutSβ heterodimer subunits (msh2Δ, msh3Δ, msh6Δ) of fission yeast. Selected negative interactors with MutSβ (msh2Δ/msh3Δ) were directly analyzed in budding yeast, and the CGI with SUMO-protease Ulp2 further examined after RNA interference/drug treatment in MSH2-deficient and -proficient human cells.
Results: This study identified distinct genetic profiles for MutSα and MutSβ, and supports a role for the latter in recombinatorial DNA repair. Approximately 28% of orthologous genetic interactions with msh2Δ/msh3Δ are conserved in both yeasts, a degree consistent with global trends across these species. Further, the CGI between budding/fission yeast msh2 and SUMO-protease Ulp2 is maintained in human cells (MSH2/SENP6), and enhanced by Olaparib, a PARP inhibitor that induces the accumulation of single-strand DNA breaks. This identifies SENP6 as a promising new target for the treatment of MMR-deficient cancers.
Conclusion: Our findings demonstrate the utility of employing evolutionary distance in tractable lower eukaryotes to predict orthologous genetic relationships in higher eukaryotes. Moreover, we provide novel insights into the genome maintenance functions of a critical DNA repair complex and propose a promising targeted treatment for MMR deficient tumors.

Cancer Genome Landscapes

B Vogelstein, N Papadopoulos, VE Velculescu, S Zhou, LA Diaz Jr., KW Kinzler, et al.
Science 339, 1546 (2013); http://dx.doi.org:/10.1126/science.1235122

Over the past decade, comprehensive sequencing efforts have revealed the genomic landscapes of common forms of human cancer. For most cancer types, this landscape consists of a small number of “mountains” (genes altered in a high percentage of tumors) and a much larger number of “hills” (genes altered infrequently). To date, these studies have revealed ~140 genes that, when altered by intragenic mutations, can promote or “drive” tumorigenesis. A typical tumor contains two to eight of these “driver gene” mutations; the remaining mutations are passengers that confer no selective growth advantage. Driver genes can be classified into 12 signaling pathways that regulate three core cellular processes: cell fate, cell survival, and genome maintenance. A better understanding of these pathways is one of the most pressing needs in basic cancer research. Even now, however, our knowledge of cancer genomes is sufficient to guide the development of more effective approaches for reducing cancer morbidity and mortality.

Approaches for establishing the function of regulatory genetic variants involved in disease

Julian Charles Knight
Genome Medicine 2014, 6:92. http://genomemedicine.com/content/6/10/92

The diversity of regulatory genetic variants and their mechanisms of action reflect the complexity and context-specificity of gene regulation. Regulatory variants are important in human disease and defining such variants and establishing mechanism is crucial to the interpretation of disease-association studies. This review describes approaches for identifying and functionally characterizing regulatory variants, illustrated using examples from common diseases. Insights from recent advances in resolving the functional epigenomic regulatory landscape in which variants act are highlighted, showing how this has enabled functional annotation of variants and the generation of hypotheses about mechanism of action. The utility of quantitative trait mapping at the transcript, protein and metabolite level to define association of specific genes with particular variants and further inform disease associations are reviewed. Establishing mechanism of action is an essential step in resolving functional regulatory variants, and this review describes how this is being facilitated by new methods for analyzing allele-specific expression, mapping chromatin interactions and advances in genome editing. Finally, integrative approaches are discussed together with examples highlighting how defining the mechanism of action of regulatory variants and identifying specific modulated genes can maximize the translational utility of genome-wide association studies to understand the pathogenesis of diseases and discover new drug targets or opportunities to repurpose existing drugs to treat them.

Biomarkers

TRIM29 as a Novel Biomarker in Pancreatic Adenocarcinoma

Hongli Sun, Xianwei Dai, and Bing Han
Disease Markers 2014, Article ID 317817, 7 pages
http://dx.doi.org/10.1155/2014/317817

Background and Aim. Tripartite motif-containing 29 (TRIM29) is structurally a member of the tripartite motif family of proteins and is involved in diverse human cancers. However, its role in pancreatic cancer remains unclear.
Methods. The expression pattern of TRIM29 in pancreatic ductal adenocarcinoma was assessed by immunocytochemistry. Multivariate logistic regression analysis was used to investigate the association between TRIM29 and clinical characteristics. In vitro analyses by scratch wound healing assay and invasion assays were performed using the pancreatic cancer cell lines.
Results. Immunohistochemical analysis showed TRIM29 expression in pancreatic cancer tissues was significantly higher (𝑛 = 186) than that in matched adjacent nontumor tissues. TRIM29 protein expression was significantly correlated with lymph node metastasis (𝑃 = 0.019). Patients with positive TRIM29 expression showed both shorter overall survival and shorter recurrence-free survival than those with negative TRIM29 expression. Multivariate analysis revealed that TRIM29 was an independent factor for pancreatic cancer over survival (HR = 2.180, 95% CI: 1.324–4.198, 𝑃 = 0.011). In vitro, TRIM29 knockdown resulted in inhibition of pancreatic cancer cell proliferation, migration, and invasion.
Conclusions. Our results indicate that TRIM29 promotes tumor progression and may be a novel prognostic marker for pancreatic ductal adenocarcinoma.

Bioinformatic identification of proteins with tissue-specific expression for biomarker discovery

I Prassas, CC Chrystoja, S Makawita1, and EP Diamandis
BMC Medicine 2012, 10:39. http://www.biomedcentral.com/1741-7015/10/39

Background: There is an important need for the identification of novel serological biomarkers for the early detection of cancer. Current biomarkers suffer from a lack of tissue specificity, rendering them vulnerable to nondisease-specific increases. The present study details a strategy to rapidly identify tissue-specific proteins using bioinformatics.
Methods: Previous studies have focused on either gene or protein expression databases for the identification of candidates. We developed a strategy that mines six publicly available gene and protein databases for tissue-specific proteins, selects proteins likely to enter the circulation, and integrates proteomic datasets enriched for the cancer secretome to prioritize candidates for further verification and validation studies.
Results: Using colon, lung, pancreatic and prostate cancer as case examples, we identified 48 candidate tissuespecific biomarkers, of which 14 have been previously studied as biomarkers of cancer or benign disease. Twenty six candidate biomarkers for these four cancer types are proposed.
Conclusions: We present a novel strategy using bioinformatics to identify tissue-specific proteins that are potential cancer serum biomarkers. Investigation of the 26 candidates in disease states of the organs is warranted

The Serum Glycome to Discriminate between Early-Stage Epithelial Ovarian Cancer and Benign Ovarian Diseases

K Biskup, E Iona Braicu, J Sehouli, R Tauber, and V Blanchard
Disease Markers 2014, Article ID 238197, 10 pages
http://dx.doi.org/10.1155/2014/238197

Epithelial ovarian cancer (EOC) is the sixth most common cause of cancer deaths in women because the diagnosis occurs mostly when the disease is in its late-stage. Current diagnostic methods of EOC show only a moderate sensitivity, especially at an early-stage of the disease; hence, novel biomarkers are needed to improve the diagnosis. We recently reported that serum glycome modifications observed in late-stage EOC patients by MALDI-TOF-MS could be combined as a glycan score named GLYCOV that was calculated from the relative areas of the 11 N-glycan structures that were significantly modulated. Here, we evaluated the ability of GLYCOV to recognize early-stage EOC in a cohort of 73 individuals comprised of 20 early-stage primary serous EOC, 20 benign ovarian diseases (BOD), and 33 age-matched healthy controls. GLYCOV was able to recognize stage I EOC whereas CA125 values were statistically significant only for stage II EOC patients. In addition, GLYCOV was more sensitive and specific compared to CA125 in distinguishing early-stage EOC from BOD patients, which is of high relevance to clinicians as it is difficult for them to diagnose malignancy prior to operation.

The Clinicopathological Significance of miR-133a in Colorectal Cancer

Timothy Ming-Hun Wan, Colin Siu-Chi Lam, Lui Ng, Ariel Ka-Man Chow, et al.
Disease Markers 2014, Article ID 919283, 8 pages http://dx.doi.org/10.1155/2014/919283

This study determined the expression of microRNA-133a (MiR-133a) in colorectal cancer (CRC) and adjacent normal mucosa samples and evaluated its clinicopathological role in CRC. The expression of miR-133a in 125 pairs of tissue samples was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and correlated with patient’s clinicopathological data by statistical analysis. Endogenous expression levels of several potential target genes were determined by qRT-PCR and correlated using Pearson’s method. MiR-133a was downregulated in 83.2% of tumors compared to normal mucosal tissue. Higher miR-133a expression in tumor tissues was associated with development of distant metastasis, advanced Dukes and TNM staging, and poor survival. The unfavorable prognosis of higher miR-133a expression was accompanied by dysregulation of potential miR-133a target genes, LIM and SH3 domain protein 1 (LASP1), Caveolin-1 (CAV1), and Fascin-1 (FSCN1). LASP1 was found to possess a negative correlation (𝛾 = −0.23), whereas CAV1 exhibited a significant positive correlation (𝛾 = 0.27), and a stronger correlation was found in patients who developed distant metastases (𝛾 = 0.42). In addition, a negative correlation of FSCN1 was only found in nonmetastatic patients. In conclusion, miR-133a was downregulated in CRC tissues, but its higher expression correlated with adverse clinical characteristics and poor prognosis.

The Clinical Significance of PR, ER, NF-𝜅B, and TNF-𝛼 in Breast Cancer

Xian-Long Zhou, Wei Fan, Gui Yang, and Ming-Xia Yu
Disease Markers 2014, Article ID 494581, 7 pages http://dx.doi.org/10.1155/2014/494581

Objectives. To investigate the expression of estrogen (ER), progesterone receptors (PR), nuclear factor-𝜅B (NF-𝜅B), and tumor necrosis factor-𝛼 (TNF-𝛼) in human breast cancer (BC), and the correlation of these four parameters with clinicopathological features of BC.
Methods and Results. We performed an immunohistochemical SABC method for the identification of ER, PR, NF-𝜅B, and TNF-𝛼 expression in 112 patients with primary BC.The total positive expression rate of ER, PR, NF-𝜅B, and TNF-𝛼 was 67%, 76%, 84%, and 94%, respectively. The expressions of ER and PR were correlated with tumor grade, TNM stage, and lymph node metastasis (𝑃 < 0.01, resp.), but not with age, tumor size, histological subtype, age at menarche, menopause status, number of pregnancies, number of deliveries, and family history of cancer. Expressions of ER and PR were both correlated with NF-𝜅B and TNF-𝛼 expression (𝑃 < 0.05, resp.). Moreover, there was significant correlation between ER and PR (𝑃 < 0.0001) as well as between NF-𝜅B and TNF-𝛼 expression (𝑃 < 0.05).
Conclusion. PR and ER are highly expressed, with significant correlation with NF-𝜅B and TNF-𝛼 expression in breast cancer. The important roles of ER and PR in invasion and metastasis of breast cancer are probably associated with NF-𝜅B and TNF-𝛼 expression.

Serum Protein Profile at Remission Can Accurately Assess Therapeutic Outcomes and Survival for Serous Ovarian Cancer

J Wang, A Sharma, SA Ghamande, S Bush, D Ferris, W Zhi, et la.
PLoS ONE 8(11): e78393. http://dx.doi.org:/10.1371/journal.pone.0078393

Background: Biomarkers play critical roles in early detection, diagnosis and monitoring of therapeutic outcome and recurrence of cancer. Previous biomarker research on ovarian cancer (OC) has mostly focused on the discovery and validation of diagnostic biomarkers. The primary purpose of this study is to identify serum biomarkers for prognosis and therapeutic outcomes of ovarian cancer. Experimental Design: Forty serum proteins were analyzed in 70 serum samples from healthy controls (HC) and 101 serum samples from serous OC patients at three different disease phases: post diagnosis (PD), remission (RM) and recurrence (RC). The utility of serum proteins as OC biomarkers was evaluated using a variety of statistical methods including survival analysis.
Results: Ten serum proteins (PDGF-AB/BB, PDGF-AA, CRP, sFas, CA125, SAA, sTNFRII, sIL-6R, IGFBP6 and MDC) have individually good area-under-the-curve (AUC) values (AUC = 0.69–0.86) and more than 10 three-marker combinations have excellent AUC values (0.91–0.93) in distinguishing active cancer samples (PD & RC) from HC. The mean serum protein levels for RM samples are usually intermediate between HC and OC patients with active cancer (PD & RC). Most importantly, five proteins (sICAM1, RANTES, sgp130, sTNFR-II and sVCAM1) measured at remission can classify, individually and in combination, serous OC patients into two subsets with significantly different overall survival (best HR = 17, p,1023).
Conclusion: We identified five serum proteins which, when measured at remission, can accurately predict the overall survival of serous OC patients, suggesting that they may be useful for monitoring the therapeutic outcomes for ovarian cancer.

Serum Clusterin as a Tumor Marker and Prognostic Factor for Patients with Esophageal Cancer

Wei Guo, Xiao Ma, Christine Xue, Jianfeng Luo, Xiaoli Zhu, et al.
Disease Markers 2014, Article ID 168960, 7 pages http://dx.doi.org/10.1155/2014/168960

Background. Recent studies have revealed that clusterin is implicated in many physiological and pathological processes, including tumorigenesis. However, the relationship between serum clusterin expression and esophageal squamous cell carcinoma (ESCC) is unclear.
Methods. The serum clusterin concentrations of 87 ESCC patients and 136 healthy individuals were examined. An independent-samples Mann-Whitney 𝑈 test was used to compare serum clusterin concentrations of ESCC patients to those of healthy controls. Univariate analysis was conducted using the log-rank test and multivariate analyses were performed using the Cox proportional hazards model. Results. In healthy controls, the mean clusterin concentration was 288.8 ± 75.1 𝜇g/mL, while in the ESCC patients, the mean clusterin concentration was higher at 412.3±159.4 𝜇g/mL (𝑃 < 0.0001). The 1-, 2-, and 4-year survival rates for the 87 ESCC patients were 89.70%, 80.00%, and 54.50%. Serum clusterin had an optimal diagnostic cut-off point (serum clusterin concentration = 335.5 𝜇g/mL) for esophageal squamous cell carcinoma with sensitivity of 71.26% and specificity of 77.94%. And higher serum clusterin concentration (>500 𝜇g/mL) indicated better prognosis (𝑃 = 0.030).
Conclusions. Clusterin may play a key role during tumorigenesis and tumor progression of ESCC and it could be applied in clinical work as a tumor marker and prognostic factor.

Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer

JD Warren, Wei Xiong, AM Bunker, CP Vaughn, LV Furtado, et al.
BMC Medicine 2011, 9:133. http://www.biomedcentral.com/1741-7015/9/133

Background: About half of Americans 50 to 75 years old do not follow recommended colorectal cancer (CRC) screening guidelines, leaving 40 million individuals unscreened. A simple blood test would increase screening compliance, promoting early detection and better patient outcomes. The objective of this study is to demonstrate the performance of an improved sensitivity blood-based Septin 9 (SEPT9) methylated DNA test for colorectal cancer. Study variables include clinical stage, tumor location and histologic grade.
Methods: Plasma samples were collected from 50 untreated CRC patients at 3 institutions; 94 control samples were collected at 4 US institutions; samples were collected from 300 colonoscopy patients at 1 US clinic prior to endoscopy. SEPT9 methylated DNA concentration was tested in analytical specimens, plasma of known CRC cases, healthy control subjects, and plasma collected from colonoscopy patients.
Results: The improved SEPT9 methylated DNA test was more sensitive than previously described methods; the test had an overall sensitivity for CRC of 90% (95% CI, 77.4% to 96.3%) and specificity of 88% (95% CI, 79.6% to 93.7%), detecting CRC in patients of all stages. For early stage cancer (I and II) the test was 87% (95% CI, 71.1% to 95.1%) sensitive. The test identified CRC from all regions, including proximal colon (for example, the cecum) and had a 12% false-positive rate. In a small prospective study, the SEPT9 test detected 12% of adenomas with a false-positive rate of 3%.
Conclusions: A sensitive blood-based CRC screening test using the SEPT9 biomarker specifically detects a majority of CRCs of all stages and colorectal locations. The test could be offered to individuals of average risk for CRC who are unwilling or unable to undergo colonoscopy.

Matrix Metalloproteinases in Cancer: Prognostic Markers and Therapeutic Targets

Pia Vihinen And Veli-Matti K¨Ah¨Ari
Int. J. Cancer 2002; 99: 157–166 http://dx.doi.org:/10.1002/ijc.10329

Degradation of extracellular matrix is crucial for malignant tumour growth, invasion, metastasis and angiogenesis. Matrix metalloproteinases (MMPs) are a family of zinc-dependent neutral endopeptidases collectively capable of degrading essentially all matrix components. Elevated levels of distinct MMPs can be detected in tumour tissue or serumof patients with advanced cancer and their role as prognostic indicators in cancer is studied. In addition, therapeutic intervention of tumour growth and invasion based on inhibition of MMP activity is under intensive investigation and several MMP inhibitors are in clinical trials in cancer. In this review, we discuss the current view on the feasibility of MMPs as prognostic markers and as targets for therapeutic intervention in cancer.

Mass Spectrometric Screening of Ovarian Cancer with Serum Glycans

Jae-Han Kim, Chang Won Park, Dalho Um, Ki Hwang Baek, Yohahn Jo, et al.
Disease Markers 2014, Article ID 634289, 9 pages
http://dx.doi.org/10.1155/2014/634289

development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between 𝑃 value of ANOVA and area under the curve (AUC) of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multi-biomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80∼90% sensitivity, and 70∼83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81∼84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74%in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.

Large, Collaborative Lung Cancer Trial Goes for Precision Medicine Goal

News | June 30, 2014 | Lung Cancer Targets

By Anna Azvolinsky, PhD

In a new biomarker-focused clinical trial, five therapies will be tested to develop new, precision medicine approaches to treat squamous cell lung cancer. The Lung Cancer Master Protocol (Lung-MAP)/SWOG S1400 phase 2/3 clinical trial, brings together the National Cancer Institute (NCI), the Foundation for the National Institutes of Health (FNIH), SWOG Cancer Research, five pharmaceutical companies (Amgen, AstraZeneca, Genentech, MedImmune, and Pfizer), Foundation Medicine (a molecular informatics company), and Friends of Cancer Research, a non-profit foundation.

The trial aims to enroll about 10,000 patients total and will cost about $160 million, of which the NCI is contributing $25 million.

Lung-MAP is unique as this is the first public-private partnership in drug development that includes the NCI, the Food and Drug Administration (FDA), U.S. oncology cooperative groups, and a number of patient advocacy groups according to one of the study investigators, David Gandara, MD, chair of the SWOG lung committee, and thoracic oncologist at the UC Davis Cancer Center. “Funds are made available for every aspect of the trial,” said Gandara. “There is nothing in the history of oncology or drug development like it.”

The clinical trial seeks to identify molecular aberrations in patients with advanced squamous cell lung cancer that can be targeted either by existing therapies or through the development of new ones. The innovation of this trial is a master protocol that will rely on the strength of numbers—up to 1000 patients per year at more than 200 sites throughout the U.S. for more than 200 cancer-related genetic alterations. Testing results will then dictate which experimental trial arm is most appropriate for which patient. Unlike a trial that seeks to enroll patients harboring just one mutation, which limits the access for many patients, the Lung-MAP design better ensures that a patient who is screened will be eligible for a targeted therapy trial arm.

This type of umbrella trial design is particularly suitable for squamous cell lung cancer. Thus far, has not been defined by one or several driver mutations. Instead, these tumors are made of a spectrum of genetic aberrations that are each relatively rare within the squamous lung cancer patient population, making enrollment into targeted therapy clinical trials difficult. According to the NCI, Lung-MAP “aims to establish a model of clinical testing that more efficiently meets the needs of both patients and drug developers,” facilitating more efficient matching of a patient to an investigational targeted therapy trial.

Lung-MAP was specifically designed for squamous cell lung cancer because this lung cancer subtype represents the greatest unmet need for new treatment, Gandara told OncoTherapy Network:

“All of the dramatic advances that have been made in the treatment of lung cancer over the last ten years have occurred in adenocarcinoma, a lung cancer subtype with several recently recognized and ‘druggable oncogenes’ such as EGFR mutations or ALK translocations. However, there have been essentially no advances in squamous cell lung cancer.”

But, recent genome-wide studies have identified several gene alterations in squamous cell lung cancer that are also druggable, including PI3K, FGFR, and CDK mutations, said Gandara. The trial is initially testing four targeted therapies: Genentech’s GDC-0032 (a PI3 kinase inhibitor), Pfizer’s palbociclib (an oral cyclin-dependent-kinase 4/6 inhibitor, AZD4547), an oral fibroblast growth factor receptor inhibitor from AstraZeneca, and rilotumumab, Amgen’s antibody against the human hepatocyte growth factor.

The fifth agent is, MEDI4736, an immune checkpoint inhibitor antibody targeting PD-L1. Patients whose tumors do not harbor a mutation suitable for targeting with one of the four targeted therapies will be enrolled in the MED4736 sub-study.

Once a patient is matched to a specific trial sub-study, randomization will determine whether the patient receives the experimental therapy or standard of care chemotherapy. The planned trial endpoints for each sub-study are overall survival and progression-free survival.

“I cannot overemphasize the importance of the FDA’s participation in this project, since each of these sub-studies is designed to result in approval of a paired biomarker and new drug if that sub-study meets the requirements for improved effectiveness,” said Gandara.

– See more at: http://www.oncotherapynetwork.com/lung-cancer-targets/large-collaborative-lung-cancer-trial-goes-precision-medicine-goal

The BATTLE Trial: Personalizing Therapy for Lung Cancer

Kim, RS. Herbst, II. Wistuba, JJ Lee, GR. Blumenschein Jr., A Tsao, DJ. Stewart, et al.

Authors’ Affiliations: 1Departments of Thoracic/Head and Neck Medical Oncology, 2Pathology, 3Biostatistics, and 4Diagnostic Radiology, The University of Texas MD Anderson Cancer Center, Houston, Texas; 5Winship Cancer Center, Emory University, Atlanta, Georgia; 6Dana-Farber Cancer Institute, Boston, Massachusetts; and 7University of Maryland, Baltimore, Maryland.

Corresponding Author:

Waun K. Hong, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: 713-794-1441; Fax: 1-713-792-4654; E-mail:whong@mdanderson.org

The Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) trial represents the first completed prospective, biopsy-mandated, biomarker-based, adaptively randomized study in 255 pretreated lung cancer patients. Following an initial equal randomization period, chemorefractory non–small cell lung cancer (NSCLC) patients were adaptively randomized to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib, based on relevant molecular biomarkers analyzed in fresh core needle biopsy specimens. Overall results include a 46% 8-week disease control rate (primary end point), confirm prespecified hypotheses, and show an impressive benefit from sorafenib among mutant-KRAS patients. BATTLE establishes the feasibility of a new paradigm for a personalized approach to lung cancer clinical trials.

(ClinicalTrials.gov numbers:NCT00409968, NCT00411671, NCT00411632, NCT00410059, and NCT00410189.

Significance: The BATTLE study is the first completed prospective, adaptively randomized study in heavily pretreated NSCLC patients that mandated tumor profiling with “real-time” biopsies, taking a substantial step toward realizing personalized lung cancer therapy by integrating real-time molecular laboratory findings in delineating specific patient populations for individualized treatment. Cancer Discovery; 1(1); 44–53. © 2011 AACR.

Read the Commentary on this article by Sequist et al., p. 14
Read the Commentary on this article by Rubin et al., p. 17
This article is highlighted in the In This Issue feature, p. 4

Pharmacometabolomics in Drug Discovery & Development: Applications and Challenges

Yang and F. Marotta
Metabolomics 2012, 2:5 http://dx.doi.org/10.4172/2153-0769.1000e122

Recently, the concept of pharmaco-metabolomics is mentioned more frequently as an emerging discipline to study the effect of drugs on the whole pattern of small endogenous molecules and in applying the profiles of metabolomics for drug development. For the latter part, metabolomics is majorly used to differentiate patients into responder or non-responder groups in an effort to decrease large inter-individual variation in clinical trials. It is a novel approach that combines metabolite profile and chemo-metrics to model and predict drug targets, efficacy, pharmacokinetics and toxicity on both individual and population basis. It attracts many scientists’ attention because of its intrinsic advantages and promising potentials in drug discovery and development. Considering personalized drug treatment is the desired goal for current drug development, pharmaco-metabolomics provide an effective and inexpensive strategy to evaluate drug efficacy and toxicology, which may make personalized medicine realistic both from scientific and financial perspectives. Furthermore, the FDA also realized that metabolomics coupling with other “Omics” approaches could be a valuable tool in evaluating general toxicology and could eventually replace the use of animals after addressing certain challenges.

Networking metabolites and diseases

Pascal Braun, Edward Rietman, and Marc Vidal
PNAS July 22, 2008; 105(29): 9849–9850

Diseasome and Drug-Target Network

Recently, Goh et al. constructed a ‘‘diseasome’’ network in which two diseases are linked to each other if they share at least one gene, in which mutations are associated with both diseases. In the resulting network, related disease families cluster tightly together, thus phenotypically defining functional modules. Importantly, for the first time this study applied concepts from network biology to human diseases, thus opening the door for discovering causal relationships between disregulated networks and resulting ailments.

Subsequently Yilderim et al. linked drugs to protein targets in a drug–target network, which could then be overlaid with the diseasome network. One notable finding was the recent trend toward the development of new compounds directly targeted at disease gene products, whereas previous drugs, often found by trial and error, appear to target proteins only indirectly related to the actual disease molecular mechanisms. An important question that remains in this emerging field of network analysis consists of investigating the extent to which directly targeting the product of mutated genes is an efficient approach or whether targeting network properties instead, and thereby accounting for indirect nonlinear effects of system perturbations by drugs, may prove more fruitful. However, to answer such questions it is important to have a good understanding of the various influences that can lead to diseases.

UPDATED 6/01/2019

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

from Nat Genet. 2015 Mar;47(3):257-62. doi: 10.1038/ng.3202. Epub 2015 Feb 2.

Shlien A¹, Campbell BB², de Borja R³, Alexandrov LB⁴, Merico D⁵, Wedge D⁴, Van Loo P⁶, Tarpey PS⁴, Coupland P⁷, Behjati S⁴, Pollett A⁸, Lipman T⁹, Heidari A⁹, Deshmukh S⁹, Avitzur N⁹, Meier B¹⁰, Gerstung M⁴, Hong Y¹⁰, Merino DM³, Ramakrishna M⁴, Remke M¹¹, Arnold R³, Panigrahi GB³, Thakkar NP¹², Hodel KP¹³, Henninger EE¹³, Göksenin AY¹³, Bakry D¹⁴, Charames GS¹⁵, Druker H¹⁶, Lerner-Ellis J¹⁷, Mistry M², Dvir R¹⁸, Grant R¹⁴, Elhasid R¹⁸, Farah R¹⁹, Taylor GP²⁰, Nathan PC¹⁴, Alexander S¹⁴, Ben-Shachar S²¹, Ling SC²², Gallinger S²³, Constantini S²⁴, Dirks P²⁵, Huang A²⁶, Scherer SW²⁷, Grundy RG²⁸, Durno C²⁹, Aronson M³⁰, Gartner A¹⁰, Meyn MS³¹, Taylor MD²⁵, Pursell ZF¹³, Pearson CE¹², Malkin D³², Futreal PA⁴, Stratton MR⁴, Bouffet E²⁶, Hawkins C³³, Campbell PJ³⁴, Tabori U³⁵; Biallelic Mismatch Repair Deficiency Consortium.

Abstract: DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation/consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Genetic changes which occur in spontaneous arising somatic cancers include point mutations, copy number alterations and rearrangements and in general result from a defective DNA repair mechanisms during proliferation/replication over many years however as most somatic cancers are heterogeneous it is difficult to pinpoint the exact repair defects which may be ultimately responsible for such genetic aberrations.

However, early-onset cancers (e.g. pediatric cancers) in patients with hereditary DNA repair defects offer a good view of the mutation types and secondary pathways that drive oncogenesis. bMMRD is a childhood cancer syndrome characterized by early-onset cancers in various organs and caused by biallelic mutations. In this study, genomes from 17 inherited cancers, by exomic sequencing and microarrays, were analyzed and compared to non-neoplastic tissue genomes from matched patients. Brain cancers from these patients had an extremely high number of point mutations compared to other childhood cancers and adult cancers.

Mismatch repair was defective in all these cancers therefore it appeared that secondary mutations are required to cause the ultrahypermutated state. The most frequently mutated gene was POLE (polymerase epsilon), affecting the proofreading ability of this DNA polymerase. The genomes of tumors with mutant POLE had signature mutational spectrum and the signature occurred early but these signatures had been found in endometrial and colorectal cancers. The authors concluded, based on serial analysis of other brain cancers with bMMRD and the observation that recurrent brain cancers accumulated mutations over a relatively short period, once the proofreading capability of pol epsilon is compromised in MMR deficient cells there is no defense against rapid and catastrophic accumulations of mutations. This rapid accumulation of mutations apparently do not lead to apoptosis but rather rapid tumor initiation, and generating multiple subclones of tumor cells.

UPDATED 9/26/2021

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism

Source: https://pubmed.ncbi.nlm.nih.gov/31941752/

Abstract

Breast cancer is the most common cancer among American women and a major cause of mortality. To identify metabolic pathways as potential targets to treat metastatic breast cancer, we performed metabolomics profiling on the breast cancer cell line MDA-MB-231 and its tissue-tropic metastatic subclones. Here, we report that these subclones with increased metastatic potential display an altered metabolic profile compared with the parental population. In particular, the mitochondrial serine and one-carbon (1C) unit pathway is upregulated in metastatic subclones. Mechanistically, the mitochondrial serine and 1C unit pathway drives the faster proliferation of subclones through enhanced de novo purine biosynthesis. Inhibition of the first rate-limiting enzyme of the mitochondrial serine and 1C unit pathway, serine hydroxymethyltransferase (SHMT2), potently suppresses proliferation of metastatic subclones in culture and impairs growth of lung metastatic subclones at both primary and metastatic sites in mice. Some human breast cancers exhibit a significant association between the expression of genes in the mitochondrial serine and 1C unit pathway with disease outcome and higher expression of SHMT2 in metastatic tumor tissue compared with primary tumors. In addition to breast cancer, a few other cancer types, such as adrenocortical carcinoma and kidney chromophobe cell carcinoma, also display increased SHMT2 expression during disease progression. Together, these results suggest that mitochondrial serine and 1C unit metabolism plays an important role in promoting cancer progression, particularly in late-stage cancer. IMPLICATIONS: This study identifies mitochondrial serine and 1C unit metabolism as an important pathway during the progression of a subset of human breast cancers.

ntroduction

The majority of breast cancer patients die from metastatic disease. The process of cancer metastasis involves local invasion into surrounding tissue, dissemination into the bloodstream, extravasation, and eventual colonization of a new tissue. Following a period of dormancy, small numbers of micrometastases eventually proliferate into large macrometastases, or secondary tumors.

Previous studies have illuminated several themes of metabolic reprogramming that occur during metastasis (1–8). However, the majority of these reported site-specific metabolic features of metastatic cancer cells. We reason that breast cancer cells that leave the primary tumor and successfully establish new lesions at distal sites would encounter similar metabolic stresses during metastasis. By performing comparative metabolomics on the MDA-MB-231 human breast cancer cell line and its tissue-tropic metastatic subclones, we uncovered that the catabolism of the non-essential amino acid serine through the mitochondrial one-carbon (1C) unit pathway is an important driver of proliferation in a subset of metastatic breast cancers that closely resembles the molecular features of MDA-MB-231 cells. Emerging evidence shows that the non-essential amino acid serine is essential for cancer cell survival and proliferation. The genomic regions containing PHGDH are amplified in breast cancer and melanoma, diverting 3PG to serine synthesis (9,10). We also reported that PHGDH is upregulated upon amino acid starvation by the transcription factor ATF4 (11). On one hand, serine serves as a precursor for the synthesis of protein, lipids, nucleotides and other amino acids, which are necessary for cell division and growth. On the other hand, serine catabolism through the mitochondrial 1C unit pathway is critical for maintaining cellular redox control under stress conditions (12,13). In mitochondria, serine catabolism is initiated by serine hydroxymethyltransferase 2 (SHMT2). SHMT2 catalyzes a reversible reaction converting serine to glycine, with concurrent generation of the 1C unit donor methylene-THF, which is further oxidized by downstream enzymes MTHFD2 and MTHFD1L to produce NAD(P)H and formate. Subsequent export of formate from the mitochondria can then be re-assimilated into the cytosolic folate pool to support anabolic reactions. All three mitochondrial serine and 1C unit pathway enzymes (SHMT2, MTHFD2 and MTHFD1L) are upregulated in breast tumor samples compared to normal tissues (13,14). However, due to lack of functional investigations targeting this pathway in in vitro and in vivo breast cancer models, it remains unclear whether the mitochondrial 1C unit pathway represents a good target for treating metastatic breast cancer.

In this study, we report that enzymes in the mitochondrial serine and 1C unit pathway are even further upregulated specifically in subclones of the aggressive breast cancer cell line MDA-MB-231 that have been selected in vivo for the ability to preferentially metastasize to specific organs. We demonstrate that SHMT2 inhibition suppresses proliferation more strongly in these highly metastatic subclones compared to the parental population in vitro. Knockdown of SHMT2 also impairs breast cancer growth in vivo at both the primary and metastatic sites. In addition, we find that the expression of mitochondrial 1C unit pathway enzymes significantly associates with poor disease outcome in a subset of human breast cancer patients, potentiating its role as a therapeutic target or biomarker in advanced cancer. Finally, SHMT2 expression increases in breast invasive carcinoma, adrenocortical carcinoma, chromophobe renal cell carcinoma and papillary renal cell carcinoma during tumor progression, particularly in late stage tumors, suggesting that inhibitors targeting SHMT2 may hold promise for treating these late stage cancers when other therapeutic options become limited.

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Materials and Methods

Cell lines

All of the paired parental and metastatic subclones were generated in Dr. Joan Massagué’s laboratory (Memorial Sloan-Kettering Cancer Center) (15–17). Cells were cultured in DMEM/F12 with 10% fetal bovine serum (Sigma) with 1% penicillin/streptomycin. All cells lines were tested every three to six months and found negative for mycoplasma (MycoAlert Mycoplasma Detection Kit; Lonza). These cell lines were not authenticated by the authors. All cell lines used in experiments were passaged no more than ten times from time of thawing.

RNAi

Stable 831-BrM,1833-BoM, and 4175-LM cell lines expressing shRNA against SHMT2, MTHFD2, and c-Myc were generated through infection with lentivirus and 1 μg/mL puromycin selection. shRNA-expressing virus was obtained using a previously published method (13). Pooled populations were tested for on-target knockdown by immunoblot.

Immunoblot

The following antibodies were used: SHMT1, SHMT2 (Sigma), MTHFD2, MTHFD1L, c-Myc, Actin (Cell Signaling Technologies).

RNA Isolation, Reverse Transcription, and Real-Time PCR

Total RNA was isolated from tissue culture plates according to the TRIzol Reagant (Invitrogen) protocol. 3 μg of total RNA was used in the reverse transcription reaction using the SuperScript III (Invitrogen) protocol. Quantitative PCR amplification was performed on the Prism 7900 Sequence Detection System (Applied Biosystems) using Taqman Gene Expression Assays (Applied Biosystems). Gene expression data were normalized to 18S rRNA.

In vivo Tumor Growth Assays

All procedures involving animals and their care were approved by the Institutional Animal Care and Use Committee of Stanford University in accordance with institutional and National Institutes of Health guidelines. For orthotopic growth studies, 4175-LM shNT and 4175-LM shSHMT2 cells (1 × 10⁶ cells in 0.1 mL of PBS, n = 8 per group) were injected into the flanks of NU/J 10-week-old female mice (The Jackson Laboratory). Tumors were measured with calipers over a 50-day time course. Volumes were calculated using the formula width² × length × 0.5.

For lung metastasis assays, 4175-LM shNT and 4175-LM shSHMT2 cells (0.2 × 10⁵ cells, n = 8 per group) were injected via tail vein into 6–8 week-old female NOD SCID mice. Mice were imaged weekly using the Xenogen IVIS 200 (PerkinElmer, Waltham, MA). Briefly, mice were injected intraperitoneally with 100 μg/g of D-luciferin (potassium salt; PerkinElmer) on the day of imaging. 8 min later, mice were anesthetized in an anesthesia-induction chamber using a mixture of 3% isoflurane (Fluriso, VetOne) in O₂. Anesthesia was maintained with a mixture of 2% isoflurane in O₂ inside the imaging chamber. Using Living Image (PerkinElmer, Waltham, MA), images were acquired (Exposure time, auto; F stop. 1.2; Binning, medium) from both dorsal and ventral sides of mice and a total photon flux (p/sec/cm²/sr) per animal was calculated by averaging the signal acquired from the dorsal and ventral side. After 4 weeks, surviving mice were sacrificed and lungs snap frozen in liquid N₂ prior to homogenization in TRIzol for RNA extraction.

Metabolite Profiling and Mass Spectrometry

For total metabolite analysis, parental and metastatic cell lines were seeded in 60mm culture dishes in DMEM/F12 supplemented with 10% dialyzed fetal bovine serum. Media was refreshed 2 hours prior to harvesting by washing 3x with PBS before quenching with 800mL of −80 C 80:20 methanol:water. Extracts were spun down, supernatants collected, dried and resuspended in water before LC-MS analysis. Samples were analyzed by reversed-phase ion-pairing chromatography coupled with negative-mode electrospray-ionization high-resolution MS on a stand-alone ThermoElectron Exactive orbitrap mass spectrometer (18). Peak picking and quantification were conducted using MAVEN analysis software. Heatmap was generated in R. Multiple testing correction and q-value generation were performed in PRISM software (GraphPad).

For [2,3,3-²H]serine labeling experiments, parental and metastatic cells were cultured in RPMI medium lacking glucose, serine, and glycine (TEKnova) supplemented with 2 g/L glucose and 0.03 g/L [2,3,3-²H]serine (Cambridge Isotope Laboratories) for up to 24 hours before harvesting. Cells were washed twice with ice-cold PBS prior to extraction with 400 μL of 80:20 acetonitrile:water over ice for 15 min. Cells were scraped off plates to be collected with supernatants, sonicated for 30s, then spun down at 1.5 × 10⁴ RPM for 10 min. 200 μL of supernatant was taken out for LC-MS/MS analysis immediately.

Quantitative LC-ESI-MS/MS analysis of [2,3,3-²H]serine-labeled cell extracts was performed using an Agilent 1290 UHPLC system equipped with an Agilent 6545 Q-TOF mass spectrometer (Santa Clara, CA, US). A hydrophilic interaction chromatography method (HILIC) with an BEH amide column (100 × 2.1 mm i.d., 1.7 μm; Waters) was used for compound separation at 35 °C with a flow rate of 0.3ml/min. The mobile phase A consisted of 25 mM ammonium acetate and 25mM ammonium hydroxide in water and mobile phase B was acetonitrile. The gradient elution was 0–1 min, 85 % B; 1–12 min, 85 % B → 65 % B; 12– 12.2 min, 65 % B-40%B; 12.2–15 min, 40%B. After the gradient, the column was re-equilibrated at 85%B for 5min. The overall runtime was 20 min and the injection volume was 5 μL. Agilent Q-TOF was operated in negative mode and the relevant parameters were as listed: ion spray voltage, 3500 V; nozzle voltage, 1000 V; fragmentor voltage, 125 V; drying gas flow, 11 L/min; capillary temperature, 325 °C, drying gas temperature, 350 °C; and nebulizer pressure, 40 psi. A full scan range was set at 50 to 1600 (m/z). The reference masses were 119.0363 and 980.0164. The acquisition rate was 2 spectra/s. Isotopologues extraction was performed in Agilent Profinder B.08.00 (Agilent Technologies). Retention time (RT) of each metabolite was determined by authentic standards (Supplementary Table S1). The mass tolerance was set to +/−15 ppm and RT tolerance was +/− 0.2 min. Natural isotope abundance was corrected using Agilent Profinder software (Agilent Technologies).

Cell Line Classification

Cell line expression and copy number data were downloaded from the COSMIC cell line dataset (https://cancer.sanger.ac.uk/cell_lines), and all cell lines were classified using different cell line classifiers, including PAM50 and scmod2 using the package genefu from Bioconductor; and iC10 using package iC10 (19–22). The MDA-MB-231 parental and metastatic subclones were classified as Basal (posterior probability of 0.516), ER-Her2- (posterior probability of 0.997), IC4 (posterior probability of 0.999).

Outcome Analysis

METABRIC clinical and expression data was downloaded from EGA (EGAS00000000083) (21). Outcome analysis was performed in IC4 samples only (N=342) in order to mimic the phenotype of the MDA-MB-231 breast cancer cell line. Survival analysis was performed over disease specific survival (DSS) censored to 20 years. Gene high/low categorization was performed using the maxstat algorithm, which determines the optimal threshold for separating high and low expression (from the surv cutpoint function of package survminer). Cox Proportional Hazard multivariate models use continuous expression adjusted by age, grade, size, number of lymph nodes, ER, PR and Her2 status. Kaplan-Meier plots were generated using the package survcomp, and Cox Proportional Hazards were generated using the package rms.

Immunohistochemical Staining and Quantification for SHMT2

Human primary breast cancer tissue and paired lymph node metastases were obtained from Biomax.us. Tumors were graded by Biomax.us pathologists according to the Nottingham grading system with respect to degree of glandular duct formation, nuclear pleomorphism, and nuclear fission counting. Each feature was scored from 1–3, and the total score was used to determine the following grades: Grade 1 (total score 3–5; low grade or well differentiated), Grade 2 (total score 6–7; intermediate grade or moderately differentiated), Grade 3 (total score 8–9; high grade or poorly differentiated). Standard immunohistochemical methods were performed as previously described (23). The primary anti-human SHMT2 antibody (Sigma) was used at a concentration of 1:3000. Images were acquired on a Leica DMi8 system (Leica Microsystems) and quantified for positive SHMT2 signal intensity by ImageJ software.

SHMT2 Expression Analysis by Individual Cancer Stage

SHMT2 expression data across every annotated TCGA cancer data set was queried and downloaded from the UALCAN database (http://ualcan.path.uab.edu/index.html) (24).

Statistical Analyses

All statistical tests were performed using the paired or unpaired Student’s t test by PRISM software. Values with a p value of < 0.05 were considered significant.

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Results

Metastatic breast cancer cells exhibit altered metabolic profiles

To identify common metabolic pathways reprogrammed in metastatic breast cancer cells during cancer progression, we performed metabolomic profiling of the human triple negative breast cancer cell line MDA-MB-231 and its metastatic subpopulations (Fig. 1A and andB).B). This cell line was derived from the pleural effusion of a patient with widespread metastatic disease years after primary tumor removal (25), and the subclones of this cell line with higher metastasis rate and preference to the bone, lung, or brain were previously isolated by in vivo selection (15–17) (831-BrM: brain metastasis. 1833-BoM: bone metastasis. 4175-LM: lung metastasis).

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Figure 1.

Metastatic breast cancer subclones display an altered metabolic profile. (A) Schematic of targeted metabolomics workflow. Brain (831-BrM), bone (1833-BoM), and lung (4175-LM) metastatic subclones from tissue-tropic subpopulations were generated following IV injection of a parental population of MDA-MB-231 (231-Parental) cells into the tail vein or heart. Stable cell lines were passaged in culture prior to metabolite extraction for LC-MS/MS. (B) LC-MS profile of the 231-Parental, 831-BrM, and 1833-BoM cell lines. Cell lines were plated in biological triplicates prior to metabolite extraction. Signals were normalized to the mean signal of each metabolite across all samples, log2 transformed, and clustered.

At the time of initial metabolomics comparison, the lung metastatic subclone 4175-LM did not recover well in culture, so we profiled the 831-BrM and 1833-BoM metastatic subclones along with the parental population. We observed multiple metabolites involved in a plethora of metabolic pathways that were differentially enriched or depleted in the metastatic 831-BrM and 1833-BoM subclones compared to the parental population of MDA-MB-231 (231-Parental) cells (Fig. 1B). Following correction for false discovery rate, the levels of twenty-four metabolites were significantly altered in both 831-BrM and 1833-BoM cells compared to 231-Parental cells (Supplementary Table S2). Metabolites significantly enriched in metastatic subclones included the glycolytic intermediate dihydroxyacetone-phosphate (which is reversibly isomerized to glyceraldehyde-3-phosphate), the tricarboxylic acid (TCA) cycle intermediate succinate, amino acids such as proline and asparagine, and the pentose-phosphate pathway product 5-phosphoribosyl-1-pyrophosphate. These observations are consistent with prior observations of perturbations in lower glycolysis and the TCA cycle observed in other cell line models (notably murine 4T1 cells), suggesting common metabolic developments during metastasis of breast cancers in both mice and humans (1–3,5,6). Additionally, enrichment of asparagine has been reported to promote metastatic cancer cell phenotypes by epithelial-to-mesenchymal transition (8). Nonetheless, the most significantly depleted class of metabolites in 831-BrM and 1833-BoM cells compared to 231-Parental cells were free purine nucleotides, suggesting alterations in purine metabolism in metastatic cells (Fig. 1B).

c-Myc is important for breast cancer cell proliferation

We wondered whether reduced levels of purines reflected decreased synthesis or higher consumption in the metastatic subclones. Because it was previously reported that the oncogenic transcription factor c-Myc induces the expression of nucleotide biosynthesis genes and that c-Myc amplification and overexpression is a common event in triple-negative breast cancer (26–28), we wondered if the relative differences in purine abundance could be explained by altered c-Myc protein levels in our cell line system. Indeed, 831-BrM, 1833-BoM, and 4175-LM cells overexpressed c-Myc compared to 231-Parental cells (Fig. 2A). Since sufficiency of free nucleotides can act as an important checkpoint for cell division (29), we then compared the proliferation rates of parental and metastatic subclones. Accordingly, 831-BrM, 1833-BoM, and 4175-LM cells proliferated faster than 231-Parental cells in vitro (Fig. 2B), suggesting that the higher consumption rate is the cause of lower purine levels in the metastatic subclones.

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Figure 2.

c-Myc drives proliferation in metastatic breast cancer cell subclones. (A) IB for c-Myc from whole-cell extracts of parental and metastatic subclones. (B) Proliferation of parental cells and metastatic subclones over 3 days (mean ± SD, n = 3). (C) 3 day proliferation of 231-Parental, 831-BrM, 1833-BoM, and 4175-LM cells expressing either a nontargeting (shNT) or c-Myc targeting (shMyc) vectors. (mean ± SD, n = 3).

Because the role of c-Myc in metastasis is still unclear, with evidence suggesting it plays both pro-metastatic and anti-metastatic functions in breast cancer depending on the genetic context (30,31), we tested the sensitivity of parental and metastatic subclones to c-Myc inhibition. Small hairpin RNA (shRNA)–mediated knockdown of c-Myc reduced cell proliferation in all four cell lines, although the degree of inhibition was stronger in 831-BrM and 1833-BoM cells (Fig. 2C, Supplementary Fig. S1). Parental cells expressing a non-targeting shRNA showed elevated c-Myc expression, possibly due to puromycin selection. These data suggest that c-Myc is an important mediator of cell proliferation, and c-Myc overexpression provided a proliferative advantage at least in brain and bone-metastatic subclones.

Identification of serine and one-carbon unit pathway elevation in metastatic subclones

The products of several metabolic pathways feed into nucleotide synthesis, including ribulose-5-phosphate from the pentose phosphate pathway, and one-carbon (1C) units and glycine from the serine and 1C unit pathway. It is also known that c-Myc can promote the expression of serine and glycine metabolism genes in cancer cells (32,33). We performed expression analyses of the metastatic subclones and found elevated levels of the key mitochondrial enzymes serine hydroxymethyltransferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), and methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L), in contrast to the downregulated expression of the cytosolic isoenzyme serine hydroxymethyltransferase 1 (SHMT1) (Fig. 3A–C). Consistent with previous reports in other cell types, knockdown of c-Myc in parental and metastatic breast cancer subclones diminished MTHFD2 and MTHFD1L protein expression, suggesting these enzymes are c-Myc-regulated (Supplementary Fig. S1). SHMT2 expression did not reduce upon c-Myc knockdown, suggesting that SHMT2 expression was regulated by other transcription factors. To determine whether c-Myc and mitochondrial 1C unit pathway enzyme overexpression was a common co-occurrence in other cancer metastasis models, we checked protein expression levels in the parental and metastatic subpopulations of other human cell line systems derived from lung adenocarcinoma or ER⁺ breast carcinoma patients (34,35). There was a clear correlation of SHMT2, MTHFD2, and MTHFD1L expression with c-Myc expression among all the cell lines tested. The brain metastatic subclones of lung adnocacinoma cell lines PC9 and H2030 had increased MTHFD2 expression, though we could not find another system that also displayed overexpression of c-Myc and all the three mitochondrial 1C unit pathway enzymes in metastatic subclones relative to their corresponding parental cells (Supplementary Fig. S2). Taken together with the observations of higher serine and glycine levels in 831-BrM and 1833-BoM cells compared to 231-Parental cells (Fig. 1B), these data suggest that the role of c-Myc in regulating mitochondrial serine and 1C unit metabolism in metastatic cancer may be tissue-specific.

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Figure 3.

The mitochondrial serine and one-carbon unit pathway is upregulated in metastatic breast cancer subclones. (A) Schematic of the cytosolic and mitochondrial serine and one-carbon unit pathway. (B) qPCR for serine and one-carbon unit pathway genes (mean ± SD, n = 3, *P < 0.05 **P < 0.01 ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t test, compared to expression in parental cells). (C) IB for serine and one-carbon unit pathway enzymes from whole-cell extracts of parental cells and metastatic subclones. (D) Schematic diagram of incorporation of ²H (D) from [2,3,3-²H]serine onto glycine, one-carbon units, and purines. (E) SHMT flux estimated by relative abundance of labeled glycine from serine (mean ± SD, n = 3, **P < 0.01 by two-tailed Student’s t test). (F) Fractional labeling of [2,3,3-²H]serine onto GTP and ATP (mean ± SD, n = 3, *P < 0.05 **P < 0.01 ***P < 0.001 by two-tailed Student’s t test).

Metastatic subclones display increased mitochondrial serine and one-carbon unit pathway activity

We next asked if higher expression of mitochondrial serine and 1C unit pathway enzymes might indeed reflect higher pathway activity. Serine can be catabolized in both the mitochondrial and cytosolic branch of the 1C unit pathway. Since cancer cells predominately express the mitochondrial serine catabolic enzymes over the cytosolic enzymes, serine is generally catabolized in the mitochondria in cancer cells (13,14,36). Serine hydroxyl-methyltransferase 2 (SHMT2) initiates this reaction by converting serine to glycine while donating a carbon group to tetrahydrafolate (THF) to generate methylene-THF. Subsequent oxidation of methylene-THF by MTHFD2 and MTHFD1L generates NAD(P)H and formate. Formate can cross the mitochondrial membrane to provide 1C units for anabolic reactions such as nucleotide synthesis (37).

We hypothesized that the reason metastatic cells upregulate the serine and 1C unit pathway is to enhance nucleotide synthesis to fuel cell proliferation. Indeed, most cancer cells have been reported to utilize serine as the predominant source of 1C units for biosynthesis (38). We performed [2,3,3-²H]serine tracing to examine 1C unit pathway flux to glycine and purine nucleotides. In cells grown in media containing [2,3,3-²H]serine, the cytosolic pathway generates methylene-THF (me-THF) mass heavy by 2 (M+2) and 10-formyl-THF mass heavy by 1 (M+1), while 10-formyl-THF derived from mitochondrial formate exchange to the cytosol is strictly M+1. [2,3,3-²H]serine labeling onto the metabolites glycine and purine nucleotide triphosphates produced from the mitochondrial pathway thereby produces glycine M+1 and purines either M+1 or M+2 (Fig. 3D). Time course experiments were performed in 4175-LM cells to determine the optimal steady state labeling conditions for glycine and ATP from serine: 2 hours and 24 hours respectively (Supplementary Fig. S3). We observed higher SHMT flux in metastatic subclones, as the relative abundance of M+1 glycine was approximately 1.5-fold higher in 4175-LM cells compared to 231-Parental cells, indicating that higher purine turnover in metastatic cells was fueled by higher SHMT flux (Fig. 3E). Importantly, while robust fractions of ATP and GTP were labeled in parental cells, the metastatic subclones displayed even higher labeling fractions from serine (Fig. 3F). These results demonstrate that upregulation of serine catabolism through the mitochondrial 1C unit pathway promotes de novo purine synthesis in metastatic breast cancer cells.

Serine catabolism is necessary for metastatic cancer cell proliferation in vitro

To address the extent to which mitochondrial serine catabolism is necessary for cell proliferation, 231-Parental, 831-BrM, 1833-BoM, and 4175-LM cells were infected with lentivirus expressing shRNAs against SHMT2 (shSHMT2) or a nontargeting control (shNT). Intriguingly, knockdown of SHMT2 protein expression with two different shRNAs drastically suppressed proliferation of the metastatic subclones significantly, with a reduced effect in 231-Parental cells (Fig. 4A and andB).B). In contrast, knockdown of the downstream enzyme of the mitochondrial serine and 1C unit pathway, MTHFD2, suppressed proliferation to a lesser extent (Supplementary Fig. S4A and B). To evaluate the therapeutic potential of targeting 1C unit metabolism to block metastatic growth, we treated cells with a small-molecule inhibitor of SHMT called SHIN1 (39). In vitro, metastatic subclones were sensitive to SHIN1 with an EC50 in the 100–500 nM range (Supplementary Fig. S5). There was no obvious enhancement of SHIN1 sensitivity in 831-BrM, 1833-BoM, and 4175-LM cells compared to 231-Parental cells, possibly because SHIN1 inhibits both SHMT2 and SHMT1 (Fig. 4C). Importantly, inhibition of cell proliferation in the presence of SHIN1 could be rescued by the supplementation of formate (2 mM), a source of cellular 1C units (Fig. 4C). These results indicate that the major role of elevated mitochondrial serine catabolism is to generate 1C units for cytosolic purine biosynthesis in the metastatic subclones. Thus, targeting SHMT activity may be a promising way to restrict nucleotide availability to block metastatic breast cancer cell proliferation.

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Figure 4.

Metastatic subclones are particularly sensitive to SHMT2 inhibition. (A) 3 day proliferation of 231-Parental, 831-BrM,1833-BoM, and 4175-LM cells expressing either a nontargeting (shNT) or SHMT2 targeting (shSHMT2) vectors. Relative proliferation was calculated relative to average proliferation of shNT cells (mean ± SD, n = 3). (B) IB for SHMT2 in parental and metastatic subclones. (C) 3 day proliferation of parental and metastatic cells with 2 μM SHIN1, in RPMI with or without 2 mM formate and dialyzed FBS (mean ± SD, n = 3, ***P < 0.001 ****P < 0.0001 by two-tailed Student’s t test). Counts were normalized to the proliferation of 231-Parental cells in media without SHIN1 and formate treatment. (D) Growth of 4175-LM shNT and shSHMT2 tumors in the mammary fat pad of nude mice (mean ± SEM, n = 8, **P < 0.01 by two-tailed Student’s t test). (E) Quantification of luminescence signal in the lungs of mice 3 weeks post injection of either 4175-LM shNT or shSHMT2 cells (mean ± SEM, **P < 0.01 by two-tailed Student’s t test, shNT;n = 8 shSHMT2;n = 7). (F) qPCR analysis of hGAPDH expression in the lungs of mice 4 weeks post injection of either 4175-LM shNT or shSHMT2 cells (mean ± SEM, *P < 0.05 by two-tailed Student’s t test, shNT;n = 6 shSHMT2;n = 7).

SHMT2 knockdown impairs primary and metastatic growth in vivo

We then interrogated the effect of reducing mitochondrial 1C unit pathway activity in two different models of cancer growth in vivo. 4175-LM cells were chosen due to the relative ease of monitoring, measuring, and collecting tissue from lung metastasis compared to brain and bone metastasis. For the first model, we monitored breast cancer growth at the primary tumor site. SHMT2 knockdown significantly impaired the growth of 4175-LM cells in the mammary fat pads of immunodeficient mice (Fig. 4D, Supplementary Fig. S6). For the second model, we induced breast cancer metastasis to the lung by intravenous tail vein injection. Because 4175-LM cells express firefly luciferase (16), we tracked tumor growth in the lung by bioluminescence imaging (BLI). Both BLI and quantification of human GAPDH (hGAPDH) expression from resected mouse lungs revealed a roughly two-fold reduction of lung tumor burden in mice injected with shSHMT2 cells compared to shNT cells (Fig. 4E and andF,F, Supplementary Fig. S7A). While on average, shSHMT2 tumors had reduced human SHMT2 (hSHMT2) expression compared to shNT tumors, some shSHMT2 tumors appeared to have reacquired hSHMT2 expression (Supplementary Fig. S7B and C). These data suggest that SHMT2 is necessary for metastatic growth in vivo.

Mitochondrial serine and 1C unit pathway genes are associated with more aggressive metastatic disease in some human breast cancer patients

To further explore the relevancy of mitochondrial one-carbon unit metabolism in human breast cancer metastasis, we examined the expression of SHMT1, SHMT2, MTHFD2, and MTHFD1L in the METABRIC dataset of human breast cancer patients (21). We retrospectively inferred metastatic recurrence in patients by examining the frequency of disease-specific survival (DSS) up to 20 years. Patients were separated into two groups based on the maxstat algorithm (see Materials and Methods). Patients with high SHMT2 expression were significantly more likely to succumb to metastatic recurrent disease, while patients with high expression of the cytosolic isozyme SHMT1 were significantly protected from metastatic relapse (Fig. 5A, Supplementary Fig. S8). Using three different breast cancer subtype clustering analyses based on gene expression (PAM50, IC10, SCMOD2), we classified the MDA-MB-231 cell line as basal, IC4 (copy number flat), and ER⁻Her2⁻ (20,21). We have previously described IC4 as consisting of a mixture of ER⁻ tumors with lymphocytic infiltration and ER⁺ tumors with abundant stroma. Accordingly, further analysis of the IC4 patient subgroup following adjustment for covariates of age, grade, size, number of lymph nodes, ER, PR and Her2 status revealed a significant association of MTHFD1, MTHFD1L, MTHFD2, and SHMT2 expression with worse survival and SHMT1 expression with better survival (Fig. 5B). Finally, we stained a tissue microarray panel of human breast invasive ductal carcinoma and matched lymph node metastases and found significantly higher expression of SHMT2 in metastatic cancer cells comparing to the primary tumors (Fig. 5C and andD).D). Together, these data suggest that SHMT2 and other mitochondrial 1C unit pathway enzymes may be used as prognostic markers that indicate worse patient outcome, while cytosolic SHMT1 expression may indicate better survival rate in the IC4 patient subgroup.

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Figure 5.

Mitochondrial serine and one-carbon unit pathway enzyme expression correlates with poor survival in human breast cancer. (A) Kaplan-Meier plot for SHMT1 (left) and SHMT2 (right) expression associated with disease-specific survival (DSS) in the human IC4 patient subgroup (METABRIC). (B) Forest plot for the hazard of individual 1C unit pathway genes adjusted for covariates (age, grade, size, number of lymph nodes, ER, PR and Her2 status) in the IC4 subgroup (n=343). (C) Representative SHMT2 staining (at 40x) of human breast invasive ductal carcinoma and matched metastatic carcinoma tissue samples (LN = lymph node). (D) Quantification of SHMT2 intensity by IHC in metastatic lesions compared to primary tumors (mean ± SD, n = 33 per group, *P < 0.05 by two-tailed Student’s t test).

Relevance of SHMT2 expression in the progression and aggressiveness of other cancer types

To evaluate the contribution of mitochondrial 1C unit metabolism to the progression of other cancer types, we queried SHMT2 expression in TCGA datasets through the UALCAN portal (24). In addition to breast invasive carcinoma (BRCA), we identified adrenocortical carcinoma (ACC), head and neck squamous cell carcinoma (HNSC), kidney chromophobe cell carcinoma (KICH), and kidney renal papillary cell carcinoma (KIRP) as cancer types in which SHMT2 expression progressively increased as a function of stage (Fig. 6). Notably, gain of SHMT2 expression in BRCA and HNSC tended to occur early on in cancer progression, whereas in KICH, SHMT2 upregulation may occur only during the very late stage. A few cancer types such as mesothelioma (MESO) and ovarian serous cystadenocarcinoma (OV) showed the opposite trend: a progressive loss of SHMT2 expression with increasing cancer stage (Supplementary Fig. 9). Collectively, these data present the possibility that there exist additional cancer types in which mitochondrial 1C unit metabolism promotes progression and aggressiveness.

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Figure 6.

SHMT2 expression increases with stage in various cancers.

Box plots depicting the average expression level (transcripts per million) of SHMT2 in normal tissue (N) and as a function of cancer stage (stage 1 = S1; stage 2 = S2; stage 3 = S4; stage 4 = S4). Statistically significant differences between pairwise comparisons are highlighted in red. Abbreviations for cancer types are explained as follows: ACC (adrenocortical carcinoma), BRCA (breast invasive carcinoma), HNSCC (head and neck squamous cell carcinoma), KICH (kidney chromophobe carcinoma), KIRP (kidney renal papillary cell carcinoma).

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Discussion

For breast cancer, common metastatic sites include the brain, bone, liver, and lung. At the cellular level, the original heterogeneous population of cancer cells from the primary tumor undergo a selection process whereby those clones with alterations (carrying both genetic lesions and epigenetic modifications) favoring fitness and plasticity are enriched. These adaptations, in turn, equip cells with the ability to withstand standard treatments such as chemotherapy and radiation therapy, ultimately leading to cancer progression and metastatic recurrence (40). While many previous studies have elucidated a role for molecular processes such as epithelial to mesenchymal transition and invasion and migration of cancer cells, our understanding of how metabolic pathway alterations shape metastatic growth is still limited. It is important to note that the MDA-MB-231 cells we studied were isolated from a pleural population that already metastasizes well in vivo. Our metabolomics profiling of the even more highly metastatic triple-negative breast cancer subclones suggested alterations in both glycolysis and the TCA cycle during the late stages of cancer progression, consistent with findings from other groups of heightened mitochondrial metabolism in metastatic cells (2,3,5,6). We further discovered elevated catabolism of serine in the mitochondria of our metastatic subclones. A previous study in isogenic murine 4T1 breast cancer cell lines found that transformed cells showed higher levels of nucleotides than nontransformed cells, and that “more metastatic” lines had even more nucleotides than “less metastatic” ones (1). In contrast, we found lower levels of free purines in metastatic variants of human MDA-MB-231 cell lines compared to the parental population (Fig. 1B). This discrepancy may be attributed to different oncogenic contexts in 4T1 cells versus MDA-MB-231 cells or inherent differences in purine metabolism between murine and human cells. Due to the difficulty of obtaining pure metastatic tumor tissue from in vivo studies, the metabolomic analysis were performed using established cell lines in vitro. Microenvironmental factors from metastatic niche, such as hypoxia and nutrient starvation, also regulate cancer cell metabolism. Since mitochondrial 1C unit metabolism can utilize both NAD⁺ and NADP⁺, cancer cells with upregulation of mitochondrial 1C unit metabolism may gain metabolic flexibility to sustain proliferation under stress conditions. When cells engage active respiration, the mitochondrial 1C unit pathway can utilize NAD⁺ to generate 1C units; under hypoxia or starvation conditions, when the NAD⁺/NADH ratio decreases, elevated mitochondrial ROS leads to an increased NADP⁺/NADPH ratio, which can also drive the 1C unit pathway and purine synthesis. Further investigations comparing the metabolic profile changes under these stress conditions may provide more insight into potential links between metabolic stresses and the evolution of metastatic cancer cells.

The role of serine in cancer growth has drawn increasing interest over the years ever since the identification of PHGDH amplifications in melanoma and breast cancer (9,10). A variety of mechanisms have been proposed to explain why increased serine synthesis and serine catabolism could promote tumorigenesis, including rerouting glucose carbon flux, maintenance of compartment-specific NAD(P)⁺/NAD(P)H ratios, and the control of metabolites such as acetyl-coA, α-ketoglutarate, or 2-hydroxyglutarate (12,41,42). Moreover, a previous study had implicated SHMT2 and a neutral amino acid importer of serine and glycine (ASCT2) as prognostic biomarkers for breast cancer (43). Our study is the first to directly evaluate the therapeutic potential of targeting SHMT2 in metastatic breast cancer using both genetic and pharmaceutical approaches. Intriguingly, genetic knockdown of SHMT2 strongly inhibited the proliferation of metastatic cells, while treatment with a dual SHMT1/SHMT2 inhibitor suppressed proliferation of both parental and metastatic subclones. This discrepancy may be explained by prior observations that while MDA-MB-231 cells preferentially utilize the mitochondrial pathway for 1C unit production, inhibition of individual mitochondrial enzymes can lead to a switch to the cytosolic pathway (36). We thus speculate that 231-Parental cells may be more adept at switching to cytosolic serine catabolism, and for reasons still unclear, the metastatic subclones are less flexible. Consistent with observations in colon cancer xenografts (36), SHMT2 knockdown in the lung metastatic subclone slowed, but not completely suppressed, tumor growth in the mammary fat pad and lung. In addition, we found that in the IC4 subset of human breast cancer patients, the expression of mitochondrial one-carbon unit enzymes is positively associated with more aggressive disease. Thus, interrogating the expression status of mitochondrial one-carbon unit enzymes through transcriptional or proteomic methods holds prognostic value in the metastatic setting, and warrants the need for further development of drugs that selectively inhibit serine catabolism for treating the metastasis of triple-negative breast cancer.

What causes the upregulation of mitochondrial serine catabolic flux in highly metastatic cancer cells? We provide evidence that a crucial oncogenic event promotes the ability of metastatic breast cancer subclones to catabolize serine faster than parental cells: c-Myc activation. c-Myc overexpression is known to be associated with up to 40% of breast cancers, with hyperactive c-Myc enriched particularly in the basal-like subtype (27,44). These observations are consistent with our findings of the MDA-MB-231 cell line as basal-like and its metastatic subclones expressing even higher levels of c-Myc than the parental population (Fig. 2A). We found that c-Myc was required for the maintenance of the mitochondrial serine and 1C unit pathway genes MTHFD2 and MTHFD1L, consistent with previous reports that c-Myc supports serine/glycine metabolism at the transcriptional level in other cell types (32,33). These results suggest a model for breast cancer metastasis in which a small fraction of c-Myc^high expressing cells from the primary tumor acquire the ability to upregulate serine catabolism to fuel growth in metastatic tissue sites. Alternatively, high c-Myc expression and the linked ability to upregulate serine catabolism may be intrinsic properties of stem-like metastasis-initiating cells that are enriched in breast cancer cell populations selected for high metastatic activity in mice. As one of the key oncogenic transcription factors, there is increasing evidence that c-Myc plays multiple roles during the metastatic process. c-Myc knockdown reduces invasion and migration of MDA-MB-231 cells (30). Moreover, a recent study corroborated our findings of elevated c-Myc levels in brain-metastatic derivatives of human breast cancer cells and demonstrated its necessity for the invasive growth of brain metastases (45). Our study highlights the role of c-Myc in enhancing 1C unit pathway activity and proliferation, which is also important for metastatic growth. Since SHMT2 expression was not reduced by c-Myc shRNA, it is likely that other tumor-promoting factors, such as ATF4 and NRF2, also play important roles in late stage cancer progression by modulating 1C unit metabolism. Intriguingly, a recent report showed that TGF-β signaling induces the expression of SHMT2 (46). Given the critical role of TGF-β in promoting metastasis (47,48), it may be interesting to further investigate whether serine and 1C unit pathway metabolic reprogramming is controlled by TGF-ß signaling in metastatic subpopulations of human breast cancer cells.

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The Colors of Respiration and Electron Transport

Posted in Academic Publishing, Biochemical pathways, Curation, Cytoskeleton, Dehydrogenase, Experimental validation, Explanatory, Fatty acids, Historical relevance, Lipid metabolism, Metabolism, Metabolomics, Phosphorylase, Phosphorylation, Proteins, Pyridine nucleotide transhydrogenase, Pyridine nucleotides, Signaling, Ubiquitinylation, tagged aerobic glycolysis, ATP, Coenzyme A, cytochrome c, electron transport chain (ETC), energy expenditure, enzymes, hydrogen transfer, inner membrane, membrane bound enzymes, metalloenzymes, mitochondria, oxidation-reduction, Pyridine nucleotides, repiration, transhydrogenase on January 20, 2015| Leave a Comment »

The Colors of Respiration and Electron Transport

Reporter & Curator: Larry H. Bernstein, MD, FCAP

Molecular Biology of the Cell. 4th edition

Electron-Transport Chains and Their Proton Pumps
http://www.ncbi.nlm.nih.gov/books/NBK26904/

Having considered in general terms how a mitochondrion uses electron
transport to create an electrochemical proton gradient, we need to
examine the mechanisms that underlie this membrane-based energy-conversion process. In doing so, we also accomplish a larger purpose.
As emphasized at the beginning of this chapter, very similar chemi-
osmotic mechanisms are used by mitochondria, chloroplasts, archea,
and bacteria. In fact, these mechanisms underlie the function of nearly
all living organisms— including anaerobes that derive all their energy
from electron transfers between two inorganic molecules. It is therefore
rather humbling for scientists to remind themselves that the existence
of chemiosmosis has been recognized for only about 40 years.

mitochondria

Overview of The Electron Transport Chain

We begin with a look at some of the principles that underlie the electron-transport process, with the aim of explaining how it can pump protons
across a membrane.

Although protons resemble other positive ions such as Na+ and K+
in their movement across membranes, in some respects they are unique.
Hydrogen atoms are by far the most abundant type of atom in living
organisms; they are plentiful not only in all carbon-containing
biological molecules, but also in the water molecules that surround
them. The protons in water are highly mobile, flickering through the
hydrogen-bonded network of water molecules by rapidly
dissociating from one water molecule to associate with its neighbor,
as illustrated in Figure 14-20A. Protons are thought to move across a
protein pump embedded in a lipid bilayer in a similar way: they
transfer from one amino acid side chain to another, following a
special channel through the protein.

Protons are also special with respect to electron transport. Whenever
a molecule is reduced by acquiring an electron, the electron (e -) brings
with it a negative charge. In many cases, this charge is rapidly
neutralized by the addition of a proton (H+) from water, so that
the net effect of the reduction is to transfer an entire hydrogen atom,
H+ + e – (Figure 14-20B). Similarly, when a molecule is oxidized,
a hydrogen atom removed from it can be readily dissociated into
its constituent electron and proton—allowing the electron to
be transferred separately to a molecule that accepts electrons,
while the proton is passed to the water. Therefore, in a membrane
in which electrons are being passed along an electron-transport
chain, pumping protons from one side of the membrane to
another can be relatively simple. The electron carrier merely
needs to be arranged in the membrane in a way that causes it to
pick up a proton from one side of the membrane when it accepts
an electron, and to release the proton on the other side of the
membrane as the electron is passed to the next carrier molecule
in the chain (Figure 14-21).

protons pumped across membranes ch14f21

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f21.gif

Figure 14-21

How protons can be pumped across membranes. As an electron
passes along an electron-transport chain embedded in a lipid-bilayer
membrane, it can bind and release a proton at each step.
In this diagram, electron carrier B picks up a proton (H+)
from one (more…)

e_transfer

The Redox Potential Is a Measure of Electron Affinities

In biochemical reactions, any electrons removed from one
molecule are always passed to another, so that whenever one
molecule is oxidized, another is reduced. Like any other chemical r
eaction, the tendency of such oxidation-reduction reactions, or
redox reactions, to proceed spontaneously depends on the free-
energy change (ΔG) for the electron transfer, which in turn
depends on the relative affinities of the two molecules for electrons.

Because electron transfers provide most of the energy for living
things, it is worth spending the time to understand them. Many
readers are already familiar with acids and bases, which donate
and accept protons (see Panel 2-2, pp. 112–113). Acids and bases
exist in conjugate acid-base pairs, in which the acid is readily
converted into the base by the loss of a proton. For example,
acetic acid (CH3COOH) is converted into its conjugate base
(CH3COO-) in the reaction:

Image ch14e3.jpg

In exactly the same way, pairs of compounds such as NADH and
NAD+ are called redox pairs, since NADH is converted to NAD+
by the loss of electrons in the reaction:

Image ch14e4.jpg

NAD+_NADH

NADH is a strong electron donor: because its electrons are held
in a high-energy linkage, the free-energy change for passing its
electrons to many other molecules is favorable (see Figure 14-9).
It is difficult to form a high-energy linkage. Therefore its redox
partner, NAD+, is of necessity a weak electron acceptor.

The tendency to transfer electrons from any redox pair can be
measured experimentally. All that is required is the formation
of an electrical circuit linking a 1:1 (equimolar) mixture of the
redox pair to a second redox pair that has been arbitrarily selected
as a reference standard, so the voltage difference can be measured
between them (Panel 14-1, p. 784). This voltage difference is
defined as the redox potential; as defined, electrons move
spontaneously from a redox pair like NADH/NAD+ with a low
redox potential (a low affinity for electrons) to a redox pair like
O2/H2O with a high redox potential (a high affinity for electrons).
Thus, NADH is a good molecule for donating electrons to the
respiratory chain, while O2 is well suited to act as the “sink” for
electrons at the end of the pathway. As explained in Panel 14-1,
the difference in redox potential, ΔE0′, is a direct measure of
the standard free-energy change (ΔG°) for the transfer of an
electron from one molecule to another.

Proteins of inner space

energetics-of-cellular-respiration

Box Icon

Panel 14-1

Redox Potentials.

Electron Transfers Release Large Amounts of Energy

As just discussed, those pairs of compounds that have the most negative
redox potentials have the weakest affinity for electrons and therefore
contain carriers with the strongest tendency to donate electrons.
Conversely, those pairs that have the most positive redox potentials
have the strongest affinity for electrons and therefore contain carriers
with the strongest tendency to accept electrons. A 1:1 mixture of NADH
and NAD+ has a redox potential of -320 mV, indicating that NADH has
a strong tendency to donate electrons; a 1:1 mixture of H2O and ½O2
has a redox potential of +820 mV, indicating that O2 has a strong
tendency to accept electrons. The difference in redox potential is
1.14 volts (1140 mV), which means that the transfer of each electron
from NADH to O2 under these standard conditions is enormously
favorable, where ΔG° = -26.2 kcal/mole (-52.4 kcal/mole for the two
electrons transferred per NADH molecule; see Panel 14-1). If we
compare this free-energy change with that for the formation of the
phosphoanhydride bonds in ATP (ΔG° = -7.3 kcal/mole, see Figure 2-75), we see that more than enough energy is released by the oxidization
of one NADH molecule to synthesize several molecules of ATP from
ADP and Pi.

Phosphate dependence of pyruvate oxidation

Living systems could certainly have evolved enzymes that would
allow NADH to donate electrons directly to O2 to make water in the reaction:

Image ch14e5.jpg

But because of the huge free-energy drop, this reaction would proceed
with almost explosive force and nearly all of the energy would be released
as heat. Cells do perform this reaction, but they make it proceed much
more gradually by passing the high-energy electrons from NADH to
O2 via the many electron carriers in the electron-transport chain.
Since each successive carrier in the chain holds its electrons more
tightly, the highly energetically favorable reaction 2H+ + 2e – + ½O2
→ H2O is made to occur in many small steps. This enables nearly half
of the released energy to be stored, instead of being lost to the
environment as heat.

Spectroscopic Methods Have Been Used to Identify Many Electron
Carriers in the Respiratory Chain

Many of the electron carriers in the respiratory chain absorb visible
light and change color when they are oxidized or reduced. In general,
each has an absorption spectrum and reactivity that are distinct enough
to allow its behavior to be traced spectroscopically, even in crude mixtures.
It was therefore possible to purify these components long before their
exact functions were known. Thus, the cytochromes were discovered
in 1925 as compounds that undergo rapid oxidation and reduction in
living organisms as disparate as bacteria, yeasts, and insects. By observing
cells and tissues with a spectroscope, three types of cytochromes were
identified by their distinctive absorption spectra and designated
cytochromes a, b, and c. This nomenclature has survived, even though
cells are now known to contain several cytochromes of each type and
the classification into types is not functionally important.

The cytochromes constitute a family of colored proteins that are
related by the presence of a bound heme group, whose iron atom
changes from the ferric oxidation state (Fe3+) to the ferrous oxidation
state (Fe2+) whenever it accepts an electron. The heme group consists
of a porphyrin ring with a tightly bound iron atom held by four nitrogen
atoms at the corners of a square (Figure 14-22). A similar porphyrin ring
is responsible for the red color of blood and for the green color of
leaves, being bound to iron in hemoglobin and to magnesium in
chlorophyll, respectively.

The structure of the heme group attached covalently to cytochrome c ch14f22

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f22.jpg

Figure 14-22. The structure of the heme group attached covalently
to cytochrome c.

Figure 14-22

The structure of the heme group attached covalently to cytochrome c.
The porphyrin ring is shown in blue. There are five different
cytochromes in the respiratory chain. Because the hemes in different
cytochromes have slightly different structures and (more…)

Iron-sulfur proteins are a second major family of electron carriers. In these
proteins, either two or four iron atoms are bound to an equal number of
sulfur atoms and to cysteine side chains, forming an iron-sulfur center
on the protein (Figure 14-23). There are more iron-sulfur centers than
cytochromes in the respiratory chain. But their spectroscopic detection
requires electron spin resonance (ESR) spectroscopy, and they are less
completely characterized. Like the cytochromes, these centers carry one
electron at a time.

structure of iron sulfur centers ch14f23

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f23.jpg

Figure 14-23. The structures of two types of iron-sulfur centers.

Figure 14-23

The structures of two types of iron-sulfur centers. (A) A center of the
2Fe2S type. (B) A center of the 4Fe4S type. Although they contain
multiple iron atoms, each iron-sulfur center can carry only one
electron at a time. There are more than seven different (more…)

The simplest of the electron carriers in the respiratory chain—and
the only one that is not part of a protein—is a small hydrophobic
molecule that is freely mobile in the lipid bilayer known as ubiquinone,
or coenzyme Q. A quinone (Q) can pick up or donate either one or
two electrons; upon reduction, it picks up a proton from the medium
along with each electron it carries (Figure 14-24).

quinone electron carriers ch14f24

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f24.jpg

Figure 14-24. Quinone electron carriers.

Figure 14-24

Quinone electron carriers. Ubiquinone in the respiratory chain picks
up one H+ from the aqueous environment for every electron it accepts,
and it can carry either one or two electrons as part of a hydrogen atom
(yellow). When reduced ubiquinone donates (more…)

In addition to six different hemes linked to cytochromes, more than
seven iron-sulfur centers, and ubiquinone, there are also two copper
atoms and a flavin serving as electron carriers tightly bound to respiratory-chain proteins in the pathway from NADH to oxygen. This pathway
involves more than 60 different proteins in all.

As one would expect, the electron carriers have higher and higher
affinities for electrons (greater redox potentials) as one moves along
the respiratory chain. The redox potentials have been fine-tuned
during evolution by the binding of each electron carrier in a particular
protein context, which can alter its normal affinity for electrons. However,
because iron-sulfur centers have a relatively low affinity for electrons,
they predominate in the early part of the respiratory chain; in contrast,
the cytochromes predominate further down the chain, where a higher
affinity for electrons is required.

The order of the individual electron carriers in the chain was
determined by sophisticated spectroscopic measurements (Figure 14-25),
and many of the proteins were initially isolated and characterized as
individual polypeptides. A major advance in understanding the
respiratory chain, however, was the later realization that most of
the proteins are organized into three large enzyme complexes.

path of electrons ch14f25

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f25.gif

Figure 14-25. The general methods used to determine the path of
electrons along an electron-transport chain.

Figure 14-25

The general methods used to determine the path of electrons along
an electron-transport chain. The extent of oxidation of electron
carriers a, b, c, and d is continuously monitored by following their
distinct spectra, which differ in their oxidized and (more…)

The Respiratory Chain Includes Three Large Enzyme Complexes
Embedded in the Inner Membrane

Membrane proteins are difficult to purify as intact complexes
because they are insoluble in aqueous solutions, and some of
the detergents required to solubilize them can destroy normal
protein-protein interactions. In the early 1960s, however, it
was found that relatively mild ionic detergents, such as deoxycholate,
can solubilize selected components of the inner mitochondrial
membrane in their native form. This permitted the identification
and purification of the three major membrane-bound respiratory
enzyme complexes in the pathway from NADH to oxygen (Figure 14-26).
As we shall see in this section, each of these complexes acts as an
electron-transport-driven H+ pump; however, they were
initially characterized in terms of the electron carriers that
they interact with and contain:

mitochondrial oxidative phosphorylation

http://www.ncbi.nlm.nih.gov/books/NBK26904/bin/ch14f26.gif

Figure 14-26. The path of electrons through the three respiratory
enzyme complexes.

Figure 14-26

The path of electrons through the three respiratory enzyme complexes.
The relative size and shape of each complex are shown. During the
transfer of electrons from NADH to oxygen (red lines), ubiquinone
and cytochrome c serve as mobile carriers that ferry (more…)

The NADH dehydrogenase complex (generally known as complex I)
is the largest of the respiratory enzyme complexes, containing more
than 40 polypeptide chains. It accepts electrons from NADH and
passes them through a flavin and at least seven iron-sulfur centers
to ubiquinone. Ubiquinone then transfers its electrons to a second
respiratory enzyme complex, the cytochrome b-c1 complex.

The cytochrome b-c1 complex contains at least 11 different
polypeptide chains and functions as a dimer. Each monomer
contains three hemes bound to cytochromes and an iron-sulfur
protein. The complex accepts electrons from ubiquinone
and passes them on to cytochrome c, which carries its electron
to the cytochrome oxidase complex.

The cytochrome oxidase complex also functions as a dimer; each
monomer contains 13 different polypeptide chains, including two
cytochromes and two copper atoms. The complex accepts one electron
at a time from cytochrome c and passes them four at a time to oxygen.

The cytochromes, iron-sulfur centers, and copper atoms can carry
only one electron at a time. Yet each NADH donates two electrons,
and each O2 molecule must receive four electrons to produce water.
There are several electron-collecting and electron-dispersing points
along the electron-transport chain where these changes in electron
number are accommodated. The most obvious of these is cytochrome
oxidase.

An Iron-Copper Center in Cytochrome Oxidase Catalyzes Efficient
O2 Reduction

Because oxygen has a high affinity for electrons, it releases a
large amount of free energy when it is reduced to form water.
Thus, the evolution of cellular respiration, in which O2 is
converted to water, enabled organisms to harness much more
energy than can be derived from anaerobic metabolism. This
is presumably why all higher organisms respire. The ability of
biological systems to use O2 in this way, however, requires a
very sophisticated chemistry. We can tolerate O2 in the air we
breathe because it has trouble picking up its first electron; this
fact allows its initial reaction in cells to be controlled closely by
enzymatic catalysis. But once a molecule of O2 has picked up one
electron to form a superoxide radical (O2 -), it becomes dangerously
reactive and rapidly takes up an additional three electrons wherever
it can find them. The cell can use O2 for respiration only because
cytochrome oxidase holds onto oxygen at a special bimetallic
center, where it remains clamped between a heme-linked iron
atom and a copper atom until it has picked up a total of four electrons.
Only then can the two oxygen atoms of the oxygen molecule be
safely released as two molecules of water (Figure 14-27).

Figure 14-27. The reaction of O2 with electrons in cytochrome oxidase.

Figure 14-27

The reaction of O2 with electrons in cytochrome oxidase. As indicated,
the iron atom in heme a serves as an electron queuing point; this
heme feeds four electrons into an O2 molecule held at the bimetallic
center active site, which is formed by the other (more…)

The cytochrome oxidase reaction is estimated to account for 90%
of the total oxygen uptake in most cells. This protein complex is
therefore crucial for all aerobic life. Cyanide and azide are extremely
toxic because they bind tightly to the cell’s cytochrome oxidase
complexes to stop electron transport, thereby greatly reducing
ATP production.

Although the cytochrome oxidase in mammals contains 13
different protein subunits, most of these seem to have a subsidiary
role, helping to regulate either the activity or the assembly of the
three subunits that form the core of the enzyme. The complete
structure of this large enzyme complex has recently been determined
by x-ray crystallography, as illustrated in Figure 14-28. The atomic
resolution structures, combined with mechanistic studies of the effect
of precisely tailored mutations introduced into the enzyme by genetic
engineering of the yeast and bacterial proteins, are revealing the
detailed mechanisms of this finely tuned protein machine.

Figure 14-28. The molecular structure of cytochrome oxidase.

Figure 14-28

The molecular structure of cytochrome oxidase. This protein
is a dimer formed from a monomer with 13 different protein
subunits (monomer mass of 204,000 daltons). The three colored
subunits are encoded by the mitochondrial genome, and they
form the functional (more…)

Electron Transfers Are Mediated by Random Collisions in
the Inner Mitochondrial Membrane

The two components that carry electrons between the three
major enzyme complexes of the respiratory chain—ubiquinone
and cytochrome c—diffuse rapidly in the plane of the inner
mitochondrial membrane. The expected rate of random collisions
between these mobile carriers and the more slowly diffusing
enzyme complexes can account for the observed rates of electron
transfer (each complex donates and receives an electron about
once every 5–20 milliseconds). Thus, there is no need to postulate
a structurally ordered chain of electron-transfer proteins in the
lipid bilayer; indeed, the three enzyme complexes seem to exist as
independent entities in the plane of the inner membrane, being
present in different ratios in different mitochondria.

The ordered transfer of electrons along the respiratory chain
is due entirely to the specificity of the functional interactions
between the components of the chain: each electron carrier is
able to interact only with the carrier adjacent to it in the sequence
shown in Figure 14-26, with no short circuits.

Electrons move between the molecules that carry them in
biological systems not only by moving along covalent bonds
within a molecule, but also by jumping across a gap as large
as 2 nm. The jumps occur by electron “tunneling,” a quantum-
mechanical property that is critical for the processes we are
discussing. Insulation is needed to prevent short circuits that
would otherwise occur when an electron carrier with a low redox
potential collides with a carrier with a high redox potential. This
insulation seems to be provided by carrying an electron deep
enough inside a protein to prevent its tunneling interactions
with an inappropriate partner.

How the changes in redox potential from one electron carrier
to the next are harnessed to pump protons out of the mitochondrial
matrix is the topic we discuss next.

A Large Drop in Redox Potential Across Each of the Three Respiratory
Enzyme Complexes Provides the Energy for H+ Pumping

We have previously discussed how the redox potential reflects
electron affinities (see p. 783). An outline of the redox potentials
measured along the respiratory chain is shown in Figure 14-29.
These potentials drop in three large steps, one across each major
respiratory complex. The change in redox potential between any
two electron carriers is directly proportional to the free energy
released when an electron transfers between them. Each enzyme
complex acts as an energy-conversion device by harnessing some
of this free-energy change to pump H+ across the inner membrane,
thereby creating an electrochemical proton gradient as electrons
pass through that complex. This conversion can be demonstrated
by purifying each respiratory enzyme complex and incorporating
it separately into liposomes: when an appropriate electron donor
and acceptor are added so that electrons can pass through the complex,
H+ is translocated across the liposome membrane.

Figure 14-29. Redox potential changes along the mitochondrial
electron-transport chain.

Figure 14-29

Redox potential changes along the mitochondrial electron-transport
chain. The redox potential (designated E′0) increases as electrons
flow down the respiratory chain to oxygen. The standard free-energy
change, ΔG°, for the transfer (more…)

The Mechanism of H+ Pumping Will Soon Be Understood in
Atomic Detail

Some respiratory enzyme complexes pump one H+ per electron
across the inner mitochondrial membrane, whereas others pump
two. The detailed mechanism by which electron transport is coupled
to H+ pumping is different for the three different enzyme complexes.
In the cytochrome b-c1 complex, the quinones clearly have a role.
As mentioned previously, a quinone picks up a H+ from the aqueous
medium along with each electron it carries and liberates it when it
releases the electron (see Figure 14-24). Since ubiquinone is freely
mobile in the lipid bilayer, it could accept electrons near the inside
surface of the membrane and donate them to the cytochrome b-c1
complex near the outside surface, thereby transferring one H+
across the bilayer for every electron transported. Two protons are
pumped per electron in the cytochrome b-c1 complex, however, and
there is good evidence for a so-called Q-cycle, in which ubiquinone
is recycled through the complex in an ordered way that makes this
two-for-one transfer possible. Exactly how this occurs can now be
worked out at the atomic level, because the complete structure of
the cytochrome b-c1 complex has been determined by x-ray
crystallography (Figure 14-30).

Figure 14-30. The atomic structure of cytochrome b-c 1.

Figure 14-30

The atomic structure of cytochrome b-c 1. This protein is a dimer.
The 240,000-dalton monomer is composed of 11 different protein
molecules in mammals. The three colored proteins form the
functional core of the enzyme: cytochrome b (green), cytochrome (more…)

Allosteric changes in protein conformations driven by electron
transport can also pump H+, just as H+ is pumped when ATP
is hydrolyzed by the ATP synthase running in reverse. For both the
NADH dehydrogenase complex and the cytochrome oxidase complex,
it seems likely that electron transport drives sequential allosteric
changes in protein conformation that cause a portion of the protein
to pump H+ across the mitochondrial inner membrane. A general
mechanism for this type of H+ pumping is presented in Figure 14-31.

Figure 14-31. A general model for H+ pumping.

Figure 14-31

A general model for H+ pumping. This model for H+ pumping
by a transmembrane protein is based on mechanisms that are
thought to be used by both cytochrome oxidase and the light-driven
procaryotic proton pump, bacteriorhodopsin. The protein
is driven through (more…)

H+ Ionophores Uncouple Electron Transport from ATP Synthesis

Since the 1940s, several substances—such as 2,4-dinitrophenol—
have been known to act as uncoupling agents, uncoupling electron
transport from ATP synthesis. The addition of these low-molecular-weight organic compounds to cells stops ATP synthesis by mitochondria
without blocking their uptake of oxygen. In the presence of an
uncoupling agent, electron transport and H+ pumping continue at
a rapid rate, but no H+ gradient is generated. The explanation for
this effect is both simple and elegant: uncoupling agents are lipid-
soluble weak acids that act as H+ carriers (H+ ionophores), and
they provide a pathway for the flow of H+ across the inner mitochondrial
membrane that bypasses the ATP synthase. As a result of this short-
circuiting, the proton-motive force is dissipated completely, and
ATP can no longer be made.

Respiratory Control Normally Restrains Electron Flow
Through the Chain

When an uncoupler such as dinitrophenol is added to cells,
mitochondria increase their oxygen uptake substantially because
of an increased rate of electron transport. This increase reflects
the existence of respiratory control. The control is thought to
act via a direct inhibitory influence of the electrochemical proton
gradient on the rate of electron transport. When the gradient is
collapsed by an uncoupler, electron transport is free to run unchecked
at the maximal rate. As the gradient increases, electron transport
becomes more difficult, and the process slows. Moreover, if an
artificially large electrochemical proton gradient is experimentally
created across the inner membrane, normal electron transport
stops completely, and a reverse electron flow can be detected in
some sections of the respiratory chain. This observation suggests
that respiratory control reflects a simple balance between the
free-energy change for electron-transport-linked proton pumping
and the free-energy change for electron transport—that is, the
magnitude of the electrochemical proton gradient affects both
the rate and the direction of electron transport, just as it affects
the directionality of the ATP synthase (see Figure 14-19).

Respiratory control is just one part of an elaborate interlocking
system of feedback controls that coordinate the rates of glycolysis,
fatty acid breakdown, the citric acid cycle, and electron transport.
The rates of all of these processes are adjusted to the ATP:ADP ratio,
increasing whenever an increased utilization of ATP causes the ratio
to fall. The ATP synthase in the inner mitochondrial membrane,
for example, works faster as the concentrations of its substrates
ADP and Pi increase. As it speeds up, the enzyme lets more H+ flow
into the matrix and thereby dissipates the electrochemical proton
gradient more rapidly. The falling gradient, in turn, enhances the
rate of electron transport.

Similar controls, including feedback inhibition of several key enzymes
by ATP, act to adjust the rates of NADH production to the rate of
NADH utilization by the respiratory chain, and so on. As a result of
these many control mechanisms, the body oxidizes fats and sugars
5–10 times more rapidly during a period of strenuous exercise than
during a period of rest.

Natural Uncouplers Convert the Mitochondria in Brown Fat into
Heat-generating Machines

In some specialized fat cells, mitochondrial respiration is normally
uncoupled from ATP synthesis. In these cells, known as brown fat
cells, most of the energy of oxidation is dissipated as heat rather
than being converted into ATP. The inner membranes of the large
mitochondria in these cells contain a special transport protein that
allows protons to move down their electrochemical gradient, by-
passing ATP synthase. As a result, the cells oxidize their fat stores
at a rapid rate and produce more heat than ATP. Tissues containing
brown fat serve as “heating pads,” helping to revive hibernating animals
and to protect sensitive areas of newborn human babies from the cold.

Bacteria Also Exploit Chemiosmotic Mechanisms to Harness Energy

Bacteria use enormously diverse energy sources. Some, like animal
cells, are aerobic; they synthesize ATP from sugars they oxidize to
CO2 and H2O by glycolysis, the citric acid cycle, and a respiratory
chain in their plasma membrane that is similar to the one in the
inner mitochondrial membrane. Others are strict anaerobes, deriving
their energy either from glycolysis alone (by fermentation) or from an
electron-transport chain that employs a molecule other than oxygen
as the final electron acceptor. The alternative electron acceptor can
be a nitrogen compound (nitrate or nitrite), a sulfur compound
(sulfate or sulfite), or a carbon compound (fumarate or carbonate),
for example. The electrons are transferred to these acceptors by a
series of electron carriers in the plasma membrane that are comparable
to those in mitochondrial respiratory chains.

Despite this diversity, the plasma membrane of the vast majority of
bacteria contains an ATP synthase that is very similar to the one in
mitochondria. In bacteria that use an electron-transport chain to
harvest energy, the electron-transport pumps H+ out of the cell and
thereby establishes a proton-motive force across the plasma membrane
that drives the ATP synthase to make ATP. In other bacteria, the
ATP synthase works in reverse, using the ATP produced by glycolysis
to pump H+ and establish a proton gradient across the plasma
membrane. The ATP used for this process is generated by
fermentation processes (discussed in Chapter 2).

Thus, most bacteria, including the strict anaerobes, maintain a proton
gradient across their plasma membrane. It can be harnessed to drive
a flagellar motor, and it is used to pump Na+ out of the bacterium via
a Na+-H+ antiporter that takes the place of the Na+-K+ pump of
eucaryotic cells. This gradient is also used for the active inward transport
of nutrients, such as most amino acids and many sugars: each nutrient is
dragged into the cell along with one or more H+ through a specific symporter
(Figure 14-32). In animal cells, by contrast, most inward transport across
the plasma membrane is driven by the Na+ gradient that is established by the
Na+-K+ pump.

Figure 14-32. The importance of H+-driven transport in bacteria.

Figure 14-32

The importance of H+-driven transport in bacteria. A proton-motive force
generated across the plasma membrane pumps nutrients into the cell and
expels Na+. (A) In an aerobic bacterium, an electrochemical proton gradient
across the plasma membrane is produced (more…)

Some unusual bacteria have adapted to live in a very alkaline
environment and yet must maintain their cytoplasm at a physiological
pH. For these cells, any attempt to generate an electrochemical H+
gradient would be opposed by a large H+ concentration gradient in
the wrong direction (H+ higher inside than outside). Presumably for
this reason, some of these bacteria substitute Na+ for H+ in all of their
chemiosmotic mechanisms. The respiratory chain pumps Na+ out of
the cell, the transport systems and flagellar motor are driven by an
inward flux of Na+, and a Na+-driven ATP synthase synthesizes
ATP. The existence of such bacteria demonstrates that the principle
of chemiosmosis is more fundamental than the proton-motive force
on which it is normally based.

Summary

The respiratory chain in the inner mitochondrial membrane contains
three respiratory enzyme complexes through which electrons pass on
their way from NADH to O2.

Each of these can be purified, inserted into synthetic lipid vesicles,
and then shown to pump H+ when electrons are transported through it.
In the intact membrane, the mobile electron carriers ubiquinone and
cytochrome c complete the electron-transport chain by shuttling between
the enzyme complexes. The path of electron flow is NADH → NADH
dehydrogenase complex → ubiquinone → cytochrome b-c1 complex →
cytochrome c → cytochrome oxidase complex → molecular oxygen (O2).

The respiratory enzyme complexes couple the energetically favorable
transport of electrons to the pumping of H+ out of the matrix. The
resulting electrochemical proton gradient is harnessed to make ATP
by another transmembrane protein complex, ATP synthase, through
which H+ flows back into the matrix. The ATP synthase is a reversible
coupling device that normally converts a backflow of H+ into ATP
phosphate bond energy by catalyzing the reaction ADP + Pi → ATP,
but it can also work in the opposite direction and hydrolyze ATP to
pump H+ if the electrochemical proton gradient is sufficiently reduced.
Its universal presence in mitochondria, chloroplasts, and procaryotes
testifies to the central importance of chemiosmotic mechanisms in cells.

By agreement with the publisher, this book is accessible by the search
feature, but cannot be browsed.

Copyright © 2002, Bruce Alberts, Alexander Johnson, Julian Lewis,
Martin Raff, Keith Roberts, and Peter Walter; Copyright © 1983, 1989,
1994, Bruce Alberts, Dennis Bray, Julian Lewis, Martin Raff, Keith
Roberts, and James D. Watson .

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The Colors of Life Function

Posted in Amino acids, Cell Biology, Curation, Discovery process, Evolutionary cognition, Experimental validation, Explanatory, Historical relevance, Metabolism, Metabolomics, Proteins, Proteomics, Signaling, tagged Amino acids, catalysts, conformation, energy metabolism, fluorescence correlation spectroscopy (FCS), ligand-binding, metalloenzymes, Near-Infrared Spectroscopy, oxidation, Proteins, Raman spectroscopy on January 20, 2015| Leave a Comment »

The Colors of Life Function

Writer and Curator: Larry H. Bernstein, MD, FCAP

2.5.1 Type 1 Copper Proteins

The Cu(II) state of this category has an intense blue color due to a thiolate ligand
to Cu(II) charge transfer, and unusual EPR properties arising from the asymmetrical
Cu site (distorted trigonal-pyramidal). The proteins all have a low molecular
mass and have, so far, rather arbitrarily been divided into sub-groups, such as
azurins, plastocyanins, pseudoazurins, amicyanins and various other blue
proteins. Of these the azurins, amicyanins, pseudo-azurins and plastocyanins
apparently have similar copper coordination by two histidine, one cysteine and
one methionine residue. Where the function of Type I copper proteins is known,
it is invariably electron transfer. As yet the names for these proteins are all trivial
and are often derived from source, function or color. The different classes are
usually discerned on the basis of their primary and tertiary structure.

The first bacterial blue proteins to be described were called azurins. Rusticyanin is
another example of a bacterial protein. It has unusual properties with a reduction
potential of 680 mV, and is functional at pH 2. The azurins have well-defined electron
-transfer functions.

The so-called pseudo-azurins differ from the azurins in the N-terminal amino acid
sequence and the optical spectra, which resemble those of plastocyanins.

The blue proteins known as plastocyanins occur in plants, blue-green and green
algae. Their electron transfer role is well defined, i.e. from the bc1 complex
(EC 1.10.2.2) to the photooxidized P-700.

Amicyanins are electron carriers between methylamine dehydrogenase and
cytochrome c, with a characteristic amino acid sequence.

Of the remaining blue proteins stellacyanin is a well- known example. Umecyanin,
plantacyanin and mavicyanin are also considered to belong to this group.
Although these proteins undergo redox reactions in vitro, their true biological
function remains unknown. Most of these proteins exhibit an unusual EPR signal
in which the copper hyperfine splitting pattern is poorly resolved. There is good
evidence that at least for stellacyanin, methionine does not function as a ligand
for copper.

2.5.2 Type 2 Copper Proteins

The copper centres in these proteins are spectroscopically consistent with square
planar or pyramidal coordination, containing oxygen and/or nitrogen ligation.
The Cu(II) is EPR active, with a ‘normal’ signal. There is no intense blue color.
This group includes the copper/zinc superoxide dismutase (EC 1.15.1.1),
dopamine b-monooxygenase (EC 1.14.17.1), galactose oxidase (EC 1.1.3.9)
and the various copper-containing amine oxidases. Some members of this last
group may also contain an organic prosthetic group, such as PQQ
(see section 10), or a modified amino-acid residue.

2.5.3 Type 3 Copper Proteins

In this group a pair of copper atoms comprise a dinuclear centre, with no EPR
activity as for single Cu’s. The best known example of an enzyme containing a
single Type 3 centre is tyrosinase (catechol oxidase, EC 1.10.3.1). This protein
contains a metal center which is a structural analogue of the dinuclear copper
center in hemocyanin (ref 31).

2.5.4 Multi-Copper Oxidases

In addition to the above, there are several proteins with catalytic activity that
contain Types 1, 2 and 3 centres in various stoichiometric ratios. These
include L-ascorbate oxidase (EC 1.10.3.3), laccase (EC 1.10.3.2) and
ceruloplasmin (ferro-oxidase, EC 1.16.3.1), the latter two having aromatic diamine
and diphenol oxidase activity. There is growing evidence that in these proteins
the Type 2 and Type 3 copper centres are juxtaposed. Recently it has been
shown that in L-ascorbate oxidase, a trinuclear copper site is present, consisting
of a type 3 copper site, very close (3.9 Å) and possibly bridged to a type 2 copper
site (ref 32). There is a view that ceruloplasmin functions as a ferro-oxidase
and the Fe(III) produced in this reaction can then oxidize the same substrates
as laccase.

2.5.5 Copper Centres in Cytochrome Oxidase

There are two copper centres that appear to be unique. Both are present in
cytochrome-c oxidase (EC 1.9.3.1). The first appears to be an isolated metal ion
and has been referred to as Cud and CuA. The second appears to be part
of a dinuclear centre with cytochrome a3. It has been referred to as Cuu,
Cua3 and CuB. At the moment the ascriptions CuA and CuB are most frequently
used; however, the recent discovery (ref 33) of a cytochrome oxidase in which
cytochrome a has been replaced by cytochrome b, leads to the recommendation
that CuB shall be referred to as Cua3.

There is a striking similarity between two of the Cu centres of N2O reductase
and CuA (ref 34, 35).

2.5.6 Molybdenum enzymes (general)

Molybdenum enzymes contain molybdenum at the catalytic center responsible
for reaction with substrate. They may be divided into those that contain
the iron-molybdenum cofactor and those that contain the pterin-molybdenum
cofactor.

2.5.7 Additional centers

If a molybdenum enzyme contains flavin, it may be called either a molybdenum
flavoprotein or a flavomolybdenum protein, as indicated above. Other centers
should be treated similarly, e.g. an iron-sulfur molybdenum protein.

2.5.8 Molybdenum enzymes containing the iron-molybdenum cofactor

The only enzymes at present known to belong to this group are the nitrogenases
(EC 1.18.6.1; and EC 1.19.6.1): see pp 89-116 in (ref 36) and pp 91-100 in (ref 37).

2.5.9 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided
into those in which the molybdenum bears a cyanide-labile sulfido (or thio
– see Note 1) ligand i.e. containing the S2- ligand as Mo=S) and those
lacking this ligand. The former group includes xanthine oxidase (EC 1.1.3.22),
xanthine dehydrogenase (EC 1.1.1.204), aldehyde oxidase (EC 1.2.3.1) and
purine hydroxylase (EC: see Note 2 and 3). These may be called ‘molybdenum-
containing hydroxylase’ as is widely done. Molybdenum enzymes lacking the
sulfide (thio) ligand include sulfite oxidase (EC 1.8.3.1), NAD(P)+-independent
aldehyde dehydrogenase and nitrate reductases (assimilatory and dissimilatory)
(EC 1.6.6.1-3).

2.5.10 Molybdenum enzymes containing the pterin-molybdenum cofactor

These enzymes [see pp 411-415 in (ref 36) and (ref 38)] may be divided into those
in which the molybdenum bears a cyanide-labile sulfido (or thio – see Note 1)
ligand i.e. containing the S2- ligand as Mo=S) and those lacking this ligand. The
former group includes xanthine oxidase (EC 1.1.3.22), xanthine dehydrogenase
(EC 1.1.1.204), aldehyde oxidase (EC 1.2.3.1) and purine hydroxylase. These
may be called ‘molybdenum-containing hydroxylase’ as is widely done.
Molybdenum enzymes lacking the sulfide (thio) ligand include sulfite oxidase
(EC 1.8.3.1), NAD(P)+-independent aldehyde dehydrogenase and nitrate
reductases (assimilatory and dissimilatory) (EC 1.6.6.1-3).

2.5.11 Metal-Substituted Metalloproteins

Scientists from several areas, dealing with spectroscopy and electron-transfer
mechanisms, often use metalloproteins in which a metal at the active site has
been substituted by another metal ion, like Co, Zn, Hg, Cd. Examples are zinc-
substituted cytochromes and cobalt-substituted ferredoxins.

The names for such modified proteins are easily given by using indications
like: ‘zinc-substituted ….’. In case of multi-metal proteins, where ambiguity might
arise about which metal has been substituted, one could easily add in parentheses
the name of the metal that has been replaced, such as: cobalt- substituted [Fe]
nitrogenase.

In formulae fragments or short names one could use the following notation:
[3Fe1Co-4S]2+, cytochrome c'[Fe[arrow right]CoFe], plastocyanin[Cu
[arrow right]Hg].

Ambler, R.P. (1980) in From Cyclotrons to Cytochromes (Kaplan, N.O. &
Robinson, A., eds) Academic Press, New York

Moore, G. & Pettigrew, F.(1987) Cytochromes c, Springer-Verlag, Berlin

Bartsch, R.G. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A. &
Vernon, L.P., ed.) p. 315, Antioch Press, Yellow Springs, Ohio.

Stiefel, E.I. & Cramer, S.P. (1985) in Molybdenum Enzymes (Spiro, T.G., ed.),
Wiley-Interscience, New York, 89-116.

Smith B.E. et al. (1988), in Nitrogen Fixation Hundred Years After (Bothe,
H., de Bruijn, F.J. & Newton, W.E., ed.), Gustav Fischer, Stuttgart, New York,
91-100

Type-2 copper-containing enzymes.
MacPherson IS1, Murphy ME.
Cell Mol Life Sci. 2007 Nov;64(22):2887-99.

Type-2 Cu sites are found in all the major branches of life and are often
involved in the catalysis of oxygen species. Four type-2 Cu protein
families are selected as model systems for review: amine oxidases,
Cu monooxygenases, nitrite reductase/multicopper oxidase, and
CuZn superoxide dismutase. For each model protein, the availability
of multiple crystal structures and detailed enzymological studies provides
a detailed molecular view of the type-2 Cu site and delineation of the
mechanistic role of the Cu in biological function. Comparison of these
model proteins leads to the identification of common properties of the
Cu sites and insight into the evolution of the trinuclear active site found
in multicopper oxidases.

Copper proteins and copper enzymes.
Cass AE, Hill HA.
Ciba Found Symp. 1980;79:71-91.
http://www.chm.bris.ac.uk/motm/caeruloplasmin/copper_proteins/t1.htm

The copper proteins that function in homeostasis, electron transport, dioxygen
transport and oxidation are discussed. Particular emphasis is placed on the
role of the ligands, their type and disposition which, in conjunction with other
residues in the active site, determine the role of the copper ion. It is proposed that
copper proteins can be considered in four groups. Those in Group I contain a
single copper ion in an approximately tetrahedral environment with nitrogen and
sulphur-containing ligands. Group II proteins have a single copper ion in a square-
planar-like arrangement. Group III proteins have two copper ions in close
proximity. Group IV consists of multi-opper proteins, composed of sites
representative of the other three groups.

Such centers owe their name to the intense blue coloration of the corresponding
Cu(II) proteins. The color is particularly distinctive since the metal centers are
so optically diluted in these metalloenzymes that only intense absorption in the
visible region, resulting from symmetry allowed electronic transitions, can give
rise to conspicuous colors. In contrast, the comparatively pale blue color of normal
Cu(II)) is the result of forbidden electronic transitions between d-orbitals of
different symmetry; in Cu2+(aq) this gives a molar extinction coefficient of
10 M-1cm-1 from a broad absorption between 10,000 cm-1 and 15,000 cm-1
compared to about 3000 M-1cm-1 observed for blue Cu(II) centers. For the
T1 centers the intense absorption is attributed to a ligand-to-metal charge
transfer between the Cu2+ and a bonded cysteinate ligand. Typically, as in
azurin or plastocyanin this occurs around 16,000 cm-1. Ceruloplasmin has
three T1 centers, and the blue absorption is at 16,400 cm-1 (610nm).

Plastocyanine geometry

around the copper Crystal structures show a very irregular ‘tetrahedral’ coordination
with two sulphurs from methionine and cysteinate, and two histidine nitrogens.
However a comparison of azurin with plastocyanin shows that the geometry
is in some ways closer to a trigonal bipyramid, with and without one extra apical
ligand, so that azurin has a weakly bound glutamine oxygen, and plastocyanine
does not. The T1 coppers in caruloplasmin are in plastocyanine-type domains.
Each of these are coordinated to two histidines and a cysteine, in two of the T1
domains there is also a methionine residue, the third T1 domain has a leucine
residue which may only have a van der Waals type contact with the copper.

T1 copper centers are functional in the reversible electron transfer:

Cu2+ + e- = Cu+

The strongly distorted geometry represents a compromise (entactic-state
situation) between d10 Cu(I), with its preferred tetrahedral or trigonal
coordination through soft sulfur ligands, and d9 Cu(II) with preferential
square planar or square pyramidal geometry and nitrogen ligand
coordination. This irregular, high energy arrangement at the metal
center resembles the transition-state geometry between the tetrahedral
and square planar equilibrium configurations of the two oxidation states
involved and permits enhanced rates of electron transfer. The potential
range for proteins with T1 copper centers runs from 180 mV in
stellacyanin to 680 mV in rusticyanin.

Zinc proteins: enzymes, storage proteins, transcription factors, and replication
proteins.
Coleman JE.
Annu Rev Biochem. 1992;61:897-946.

In the past five years there has been a great expansion in our knowledge of
the role of zinc in the structure and function of proteins. Not only is zinc
required for essential catalytic functions in enzymes (more than 300 are known
at present), but also it stabilizes and even induces the folding of protein
subdomains. The latter functions have been most dramatically illustrated
by the discovery of the essential role of zinc in the folding of the DNA-binding
domains of eukaryotic transcription factors, including the zinc
finger transcription factors, the large family of hormone receptor proteins,
and the zinc cluster transcription factors from yeasts. Similar functions are
highly probable for the zinc found in the RNA polymerases and the zinc-
containing accessory proteins involved in nucleic acid replication. The rapid
increase in the number and nature of the proteins in which zinc functions
is not unexpected since zinc is the second most abundant trace metal found in
eukaryotic organisms, second only to iron. If one subtracts the amount of iron
found in hemoglobin, zinc becomes the most abundant trace metal found
in the human body.

Zinc Coordination Spheres in Protein Structures
ACS ChemWorx
Mikko Laitaoja , Jarkko Valjakka , and Janne Jänis
Inorg. Chem., 2013, 52 (19), pp 10983–10991
http://dx.doi.org:/10.1021/ic401072d
Sept 23, 2013

Synopsis
A statistical analysis in terms of zinc coordinating amino acids, metal-to-ligand
bond lengths, coordination number, and structural classification was performed,
revealing coordination spheres from classical tetrahedral cysteine/histidine binding
sites to more complex binuclear sites with carboxylated lysine residues. According
to the results, coordination spheres of hundreds of crystal structures in the PDB
could be misinterpreted due to symmetry-related molecules or missing electron
densities for ligands.

Protein-folding location can regulate manganese-binding versus copper- or
zinc-binding.
Tottey S, Waldron KJ, Firbank SJ, Reale B, Bessant C, Sato K, Cheek TR, et al.
Nature. 2008 Oct 23;455(7216):1138-42. http://dx.doi.org:/10.1038/nature07340

Metals are needed by at least one-quarter of all proteins. Although metallo-
chaperones insert the correct metal into some proteins, they have not been
found for the vast majority, and the view is that most metalloproteins acquire
their metals directly from cellular pools. However, some metals form more
stable complexes with proteins than do others. For instance, as described
in the Irving-Williams series, Cu(2+) and Zn(2+) typically form more stable
complexes than Mn(2+). Thus it is unclear what cellular mechanisms manage
metal acquisition by most nascent proteins. To investigate this question, we
identified the most abundant Cu(2+)-protein, CucA (Cu(2+)-cupin A), and the
most abundant Mn(2+)-protein, MncA (Mn(2+)-cupin A), in the periplasm of
the cyanobacterium Synechocystis PCC 6803. Each of these newly identified
proteins binds its respective metal via identical ligands within a cupin fold.
Consistent with the Irving-Williams series, MncA only binds Mn(2+) after
folding in solutions containing at least a 10(4) times molar excess of Mn(2+)
over Cu(2+) or Zn(2+). However once MncA has bound Mn(2+), the metal
does not exchange with Cu(2+). MncA and CucA have signal peptides for
different export pathways into the periplasm, Tat and Sec respectively. Export
by the Tat pathway allows MncA to fold in the cytoplasm, which contains only
tightly bound copper or Zn(2+) (refs 10-12) but micromolar Mn(2+) (ref. 13). In
contrast, CucA folds in the periplasm to acquire Cu(2+). These results reveal
a mechanism whereby the compartment in which a protein folds overrides its
binding preference to control its metal content. They explain why the cytoplasm
must contain only tightly bound and buffered copper and Zn(2+).

Predicting copper-, iron-, and zinc-binding proteins in pathogenic species of the
Paracoccidioides genus
GB Tristão, L do Prado Assunção, LPA dos Santos, CL Borges, MG Silva-Bailão,
CM de Almeida Soares, G Cavallaro and AM Bailão*
Front. Microbiol., 9 Jan 2015 http://dx.doi.org:/10.3389/fmicb.2014.00761

Approximately one-third of all proteins have been estimated to contain at least
one metal cofactor, and these proteins are referred to as metalloproteins. These
represent one of the most diverse classes of proteins, containing metal ions that
bind to specific sites to perform catalytic, regulatory and structural functions.
Bioinformatic tools have been developed to predict metalloproteins encoded by
an organism based only on its genome sequence. Its function and the type of
metal binder can also be predicted via a bioinformatics approach. Paracoccidioides
complex includes termodimorphic pathogenic fungi that are found as saprobic
mycelia in the environment and as yeast, the parasitic form, in host tissues. They
are the etiologic agents of Paracoccidioidomycosis, a prevalent systemic mycosis
in Latin America. Many metalloproteins are important for the virulence of several
pathogenic microorganisms. Accordingly, the present work aimed to predict the
copper, iron and zinc proteins encoded by the genomes of three phylogenetic species
of Paracoccidioides (Pb01, Pb03, andPb18). The metalloproteins were identified
using bioinformatics approaches based on structure, annotation and domains. Cu-,
Fe-, and Zn-binding proteins represent 7% of the total proteins encoded by
Paracoccidioides spp. genomes. Zinc proteins were the most abundant metallo-
proteins, representing 5.7% of the fungus proteome, whereas copper and iron
proteins represent 0.3 and 1.2%, respectively. Functional classification revealed that
metalloproteins are related to many cellular processes. Furthermore, it was observed
that many of these metalloproteins serve as virulence factors in the biology of the
fungus. Thus, it is concluded that the Cu, Fe, and Zn metalloproteomes of the
Paracoccidioides spp. are of the utmost importance for the biology and virulence
of these particular human pathogens.

Zinc finger proteins: new insights into structural and functional diversity
John H Laity, Brian M Lee, Peter E Wright
Current Opinion in Structural Biology Feb 2001; 11(1): 39–46
http://epigenie.com/key-epigenetic-players/chromatin-modifying-and-dna-
binding-proteins/zinc-finger-proteins/

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes.
Their functions are extraordinarily diverse and include DNA recognition, RNA
packaging, transcriptional activation, regulation of apoptosis, protein folding
and assembly, and lipid binding. Zinc finger structures are as diverse as their
functions. Structures have recently been reported for many new zinc finger
domains with novel topologies, providing important insights into structure/function
relationships. In addition, new structural studies of proteins containing the
classical Cys2His2 zinc finger motif have led to novel insights into mechanisms
of DNA binding and to a better understanding of their broader functions in
transcriptional regulation.

Zinc Finger Proteins

Zinc finger (ZnF) proteins are a massive, diverse family of proteins that serve a
wide variety of biological functions. Due to their diversity, it is difficult to come up
with a simple definition of what unites all ZnF proteins; however, the most common
approach is to define them as all small, functional domains that require coordination
by at least one zinc ion (Laity et al., 2001). The zinc ion serves to stabilize the
integration of the protein itself, and is generally not involved in binding targets.
The “finger” refers to the secondary structures (α-helix and β-sheet) that are
held together by the Zn ion. Zinc finger containing domains typically serve
as interactors, binding DNA, RNA, proteins or small molecules (Laity et al., 2001).

ZnF Protein Families

Cys2His2 was the first domain discovered (also known as Krüppel-type). It was
initially discovered as a repeating domain in the IIIA transcription factor in
Xenopus laevis (Brown et al., 1985; Miller et al., 1985). IIIA has nine repeats
of the 30 amino acids that make up the Cys2His2 domain. Each domain forms
a left-handed ββα secondary structure, and coordinates a Zn ion between
two cysteines on the β-sheet hairpin and two histidines in the α-helix, hence
the name Cys2His2 (Lee et al., 1989). These resides are highly conserved,
as well as a general hydrophobic core that allows the helix to form. The other
residues can show great sequence diversity (Michael et al., 1992). Cys2His2
zinc fingers that bind DNA tend to have 2-4 tandem domains as part of a
larger protein. The residues of the alpha helices form specific contacts with a
specific DNA sequence motif by “reading” the nucleotides in major groove
of DNA (Elrod-Erickson et al., 1996; Pavletich and Pabo, 1991). Cys2His2
proteins are the biggest group of transcription factors in most species. Non-
DNA binding proteins can have much more flexible tertiary structure.
Examples of Cys2His2 proteins include the Inhibitor of Apoptosis (IAP) family
of proteins and the CTFC transcription factor.

Treble clef fingers are a very diverse group of ZnF protiens both in terms of
structure and function. What makes them a family is a shared fold at their core
that looks a little like a musical treble clef, especially if you squint (Grishin,
2001). Most treble clef finger motifs have a β hairpin, a variable loop region,
a β hairpin, and an α helix. The “knuckle” of the β hairpin and the α helix contain
the Cys-x-x-Cys sequence necessary to coordinate the Zn ion. Treble clef
fingers often form the core of protein structures, for example the L24E and
S14 ribosomal proteins and the RING finger family.

Zinc ribbons are a little less structurally complex than the other two major groups.
Zinc ribbons contain two zinc knuckles, often β hairpins, coordinating a zinc ion via
a two Cys residures separated by 2-4 other residues on one knuckle, and a Cys-x-x-
Cys on the other (Hahn and Roberts, 2000). Examples of zinc ribbon-containing
proteins include the basal transcription factors TFIIS and TFIIB that for a complex
with RNAPII to bind DNA, and the Npl4 nuclear core protein that uses a zinc ribbon
to bind ubiquitin (Alam et al., 2004). Cys2His2, treble clef fingers, and zinc ribbons
form the majority of zinc fingers, but there are several other smaller groups that
don’t fit neatly into these three. Green fluorescent protein as a marker for gene
expression.

Metallothionein proteins expression, copper and zinc concentrations, and lipid
peroxidation level in a rodent model for amyotrophic lateral sclerosis
E Tokuda, Shin-Ichi Ono, K Ishige, A Naganuma, Y Ito, T Suzuki
Toxicology Jan 2007; 229(1–2): 33–41

It has been hypothesized that copper-mediated oxidative stress contributes to the
pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron
disease in humans. To verify this hypothesis, we examined the copper and zinc
concentrations and the amounts of lipid peroxides, together with that of the
expression of metallothionein (MT) isoforms in a mouse model [superoxide
dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and
MT-II (MT-I/II) isoforms were measured together with Western blotting, copper
level, and lipid peroxides amounts increased in an age-dependent manner in the
spinal cord, the region responsible for motor paralysis. A significant increase was
already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not
yet exhibited motor paralysis, and showed a further increase at 16 weeks of age,
when paralysis was evident. Inversely, the spinal zinc level had significantly
decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level,
remained at the same level as an 8-week-old wild-type mouse, finally increasing
to a significant level at 16 weeks of age. It has been believed that a mutant SOD1
protein, encoded by a mutant SOD1, gains a novel cytotoxic function while
maintaining its original enzymatic activity, and causes motor neuron death
(gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable
underlying pathogenesis of gain-of-toxic function. Taking the above current
concepts and the classic functions of MT into account, MTs could have a disease
modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function
at the early stage of the disease, and the MT-III isoform at an advanced stage.

Prion protein expression level alters regional copper, iron and zinc content in
the mouse brain
MJ Pushie, IJ Pickering, GR Martin, S Tsutsui, FR Jirik and GN George
Metallomics, 2011,3, 206-214 http://dx.doi.org:/10.1039/C0MT00037J

The central role of the prion protein (PrP) in a family of fatal neurodegenerate
diseases has garnered considerable research interest over the past two decades.
Moreover, the role of PrP in neuronal development, as well as its apparent role
in metal homeostasis, is increasingly of interest. The host-encoded form of the
prion protein (PrP^C) binds multiple copper atoms via its N-terminal domain
and can influence brain copper and iron levels. The importance of PrP^C to the
regulation of brain metal homeostasis and metal distribution, however, is not
fully understood. We therefore employed synchrotron-based X-ray fluorescence
imaging to map the level and distributions of several key metals in the brains of
mice that express different levels of PrP^C. Brain sections from wild-type, prion
gene knockout (Prnp^−/−) and PrP^C over-expressing mice revealed striking
variation in the levels of iron, copper, and even zinc in specific brain regions as
a function of PrP^C expression. Our results indicate that one important function
of PrP^C may be to regulate the amount and distribution of specific metals within
the central nervous system. This raises the possibility that PrP^C levels, or its
activity, might regulate the progression of diseases in which altered metal
homeostasis is thought to play a pathogenic role such as Alzheimer’s,
Parkinson’s and Wilson’s diseases and disorders such as hemochromatosis.

Zinc & Copper Imbalances: Immense Biochemical Implications
Mar 27, 2013 by Michael McEvoy
http://metabolichealing.com/zinc-copper-imbalances-immense-biochemical-
implications/

The status of zinc and copper levels may have profound implications for
many people. Much has been written about the significance of these two
trace elements for many, many years. Many health conditions may be
directly caused by abnormal zinc and copper levels.

With all of the recent attention given to methylation status, gene mutations,
MTHFR, and the associated neurological and mental/behavioral disorders
that may ensue, zinc and copper status remains a pivotal ratio in these regards.

While zinc toxicity and copper deficiency are possible, the subject of this
article is on the more common imbalance: copper toxicity and zinc deficiency.

The Physiological Roles Of Zinc & Copper

Zinc and copper are antagonists. The balance between these two trace
elements is an example of the effects of biological dualism. While zinc
toxicity is possible, far more common is zinc deficiency and copper toxicity.
Both zinc and copper play essential roles in the body, and there can be a
number of causes for why imbalances ensue.

It may be easier to identify the roles that zinc doesn’t play in the body,
than the roles it does play. Zinc is an essential trace element that activates
several hundred enzymatic reactions. These reactions are fundamental
to life and biological activity. Some of the activities that zinc are involved in:

DNA & RNA synthesis
Gene expression
Nervous system function
Immune function & immune signaling such as cell
apoptosis
Neuronal transmission
Brain function
Zinc possesses powerful anabolic activities in the cells
Formation of zinc proteins known as “zinc fingers”
Zinc is essential for blood clotting and platelet formation
Zinc is involved in Vitamin A synthesis
Folate is made available through zinc enzyme reactions
Along with copper, Zinc makes up the antioxidant
enzyme
system, ZnCu superoxide dismutase
Steroidal hormone synthesis
Growth & development of children
Testosterone and semen formation
The highest concentration of zinc is found in the
male prostate gland

Copper is an essential trace element serving many important functions
as well. However, copper is well documented to induce several toxic effects
in the body, when elevated. Because copper is a pro-oxidant when free and
unbound, it can quickly generate free radicals.

The major sources for copper toxicity are: exposure to industrial forms
of copper such as copper pipes, copper cookware, birth control, exposure
to copper-based fungicides. Diets high in copper and low in zinc may play
a role in copper toxicity. Pyrrole disorder, which causes depletion of zinc,
may result in elevated levels of copper.

Some of the essential roles copper plays in the body:

Connective tissue formation
ATP synthesis
Iron metabolism
Brain health via neurotransmitter synthesis
Gene transcription
Synthesis of the antioxidant superoxide dismutase
Skin pigmentation
Nerve tissue: myelin sheath formation
Copper tends to rise when estrogen is dominant

Perhaps one of the first reports that zinc and copper imbalances play
a role in human health and disease was their detection in mental
disorders made by Carl Pfeiffer, MD, PhD. Dr. Pfeiffer identified a
condition known as pyrrole disorder, sometimes referred to as
pyrroluria or “mauve factor”.

As it turns out, pyrrole disorder is a major biochemical imbalance
in many people with chronic illnesses such as chronic Lyme disease,
autism, schizophrenia, depression, bi-polar, and chronic fatigue
syndrome. Pyrroles are a byproduct of hemoglobin synthesis.
Apparently, some individuals are more predisposed towards producing
higher amounts of pyrroles. When pyrroles are excessive, they irreversibly
bind to zinc and vitamin B6, causing their excretion. Consequently,
it is common that once zinc levels become depleted, copper levels tend to rise.

Copper Toxicity

Problems associated with copper toxicity include: pyrrole disorder,
estrogen dominance, schizophrenia, depression, anxiety disorder,
chronic fatigue, migraines, liver toxicity, thyroid conditions, chronic
candida yeast infections, PMS, to name a few. Some research has
even implicated copper toxicity with Alzheimer’s Disease and with
cardiovascular disease. Perhaps one of the primary mechanisms
through which copper toxicity can damage tissues is through its
initiation of oxidative stress and free radical formation. Free copper
ions that are not bound to copper proteins such as ceruloplasmin,
are pro-oxidants, and are highly reactive.

Empirical research from clinicians, indicates that there are different
types of copper imbalances. For example, if there is a lot of free,
unbound copper present, this may cause a situation of nutritive
copper deficiency. Another copper imbalance is when high pyrroles
depress zinc levels, and copper levels concomintantly rise. If high
pyrroles are present, B6 will also be lost in high amounts. In a general
but very real sense, all forms of copper excess will affect zinc status,
due to the dualistic nature of zinc and copper.

Copper & Estrogen

It has been known for many years that copper can cause a rise in
estrogen, and conversely estrogen may raise copper. Estrogen
dominance has been extensively studied in its role in breast
cancer development. One possible, critical role that can cause
estrogen to become carcinogenic, is through its oxidation induced by
copper. Once oxidized, estrogen forms volatile hydroxyl radicals and
the associated DNA damage and “mutagenesis”.

Zinc Deficiency

As mentioned previously, pyrrole disorder will directly depress
zinc status, causing high levels of its excretion. When zinc is
lost, copper rises. Because of their essential roles in neuro-
transmitter synthesis, low zinc and high copper levels can
directly effect cognition, behavior and thought processes.
Zinc has been studied in biochemical reactions involving
calcium-driven, synaptic neurotransmission, as well as in
glutamate/GABA balance and with limbic brain function.

Zinc & Reproduction

Zinc is essential for steroidal hormone synthesis, and is a
well known catalyst for testosterone synthesis, as well as
leutinizing hormone. Zinc has demonstrated its ability to
prevent miscarriage and toxicity during pregnancy. The male
prostate gland reportedly contains the highest concentration
of zinc in the body.

Zinc & Brain Function

Much attention has been given to excitotoxicity, such as the
effects induced by MSG (monosodium glutamtate). Excess
stimulation of the excitatory neurotransmitter glutamate,
may cause severe physical and psychological reactions in
certain individuals. Zinc has been studied for its ability to
enhance GABA (glutamate’s antagonistic neurotransmitter)
activity and to suppress excess glutamate.

Studies on mice demonstrated that when depleted of zinc
for two weeks, the mice developed seizures, most likely due
to GABA deficiencies and glutamate excess.

There is an emerging body of evidence that demonstrates
that Alzheimer’s disease may involve copper toxicity and
zinc deficiency. Not only can excess copper cause zinc
depletion, but so can excess lead.

The hippocampus, a major part of the limbic brain, records
memories and is responsible for processing meaningful
experiences. Numerous studies site that if hippocampal
cells are deprived of zinc, the hippocampal cells die. In
addition to hippocampus cell death induced by zinc
deprivation, the amygdala, the other major limbic gland
experiences cell death as well, when deprived of zinc.

Green Fluorescent Protein

Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC.
Science. 1994 Feb 11;263(5148):802-5.
http://www.ncbi.nlm.nih.gov/pubmed/8303295

A complementary DNA for the Aequorea victoria green fluorescent protein (GFP)
produces a fluorescent product when expressed in prokaryotic (Escherichia coli)
or eukaryotic (Caenorhabditis elegans) cells. Because exogenous substrates and
cofactors are not required for this fluorescence, GFP expression can be used
to monitor gene expression and protein localization in living organisms.

http://en.wikipedia.org/wiki/Green_fluorescent_protein

The green fluorescent protein (GFP) is a protein composed of 238 amino acid
residues (26.9 kDa) that exhibits bright green fluorescence when exposed
to light in the blue to ultraviolet range. Although many other marine organisms
have similar green fluorescent proteins, GFP traditionally refers to the protein
first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria
has a major excitation peak at a wavelength of 395 nm and a minor one at
475 nm. Its emission peak is at 509 nm, which is in the lower green portion
of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79.
The GFP from the sea pansy (Renilla reniformis) has a single major excitation
peak at 498 nm.

In cell and molecular biology, the GFP gene is frequently used as a reporter of
expression. In modified forms it has been used to make biosensors, and many
animals have been created that express GFP as a proof-of-concept that a gene
can be expressed throughout a given organism. The GFP gene can be introduced
into organisms and maintained in their genome through breeding, injection with a
viral vector, or cell transformation. To date, the GFP gene has been introduced
and expressed in many Bacteria, Yeast and other Fungi, fish (such as zebrafish),
plant, fly, and mammalian cells, including human. Martin Chalfie, Osamu Shimomura,
and Roger Y. Tsien were awarded the 2008 Nobel Prize in Chemistry on 10 October
2008 for their discovery and development of the green fluorescent protein.

http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

In Aequorea victoria a protein called aequorin releases blue light upon binding
with calcium. This blue light is then totally absorbed by the GFP, which in turn
gives off the green light as in the animation below.

In 1994 GFP was cloned. Now GFP is found in laboratories all over the world where
it is used in every conceivable plant and animal. Flatworms, algae, E. coli and pigs
have all been made to fluoresce with GFP.

The importance of GFP was recognized in 2008 when the Nobel Committee awarded
Osamu Shimomura, Marty Chalfie and Roger Tsien the Chemistry Nobel Prize ”
for the discovery and development of the green fluorescent protein, GFP.”

Why is it so popular? Well, I like to think of GFP as the microscope of the twenty-
first century. Using GFP we can see when proteins are made, and where they can go.
This is done by joining the GFP gene to the gene of the protein of interest so that
when the protein is made it will have GFP hanging off it. Since GFP fluoresces, one
can shine light at the cell and wait for the distinctive green fluorescence associated
with GFP to appear.

A variant of yellow fluorescent protein with fast and efficient maturation for
cell-biological applications
T Nagai, K Ibata, E Sun Park, M Kubota, K Mikoshiba & A Miyawaki
Nature Biotechnology 20, 87 – 90 (2002) http://dx.doi.org:/10.1038/nbt0102-87

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria
has provided a myriad of applications for biological systems. Over the last
several years, mutagenesis studies have improved folding properties of GFP.
However, slow maturation is still a big obstacle to the use of GFP variants for
visualization. These problems are exacerbated when GFP variants are expressed
at 37°C and/or targeted to certain organelles. Thus, obtaining GFP variants that
mature more efficiently is crucial for the development of expanded research
applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs)
are relatively acid-sensitive,and uniquely quenched by chloride ion (Cl−)3. For
YFP to be fully and stably fluorescent, mutations that decrease the sensitivity
to both pH and Cl− are desired. Here we describe the development of an
improved version of YFP named “Venus”. Venus contains a novel mutation,
F46L, which at 37°C greatly accelerates oxidation of the chromophore, the rate-
limiting step of maturation. As a result of other mutations, F64L/M153T/
V163A/S175G, Venus folds well and is relatively tolerant of exposure
to acidosis and Cl−. We succeeded in efficiently targeting a neuropeptide
Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion
was readily monitored by measuring release of fluorescence into the medium.
The use of Venus as an acceptor allowed early detection of reliable signals of
fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain
slices. With the improved speed and efficiency of maturation and the increased
resistance to environment, Venus will enable fluorescent labelings that were not
possible before.

Rhodopsin-like Protein from the Purple Membrane of Halobacterium halobium
DIETER OESTERHELT & WALTHER STOECKENIUS
Nature New Biology 29 Sep 1971; 233, 149-152 | http://dx.doi.org:/10.1038/
newbio233149a0

HALOPHILIC bacteria require high concentrations of sodium chloride and lower
concentrations of KCl and MgCl2 for growth. The cell membrane dissociates into
fragments of varying size when the salt is removed1. One characteristic fragment—
termed the “purple membrane” because of its characteristic deep purple colour—
has been isolated in relatively pure form from Halobacterium halobium. We can
now show that the purple colour is due to retinal bound to an opsin-like protein,
the only protein present in this membrane fragment.

References

Stoeckenius, W. , and Rowen, R. , J. Cell Biol., 34, 365 (1967).

Stoeckenius, W. , and Kunau, W. H. , J. Cell Biol., 38, 337 (1968).

Blaurock, A. E. , and Stoeckenius, W. , Nature New Biology, 233, 152 (1971).

Sehgal, S. N. , and Gibbons, N. E. , Canad. J. Microbiol., 6, 165 (1960).

Kelly, M. , Norgård, S. , and Liaach-Jensen, S. , Acta Chem. Scand., 2A, 2169 (1970).

Shapiro, A. L. , Vinnela, E. , and Maizel, jun., J. V. , Biochem. Biophys. Res.
Commun., 28, 815 (1967).

The monomerization of the Purple protein, a member of the GFP-family
Corning, Brooke

Green fluorescent protein (GFP) has been used extensively since its discovery
in the 1960s to report and visualize gene expression. For years it has been the only
known naturally occurring fluorescent pigment that is encoded by a single gene,
making it extremely useful in various fields of biology, because the expression of
this gene directly leads to the appearance of the fluorescent green color. Recently,
however, many more proteins with similar properties to GFP, and available in a
variety of colors, have been isolated from the class of marine organisms called
Anthozoa, which includes the corals. This increase in the availability of colored
proteins in GFP family in turn has expanded the number of available biotech-
nology applications. However, some of these newly discovered GFP-like
proteins do not have wild-type forms that readily allow for the creation of
fusion proteins, particularly because of oligomerization. It is widely accepted
that almost all members of the GFP-family form dimers or tetramers in their
functional forms.

This study investigates a GFP-ike protein, Purple, isolated from two species,
Galaxea fascicularis and Montipora efflorescens. Purple protein forms oligomers
when expressed, which would then interfere with the normal expression of a protein
to be tagged in gene fusion experiments. We selectively mutated 3 amino acids,
which we believed were responsible for oligomerization in Purple. These 3
residues were chosen based on sequence similarities to a very similar protein,
a mutant form of the Rtms5 chromoprotein from Montipora efflorescens. While
we had hoped that the resulting triple-mutant Purple protein would form
monomers in vivo while retaining its purple coloration, this turned out to
be incorrect. The resulting mutants had lost their ability to turn purple. However,
we also determined that we had successfully changed the oligomerization
state of Purple by examining the relative molecular mass of one our
mutant proteins, which turned out to be half the size of the original
purple protein. It is possible that by adding additional mutations in
the future, the original spectral properties could be recovered. If
successful, this would further expand the utility of the GFP family.

Rhodopsin, also known as visual purple, from Ancient Greek ῥόδον
(rhódon, “rose”), due to its pinkish color, and ὄψις (ópsis, “sight”), is
a light-sensitive receptor protein. It is a biological pigment in photo-
receptor cells of the retina. Rhodopsin is the primary pigment found
in rod photoreceptors. Rhodopsins belong to the G-protein-coupled
receptor (GPCR) family. They are extremely sensitive to light, enabling
vision in low-light conditions. Exposed to light, the pigment
immediately photobleaches, and it takes about 45 minutes to regenerate
fully in humans. Its discovery was reported by German physiologist
Franz Christian Boll in 1876.

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Archive for the ‘Proteomics’ Category

The Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics

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Depth Underwater and Underground

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Altitude Adaptation

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Neonatal Pathophysiology

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Endocrine Action on Midbrain

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Thyroid Function and Disorders

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Proteomics

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Genomics and Metabolomics Advances in Cancer

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

Metabolic Profiling Reveals a Dependency of Human Metastatic Breast Cancer on Mitochondrial Serine and One-Carbon Unit Metabolism

Abstract

ntroduction

Materials and Methods

Cell lines

RNAi

Immunoblot

RNA Isolation, Reverse Transcription, and Real-Time PCR

In vivo Tumor Growth Assays

Metabolite Profiling and Mass Spectrometry

Cell Line Classification

Outcome Analysis

Immunohistochemical Staining and Quantification for SHMT2

SHMT2 Expression Analysis by Individual Cancer Stage

Statistical Analyses

Results

Metastatic breast cancer cells exhibit altered metabolic profiles

c-Myc is important for breast cancer cell proliferation

Identification of serine and one-carbon unit pathway elevation in metastatic subclones

Metastatic subclones display increased mitochondrial serine and one-carbon unit pathway activity

Serine catabolism is necessary for metastatic cancer cell proliferation in vitro

SHMT2 knockdown impairs primary and metastatic growth in vivo

Mitochondrial serine and 1C unit pathway genes are associated with more aggressive metastatic disease in some human breast cancer patients

Relevance of SHMT2 expression in the progression and aggressiveness of other cancer types

Discussion

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The Colors of Respiration and Electron Transport

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The Colors of Life Function

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