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Miller started his education focusing on speech and language and published papers on these topics, focusing on mathematical, computational and psychological aspects of the field. He started his career at a time when the reigning theory in psychology was behaviorism, which eschewed any attempt to study mental processes and focused only on observable behavior. Working mostly at Harvard University, MIT and Princeton University, Miller introduced experimental techniques to study the psychology of mental processes, by linking the new field of cognitive psychology to the broader area of cognitive science, including computation theory and linguistics. He collaborated and co-authored work with other figures in cognitive science and psycholinguistics, such as Noam Chomsky. For moving psychology into the realm of mental processes and for aligning that move with information theory, computation theory, and linguistics, Miller is considered one of the great twentieth-century psychologists. A Review of General Psychology survey, published in 2002, ranked Miller as the 20th most cited psychologist of that era.[2]
Remembering George A. Miller
The human mind works a lot like a computer: It collects, saves, modifies, and retrieves information. George A. Miller, one of the founders of cognitive psychology, was a pioneer who recognized that the human mind can be understood using an information-processing model. His insights helped move psychological research beyond behaviorist methods that dominated the field through the 1950s. In 1991, he was awarded the National Medal of Science for his significant contributions to our understanding of the human mind.
From the days of William James, psychologists had the idea memory consisted of short-term and long-term memory. While short-term memory was expected to be limited, its exact limits were not known. In 1956, Miller would quantify its capacity limit in the paper “The magical number seven, plus or minus two”. He tested immediate memory via tasks such as asking a person to repeat a set of digits presented; absolute judgment by presenting a stimulus and a label, and asking them to recall the label later; and span of attention by asking them to count things in a group of more than a few items quickly. For all three cases, Miller found the average limit to be seven items. He had mixed feelings about the focus on his work on the exact number seven for quantifying short-term memory, and felt it had been misquoted often. He stated, introducing the paper on the research for the first time, that he was being persecuted by an integer.[1] Miller also found humans remembered chunks of information, interrelating bits using some scheme, and the limit applied to chunks. Miller himself saw no relationship among the disparate tasks of immediate memory and absolute judgment, but lumped them to fill a one-hour presentation. The results influenced the budding field of cognitive psychology.[15]
WordNet
For many years starting from 1986, Miller directed the development of WordNet, a large computer-readable electronic reference usable in applications such as search engines.[12] Wordnet is a dictionary of words showing their linkages by meaning. Its fundamental building block is a synset, which is a collection of synonyms representing a concept or idea. Words can be in multiple synsets. The entire class of synsets is grouped into nouns, verbs, adjectives and adverbs separately, with links existing only within these four major groups but not between them. Going beyond a thesaurus, WordNet also included inter-word relationships such as part/whole relationships and hierarchies of inclusion.[16] Miller and colleagues had planned the tool to test psycholinguistic theories on how humans use and understand words.[17] Miller also later worked closely with the developers at Simpli.com Inc., on a meaning-based keyword search engine based on WordNet.[18]
Language psychology and computation
Miller is considered one of the founders of psycholinguistics, which links language and cognition in psychology, to analyze how people use and create language.[1] His 1951 book Language and Communication is considered seminal in the field.[5] His later book, The Science of Words (1991) also focused on language psychology.[19] He published papers along with Noam Chomsky on the mathematics and computational aspects of language and its syntax, two new areas of study.[20][21][22] Miller also researched how people understood words and sentences, the same problem faced by artificial speech-recognition technology. The book Plans and the Structure of Behavior (1960), written with Eugene Galanter and Karl H. Pribram, explored how humans plan and act, trying to extrapolate this to how a robot could be programmed to plan and do things.[1] Miller is also known for coining Miller’s Law: “In order to understand what another person is saying, you must assume it is true and try to imagine what it could be true of”.[23]
Miller’s Language and Communication was one of the first significant texts in the study of language behavior. The book was a scientific study of language, emphasizing quantitative data, and was based on the mathematical model of Claude Shannon‘s information theory.[24] It used a probabilistic model imposed on a learning-by-association scheme borrowed from behaviorism, with Miller not yet attached to a pure cognitive perspective.[25] The first part of the book reviewed information theory, the physiology and acoustics of phonetics, speech recognition and comprehension, and statistical techniques to analyze language.[24]The focus was more on speech generation than recognition.[25] The second part had the psychology: idiosyncratic differences across people in language use; developmental linguistics; the structure of word associations in people; use of symbolism in language; and social aspects of language use.[24]
Reviewing the book, Charles E. Osgood classified the book as a graduate-level text based more on objective facts than on theoretical constructs. He thought the book was verbose on some topics and too brief on others not directly related to the author’s expertise area. He was also critical of Miller’s use of simple, Skinnerian single-stage stimulus-response learning to explain human language acquisition and use. This approach, per Osgood, made it impossible to analyze the concept of meaning, and the idea of language consisting of representational signs. He did find the book objective in its emphasis on facts over theory, and depicting clearly application of information theory to psychology.[24]
In Plans and the Structure of Behavior, Miller and his co-authors tried to explain through an artificial-intelligence computational perspective how animals plan and act.[26] This was a radical break from behaviorism which explained behavior as a set or sequence of stimulus-response actions. The authors introduced a planning element controlling such actions.[27] They saw all plans as being executed based on input using a stored or inherited information of the environment (called the image), and using a strategy called test-operate-test-exit (TOTE). The image was essentially a stored memory of all past context, akin to Tolman‘scognitive map. The TOTE strategy, in its initial test phase, compared the input against the image; if there was incongruity the operate function attempted to reduce it. This cycle would be repeated till the incongruity vanished, and then the exit function would be invoked, passing control to another TOTE unit in a hierarchically arranged scheme.[26]
Peter Milner, in a review in the Canadian Journal of Psychology, noted the book was short on concrete details on implementing the TOTE strategy. He also critically viewed the book as not being able to tie its model to details from neurophysiology at a molecular level. Per him, the book covered only the brain at the gross level of lesion studies, showing that some of its regions could possibly implement some TOTE strategies, without giving a reader an indication as to how the region could implement the strategy.[26]
Miller’s 1967 work, The Psychology of Communication, was a collection of seven previously published articles. The first “Information and Memory” dealt with chunking, presenting the idea of separating physical length (the number of items presented to be learned) and psychological length (the number of ideas the recipient manages to categorize and summarize the items with). Capacity of short-term memory was measured in units of psychological length, arguing against a pure behaviorist interpretation since meaning of items, beyond reinforcement and punishment, was central to psychological length.[28]
The second essay was the paper on magical number seven. The third, ‘The human link in communication systems,’ used information theory and its idea of channel capacity to analyze human perception bandwidth. The essay concluded how much of what impinges on us we can absorb as knowledge was limited, for each property of the stimulus, to a handful of items.[28] The paper on “Psycholinguists” described how effort in both speaking or understanding a sentence was related to how much of self-reference to similar-structures-present-inside was there when the sentence was broken down into clauses and phrases.[29] The book, in general, used the Chomskian view of seeing language rules of grammar as having a biological basis—disproving the simple behaviorist idea that language performance improved with reinforcement—and using the tools of information and computation to place hypotheses on a sound theoretical framework and to analyze data practically and efficiently. Miller specifically addressed experimental data refuting the behaviorist framework at concept level in the field of language and cognition. He noted this only qualified behaviorism at the level of cognition, and did not overthrow it in other spheres of psychology.[28]
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.
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)
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
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
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
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
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
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
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
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
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
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
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
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?
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
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.
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
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
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?
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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)
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
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
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
Bone mesenchymal development
The bone-remodeling cycle
Nuclear receptor modulation – Role of coregulators in selective estrogen receptor modulator (SERM) actions
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
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 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.
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
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
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
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.
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 HbSDPunjab disease give us an overall idea of the severe clinical presentation of the disease in different regions of India. However the HbDPunjabE cases were milder or asymptomatic and the HbSE cases were moderately symptomatic. Since most of the cases of HbSDPunjab 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 HbSDPunjab 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
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
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 bilirubinneurotoxicityIsoimmune 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
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 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
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
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 32nd 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
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
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 (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
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.
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
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.
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.
Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer
Author and Curator: Larry H. Bernstein, MD, FCAP
This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to pharmaceutical discovery and personalized medicine. We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed. I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.
Both the developmental and phylogenetic histories of an organism describe the evolution of physiology—the complex of metabolic pathways that govern the function of an organism as a whole. The necessity of establishing and maintaining homeostatic mechanisms began at the cellular level, with the very first cells, and homeostasis provides the underlying selection pressure fueling evolution.
While the events leading to the formation of the first functioning cell are debatable, a critical one was certainly the formation of simple lipid-enclosed vesicles, which provided a protected space for the evolution of metabolic pathways. Protocells evolved from a common ancestor that experienced environmental stresses early in the history of cellular development, such as acidic ocean conditions and low atmospheric oxygen levels, which shaped the evolution of metabolism.
The reduction of evolution to cell biology may answer the perennially unresolved question of why organisms return to their unicellular origins during the life cycle.
As primitive protocells evolved to form prokaryotes and, much later, eukaryotes, changes to the cell membrane occurred that were critical to the maintenance of chemiosmosis, the generation of bioenergy through the partitioning of ions. The incorporation of cholesterol into the plasma membrane surrounding primitive eukaryotic cells marked the beginning of their differentiation from prokaryotes. Cholesterol imparted more fluidity to eukaryotic cell membranes, enhancing functionality by increasing motility and endocytosis. Membrane deformability also allowed for increased gas exchange.
Acidification of the oceans by atmospheric carbon dioxide generated high intracellular calcium ion concentrations in primitive aquatic eukaryotes, which had to be lowered to prevent toxic effects, namely the aggregation of nucleotides, proteins, and lipids. The early cells achieved this by the evolution of calcium channels composed of cholesterol embedded within the cell’s plasma membrane, and of internal membranes, such as that of the endoplasmic reticulum, peroxisomes, and other cytoplasmic organelles, which hosted intracellular chemiosmosis and helped regulate calcium.
As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.
Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor. ….
As eukaryotes thrived, they experienced increasingly competitive pressure for metabolic efficiency. Engulfed bacteria, assimilated as mitochondria, provided more bioenergy. As the evolution of eukaryotic organisms progressed, metabolic cooperation evolved, perhaps to enable competition with biofilm-forming, quorum-sensing prokaryotes. The subsequent appearance of multicellular eukaryotes expressing cellular growth factors and their respective receptors facilitated cell-cell signaling, forming the basis for an explosion of multicellular eukaryote evolution, culminating in the metazoans.
Casting a cellular perspective on evolution highlights the integration of genotype and phenotype. Starting from the protocell membrane, the functional homolog for all complex metazoan organs, it offers a way of experimentally determining the role of genes that fostered evolution based on the ontogeny and phylogeny of cellular processes that can be traced back, in some cases, to our last universal common ancestor.
Given that the unicellular toolkit is complete with all the traits necessary for forming multicellular organisms (Science, 301:361-63, 2003), it is distinctly possible that metazoans are merely permutations of the unicellular body plan. That scenario would clarify a lot of puzzling biology: molecular commonalities between the skin, lung, gut, and brain that affect physiology and pathophysiology exist because the cell membranes of unicellular organisms perform the equivalents of these tissue functions, and the existence of pleiotropy—one gene affecting many phenotypes—may be a consequence of the common unicellular source for all complex biologic traits. …
The cell-molecular homeostatic model for evolution and stability addresses how the external environment generates homeostasis developmentally at the cellular level. It also determines homeostatic set points in adaptation to the environment through specific effectors, such as growth factors and their receptors, second messengers, inflammatory mediators, crossover mutations, and gene duplications. This is a highly mechanistic, heritable, plastic process that lends itself to understanding evolution at the cellular, tissue, organ, system, and population levels, mediated by physiologically linked mechanisms throughout, without having to invoke random, chance mechanisms to bridge different scales of evolutionary change. In other words, it is an integrated mechanism that can often be traced all the way back to its unicellular origins.
The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.
A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion. THE SCIENTIST STAFF
From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.
Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.
This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.
Gut Microbiome Heritability
Analyzing data from a large twin study, researchers have homed in on how host genetics can shape the gut microbiome.
By Tracy Vence | The Scientist Nov 6, 2014
Previous research suggested host genetic variation can influence microbial phenotype, but an analysis of data from a large twin study published in Cell today (November 6) solidifies the connection between human genotype and the composition of the gut microbiome. Studying more than 1,000 fecal samples from 416 monozygotic and dizygotic twin pairs, Cornell University’s Ruth Ley and her colleagues have homed in on one bacterial taxon, the family Christensenellaceae, as the most highly heritable group of microbes in the human gut. The researchers also found that Christensenellaceae—which was first described just two years ago—is central to a network of co-occurring heritable microbes that is associated with lean body mass index (BMI). …
Of particular interest was the family Christensenellaceae, which was the most heritable taxon among those identified in the team’s analysis of fecal samples obtained from the TwinsUK study population.
While microbiologists had previously detected 16S rRNA sequences belonging to Christensenellaceae in the human microbiome, the family wasn’t named until 2012. “People hadn’t looked into it, partly because it didn’t have a name . . . it sort of flew under the radar,” said Ley.
Ley and her colleagues discovered that Christensenellaceae appears to be the hub in a network of co-occurring heritable taxa, which—among TwinsUK participants—was associated with low BMI. The researchers also found that Christensenellaceae had been found at greater abundance in low-BMI twins in older studies.
To interrogate the effects of Christensenellaceae on host metabolic phenotype, the Ley’s team introduced lean and obese human fecal samples into germ-free mice. They found animals that received lean fecal samples containing more Christensenellaceae showed reduced weight gain compared with their counterparts. And treatment of mice that had obesity-associated microbiomes with one member of the Christensenellaceae family, Christensenella minuta, led to reduced weight gain. …
Ley and her colleagues are now focusing on the host alleles underlying the heritability of the gut microbiome. “We’re running a genome-wide association analysis to try to find genes—particular variants of genes—that might associate with higher levels of these highly heritable microbiota. . . . Hopefully that will point us to possible reasons they’re heritable,” she said. “The genes will guide us toward understanding how these relationships are maintained between host genotype and microbiome composition.”
The desire for temporal and spatial control of medications to minimize side effects and maximize benefits has inspired the development of light-controllable drugs, or optopharmacology. Early versions of such drugs have manipulated ion channels or protein-protein interactions, “but never, to my knowledge, G protein–coupled receptors [GPCRs], which are one of the most important pharmacological targets,” says Pau Gorostiza of the Institute for Bioengineering of Catalonia, in Barcelona.
Gorostiza has taken the first step toward filling that gap, creating a photosensitive inhibitor of the metabotropic glutamate 5 (mGlu5) receptor—a GPCR expressed in neurons and implicated in a number of neurological and psychiatric disorders. The new mGlu5 inhibitor—called alloswitch-1—is based on a known mGlu receptor inhibitor, but the simple addition of a light-responsive appendage, as had been done for other photosensitive drugs, wasn’t an option. The binding site on mGlu5 is “extremely tight,” explains Gorostiza, and would not accommodate a differently shaped molecule. Instead, alloswitch-1 has an intrinsic light-responsive element.
In a human cell line, the drug was active under dim light conditions, switched off by exposure to violet light, and switched back on by green light. When Gorostiza’s team administered alloswitch-1 to tadpoles, switching between violet and green light made the animals stop and start swimming, respectively.
The fact that alloswitch-1 is constitutively active and switched off by light is not ideal, says Gorostiza. “If you are thinking of therapy, then in principle you would prefer the opposite,” an “on” switch. Indeed, tweaks are required before alloswitch-1 could be a useful drug or research tool, says Stefan Herlitze, who studies ion channels at Ruhr-Universität Bochum in Germany. But, he adds, “as a proof of principle it is great.” (Nat Chem Biol, http://dx.doi.org:/10.1038/nchembio.1612, 2014)
To understand disease processes, scientists often focus on unraveling how gene expression in disease-associated cells is altered. Increases or decreases in transcription—as dictated by a regulatory stretch of DNA called an enhancer, which serves as a binding site for transcription factors and associated proteins—can produce an aberrant composition of proteins, metabolites, and signaling molecules that drives pathologic states. Identifying the root causes of these changes may lead to new therapeutic approaches for many different diseases.
Although few therapies for human diseases aim to alter gene expression, the outstanding examples—including antiestrogens for hormone-positive breast cancer, antiandrogens for prostate cancer, and PPAR-γ agonists for type 2 diabetes—demonstrate the benefits that can be achieved through targeting gene-control mechanisms. Now, thanks to recent papers from laboratories at MIT, Harvard, and the National Institutes of Health, researchers have a new, much bigger transcriptional target: large DNA regions known as super-enhancers or stretch-enhancers. Already, work on super-enhancers is providing insights into how gene-expression programs are established and maintained, and how they may go awry in disease. Such research promises to open new avenues for discovering medicines for diseases where novel approaches are sorely needed.
Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions (Cell, 153:307-19, 2013). They also appear to bind a large percentage of the transcriptional machinery compared to typical enhancers, allowing them to better establish and enforce cell-type specific transcriptional programs (Cell, 153:320-34, 2013).
Super-enhancers are closely associated with genes that dictate cell identity, including those for cell-type–specific master regulatory transcription factors. This observation led to the intriguing hypothesis that cells with a pathologic identity, such as cancer cells, have an altered gene expression program driven by the loss, gain, or altered function of super-enhancers.
Sure enough, by mapping the genome-wide location of super-enhancers in several cancer cell lines and from patients’ tumor cells, we and others have demonstrated that genes located near super-enhancers are involved in processes that underlie tumorigenesis, such as cell proliferation, signaling, and apoptosis.
Super-enhancers cover stretches of DNA that are 10- to 100-fold longer and about 10-fold less abundant in the genome than typical enhancer regions.
Genome-wide association studies (GWAS) have found that disease- and trait-associated genetic variants often occur in greater numbers in super-enhancers (compared to typical enhancers) in cell types involved in the disease or trait of interest (Cell, 155:934-47, 2013). For example, an enrichment of fasting glucose–associated single nucleotide polymorphisms (SNPs) was found in the stretch-enhancers of pancreatic islet cells (PNAS, 110:17921-26, 2013). Given that some 90 percent of reported disease-associated SNPs are located in noncoding regions, super-enhancer maps may be extremely valuable in assigning functional significance to GWAS variants and identifying target pathways.
Because only 1 to 2 percent of active genes are physically linked to a super-enhancer, mapping the locations of super-enhancers can be used to pinpoint the small number of genes that may drive the biology of that cell. Differential super-enhancer maps that compare normal cells to diseased cells can be used to unravel the gene-control circuitry and identify new molecular targets, in much the same way that somatic mutations in tumor cells can point to oncogenic drivers in cancer. This approach is especially attractive in diseases for which an incomplete understanding of the pathogenic mechanisms has been a barrier to discovering effective new therapies.
Another therapeutic approach could be to disrupt the formation or function of super-enhancers by interfering with their associated protein components. This strategy could make it possible to downregulate multiple disease-associated genes through a single molecular intervention. A group of Boston-area researchers recently published support for this concept when they described inhibited expression of cancer-specific genes, leading to a decrease in cancer cell growth, by using a small molecule inhibitor to knock down a super-enhancer component called BRD4 (Cancer Cell, 24:777-90, 2013). More recently, another group showed that expression of the RUNX1 transcription factor, involved in a form of T-cell leukemia, can be diminished by treating cells with an inhibitor of a transcriptional kinase that is present at the RUNX1 super-enhancer (Nature, 511:616-20, 2014).
detect pathogen-associated molecular patterns to activate immunity,
pathogens attempt to deregulate host immunity through secreted effectors.
Fungi employ LysM effectors to prevent
recognition of cell wall-derived chitin by host immune receptors
Structural analysis of the LysM effector Ecp6 of
the fungal tomato pathogen Cladosporium fulvum reveals
a novel mechanism for chitin binding,
mediated by intrachain LysM dimerization,
leading to a chitin-binding groove that is deeply buried in the effector protein.
This composite binding site involves
two of the three LysMs of Ecp6 and
mediates chitin binding with ultra-high (pM) affinity.
The remaining singular LysM domain of Ecp6 binds chitin with
low micromolar affinity but can nevertheless still perturb chitin-triggered immunity.
Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.
Mutated Genes in Schizophrenia Map to Brain Networks From www.nih.gov – Sep 3, 2013
Previous studies have shown that many people with schizophrenia have de novo, or new, genetic mutations. These misspellings in a gene’s DNA sequence
occur spontaneously and so aren’t shared by their close relatives.
Dr. Mary-Claire King of the University of Washington in Seattle and colleagues set out to
identify spontaneous genetic mutations in people with schizophrenia and
to assess where and when in the brain these misspelled genes are turned on, or expressed.
The study was funded in part by NIH’s National Institute of Mental Health (NIMH). The results were published in the August 1, 2013, issue of Cell.
The researchers sequenced the exomes (protein-coding DNA regions) of 399 people—105 with schizophrenia plus their unaffected parents and siblings. Gene variations
that were found in a person with schizophrenia but not in either parent were considered spontaneous.
The likelihood of having a spontaneous mutation was associated with
the age of the father in both affected and unaffected siblings.
Significantly more mutations were found in people
whose fathers were 33-45 years at the time of conception compared to 19-28 years.
Among people with schizophrenia, the scientists identified
54 genes with spontaneous mutations
predicted to cause damage to the function of the protein they encode.
The researchers used newly available database resources that show
where in the brain and when during development genes are expressed.
The genes form an interconnected expression network with many more connections than
that of the genes with spontaneous damaging mutations in unaffected siblings.
The spontaneously mutated genes in people with schizophrenia
were expressed in the prefrontal cortex, a region in the front of the brain.
The genes are known to be involved in important pathways in brain development. Fifty of these genes were active
mainly during the period of fetal development.
“Processes critical for the brain’s development can be revealed by the mutations that disrupt them,” King says. “Mutations can lead to loss of integrity of a whole pathway,
not just of a single gene.”
These findings support the concept that schizophrenia may result, in part, from
disruptions in development in the prefrontal cortex during fetal development.
James E. Darnell’s “Reflections”
A brief history of the discovery of RNA and its role in transcription — peppered with career advice
By Joseph P. Tiano
James Darnell begins his Journal of Biological Chemistry “Reflections” article by saying, “graduate students these days
have to swim in a sea virtually turgid with the daily avalanche of new information and
may be momentarily too overwhelmed to listen to the aging.
I firmly believe how we learned what we know can provide useful guidance for how and what a newcomer will learn.” Considering his remarkable discoveries in
RNA processing and eukaryotic transcriptional regulation
spanning 60 years of research, Darnell’s advice should be cherished. In his second year at medical school at Washington University School of Medicine in St. Louis, while
studying streptococcal disease in Robert J. Glaser’s laboratory, Darnell realized he “loved doing the experiments” and had his first “career advancement event.”
He and technician Barbara Pesch discovered that in vivo penicillin treatment killed streptococci only in the exponential growth phase and not in the stationary phase. These
results were published in the Journal of Clinical Investigation and earned Darnell an interview with Harry Eagle at the National Institutes of Health.
Darnell arrived at the NIH in 1956, shortly after Eagle shifted his research interest to developing his minimal essential cell culture medium, still used. Eagle, then studying cell metabolism, suggested that Darnell take up a side project on poliovirus replication in mammalian cells in collaboration with Robert I. DeMars. DeMars’ Ph.D.
adviser was also James Watson’s mentor, so Darnell met Watson, who invited him to give a talk at Harvard University, which led to an assistant professor position
at the MIT under Salvador Luria. A take-home message is to embrace side projects, because you never know where they may lead: this project helped to shape
his career.
Darnell arrived in Boston in 1961. Following the discovery of DNA’s structure in 1953, the world of molecular biology was turning to RNA in an effort to understand how
proteins are made. Darnell’s background in virology (it was discovered in 1960 that viruses used RNA to replicate) was ideal for the aim of his first independent lab:
exploring mRNA in animal cells grown in culture. While at MIT, he developed a new technique for purifying RNA along with making other observations
suggesting that nonribosomal cytoplasmic RNA may be involved in protein synthesis.
When Darnell moved to Albert Einstein College of Medicine for full professorship in 1964, it was hypothesized that heterogenous nuclear RNA was a precursor to mRNA.
At Einstein, Darnell discovered RNA processing of pre-tRNAs and demonstrated for the first time
that a specific nuclear RNA could represent a possible specific mRNA precursor.
In 1967 Darnell took a position at Columbia University, and it was there that he discovered (simultaneously with two other labs) that
mRNA contained a polyadenosine tail.
The three groups all published their results together in the Proceedings of the National Academy of Sciences in 1971. Shortly afterward, Darnell made his final career move
four short miles down the street to Rockefeller University in 1974.
Over the next 35-plus years at Rockefeller, Darnell never strayed from his original research question: How do mammalian cells make and control the making of different
mRNAs? His work was instrumental in the collaborative discovery of
splicing in the late 1970s and
in identifying and cloning many transcriptional activators.
Perhaps his greatest contribution during this time, with the help of Ernest Knight, was
the discovery and cloning of the signal transducers and activators of transcription (STAT) proteins.
And with George Stark, Andy Wilks and John Krowlewski, he described
cytokine signaling via the JAK-STAT pathway.
Darnell closes his “Reflections” with perhaps his best advice: Do not get too wrapped up in your own work, because “we are all needed and we are all in this together.”
GxGD proteases are a family of intramembranous enzymes capable of hydrolyzing
the transmembrane domain of some integral membrane proteins.
The GxGD family is one of the three families of
intramembrane-cleaving proteases discovered so far (along with the rhomboid and site-2 protease) and
includes the γ-secretase and the signal peptide peptidase.
Although only recently discovered, a number of functions in human pathology and in numerous other biological processes
have been attributed to γ-secretase and SPP.
Taisuke Tomita and Takeshi Iwatsubo of the University of Tokyo highlighted the latest findings on the structure and function of γ-secretase and SPP
in a recent minireview in The Journal of Biological Chemistry.
γ-secretase is involved in cleaving the amyloid-β precursor protein, thus producing amyloid-β peptide,
the main component of senile plaques in Alzheimer’s disease patients’ brains. The complete structure of mammalian γ-secretase is not yet known; however,
Tomita and Iwatsubo note that biochemical analyses have revealed it to be a multisubunit protein complex.
Its catalytic subunit is presenilin, an aspartyl protease.
In vitro and in vivo functional and chemical biology analyses have revealed that
presenilin is a modulator and mandatory component of the γ-secretase–mediated cleavage of APP.
Genetic studies have identified three other components required for γ-secretase activity:
nicastrin,
anterior pharynx defective 1 and
presenilin enhancer 2.
By coexpression of presenilin with the other three components, the authors managed to
reconstitute γ-secretase activity.
Tomita and Iwatsubo determined using the substituted cysteine accessibility method and by topological analyses, that
the catalytic aspartates are located at the center of the nine transmembrane domains of presenilin,
by revealing the exact location of the enzyme’s catalytic site.
The minireview also describes in detail the formerly enigmatic mechanism of γ-secretase mediated cleavage.
SPP, an enzyme that cleaves remnant signal peptides in the membrane
during the biogenesis of membrane proteins and
signal peptides from major histocompatibility complex type I,
also is involved in the maturation of proteins of the hepatitis C virus and GB virus B.
Bioinformatics methods have revealed in fruit flies and mammals four SPP-like proteins,
two of which are involved in immunological processes.
By using γ-secretase inhibitors and modulators, it has been confirmed
that SPP shares a similar GxGD active site and proteolytic activity with γ-secretase.
Upon purification of the human SPP protein with the baculovirus/Sf9 cell system,
single-particle analysis revealed further structural and functional details.
HLA targeting efficiency correlates with human T-cell response magnitude and with mortality from influenza A infection
Experimental and computational evidence suggests that
HLAs preferentially bind conserved regions of viral proteins, a concept we term “targeting efficiency,” and that
this preference may provide improved clearance of infection in several viral systems.
To test this hypothesis, T-cell responses to A/H1N1 (2009) were measured from peripheral blood mononuclear cells obtained from a household cohort study
performed during the 2009–2010 influenza season. We found that HLA targeting efficiency scores significantly correlated with
A further population-based analysis found that the carriage frequencies of the alleles with the lowest targeting efficiencies, A*24,
were associated with pH1N1 mortality (r = 0.37, P = 0.031) and
are common in certain indigenous populations in which increased pH1N1 morbidity has been reported.
HLA efficiency scores and HLA use are associated with CD8 T-cell magnitude in humans after influenza infection.
The computational tools used in this study may be useful predictors of potential morbidity and
identify immunologic differences of new variant influenza strains
more accurately than evolutionary sequence comparisons.
Population-based studies of the relative frequency of these alleles in severe vs. mild influenza cases
might advance clinical practices for severe H1N1 infections among genetically susceptible populations.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ.
Cold Spring Harbor Symposia on Quantitative Biology 11/2011; 76:235-46. http://dx.doi.org:/10.1101/sqb.2011.76.010694
Most diseases result in metabolic changes. In many cases, these changes play a causative role in disease progression. By identifying pathological metabolic changes,
metabolomics can point to potential new sites for therapeutic intervention.
Particularly promising enzymatic targets are those that
carry increased flux in the disease state.
Definitive assessment of flux requires the use of isotope tracers. Here we present techniques for
finding new drug targets using metabolomics and isotope tracers.
The utility of these methods is exemplified in the study of three different viral pathogens. For influenza A and herpes simplex virus,
metabolomic analysis of infected versus mock-infected cells revealed
dramatic concentration changes around the current antiviral target enzymes.
Similar analysis of human-cytomegalovirus-infected cells, however, found the greatest changes
in a region of metabolism unrelated to the current antiviral target.
Instead, it pointed to the tricarboxylic acid (TCA) cycle and
its efflux to feed fatty acid biosynthesis as a potential preferred target.
Isotope tracer studies revealed that cytomegalovirus greatly increases flux through
the key fatty acid metabolic enzyme acetyl-coenzyme A carboxylase.
Inhibition of this enzyme blocks human cytomegalovirus replication.
Examples where metabolomics has contributed to identification of anticancer drug targets are also discussed. Eventual proof of the value of
metabolomics as a drug target discovery strategy will be
successful clinical development of therapeutics hitting these new targets.
Related References
Use of metabolic pathway flux information in targeted cancer drug design. Drug Discovery Today: Therapeutic Strategies 1:435-443, 2004.
Detection of resistance to imatinib by metabolic profiling: clinical and drug development implications. Am J Pharmacogenomics. 2005;5(5):293-302. Review. PMID: 16196499
Medicinal chemistry, metabolic profiling and drug target discovery: a role for metabolic profiling in reverse pharmacology and chemical genetics.
Mini Rev Med Chem. 2005 Jan;5(1):13-20. Review. PMID: 15638788 [PubMed – indexed for MEDLINE] Related citations
Development of Tracer-Based Metabolomics and its Implications for the Pharmaceutical Industry. Int J Pharm Med 2007; 21 (3): 217-224.
Use of metabolic pathway flux information in anticancer drug design. Ernst Schering Found Symp Proc. 2007;(4):189-203. Review. PMID: 18811058
Pharmacological targeting of glucagon and glucagon-like peptide 1 receptors has different effects on energy state and glucose homeostasis in diet-induced obese mice. J Pharmacol Exp Ther. 2011 Jul;338(1):70-81. http://dx.doi.org:/10.1124/jpet.111.179986. PMID: 21471191
Single valproic acid treatment inhibits glycogen and RNA ribose turnover while disrupting glucose-derived cholesterol synthesis in liver as revealed by the
[U-C(6)]-d-glucose tracer in mice. Metabolomics. 2009 Sep;5(3):336-345. PMID: 19718458
Iron regulates glucose homeostasis in liver and muscle via AMP-activated protein kinase in mice. FASEB J. 2013 Jul;27(7):2845-54. http://dx.doi.org:/10.1096/fj.12-216929. PMID: 23515442
Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery
The change in drug discovery strategy from ‘classical’ function-first approaches (in which the assay of drug function was at the tissue or organism level),
with mechanistic studies potentially coming later, to more-recent target-based approaches where initial assays usually involve assessing the interactions
of drugs with specified (and often cloned, recombinant) proteins in vitro. In the latter cases, effects in vivo are assessed later, with concomitantly high levels of attrition.
Arguably the two chief hallmarks of the systems biology approach are:
(i) that we seek to make mathematical models of our systems iteratively or in parallel with well-designed ‘wet’ experiments, and
(ii) that we do not necessarily start with a hypothesis but measure as many things as possible (the ’omes) and
let the data tell us the hypothesis that best fits and describes them.
Although metabolism was once seen as something of a Cinderella subject,
there are fundamental reasons to do with the organisation of biochemical networks as
to why the metabol(om)ic level – now in fact seen as the ‘apogee’ of the ’omics trilogy –
is indeed likely to be far more discriminating than are
changes in the transcriptome or proteome.
The next two subsections deal with these points and Fig. 2 summarises the paper in the form of a Mind Map.
Despite the advent of new drug classes, the global epidemic of cardiometabolic disease has not abated. Continuing
unmet medical needs remain a major driver for new research.
Drug discovery approaches in this field have mirrored industry trends, leading to a recent
increase in the number of molecules entering development.
However, worrisome trends and newer hurdles are also apparent. The history of two newer drug classes—
glucagon-like peptide-1 receptor agonists and
dipeptidyl peptidase-4 inhibitors—
illustrates both progress and challenges. Future success requires that researchers learn from these experiences and
continue to explore and apply new technology platforms and research paradigms.
The global epidemic of obesity and diabetes continues to progress relentlessly. The International Diabetes Federation predicts an even greater diabetes burden (>430 million people afflicted) by 2030, which will disproportionately affect developing nations (International Diabetes Federation, 2011). Yet
existing drug classes for diabetes, obesity, and comorbid cardiovascular (CV) conditions have substantial limitations.
Currently available prescription drugs for treatment of hyperglycemia in patients with type 2 diabetes (Table 1) have notable shortcomings. In general,
Therefore, clinicians must often use combination therapy, adding additional agents over time. Ultimately many patients will need to use insulin—a therapeutic class first introduced in 1922. Most existing agents also have
issues around safety and tolerability as well as dosing convenience (which can impact patient compliance).
Pharmacometabolomics, also known as pharmacometabonomics, is a field which stems from metabolomics,
the quantification and analysis of metabolites produced by the body.
It refers to the direct measurement of metabolites in an individual’s bodily fluids, in order to
This concludes a long step-by-step journey into rediscovering biological processes from the genome as a framework to the remodeled and reconstituted cell through a number of posttranscription and posttranslation processes that modify the proteome and determine the metabolome. The remodeling process continues over a lifetime. The process requires a balance between nutrient intake, energy utilization for work in the lean body mass, energy reserves, endocrine, paracrine and autocrine mechanisms, and autophagy. It is true when we look at this in its full scope – What a creature is man?
Smith CA, O’Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, Custodio DE, Abagyan R, Siuzdak G METLIN: A Metabolite Mass Spectral Database Therapeutic Drug Monitoring, 2005, 27(6), 747-751
Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.[1] The metabolome represents the collection of all metabolites in a biological cell, tissue, organ or organism, which are the end products of cellular processes.[2]mRNAgene expression data and proteomic analyses reveal the set of gene products being produced in the cell, data that represents one aspect of cellular function. Conversely, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomic information to provide a better understanding of cellular biology.
The term “metabolic profile” was introduced by Horning, et al. in 1971 after they demonstrated that gas chromatography-mass spectrometry (GC-MS) could be used to measure compounds present in human urine and tissue extracts. The Horning group, along with that of Linus Pauling and Arthur B. Robinson led the development of GC-MS methods to monitor the metabolites present in urine through the 1970s.
Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples.This first study on muscle highlighted the value of NMR in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic angle spinning, NMR continues to be a leading analytical tool to investigate metabolism. Efforts to utilize NMR for metabolomics have been influenced by the laboratory of Dr. Jeremy Nicholson at Birkbeck College, University of London and later at Imperial College London. In 1984, Nicholson showed 1H NMR spectroscopy could potentially be used to diagnose diabetes mellitus, and later pioneered the application of pattern recognition methods to NMR spectroscopic data.
In 2005, the first metabolomics web database, METLIN, for characterizing human metabolites was developed in the Siuzdak laboratory at The Scripps Research Institute and contained over 10,000 metabolites and tandem mass spectral data. As of September 2012, METLIN contains over 60,000 metabolites as well as the largest repository of tandem mass spectrometry data in metabolomics.
On 23 January 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components. Similar projects have been underway in several plant species, most notably Medicago truncatula and Arabidopsis thaliana for several years.
As late as mid-2010, metabolomics was still considered an “emerging field”. Further, it was noted that further progress in the field depended in large part, through addressing otherwise “irresolvable technical challenges”, by technical evolution of mass spectrometry instrumentation.
Metabolome refers to the complete set of small-molecule metabolites (such as metabolic intermediates, hormones and other signaling molecules, and secondary metabolites) to be found within a biological sample, such as a single organism. The word was coined in analogy with transcriptomics and proteomics; like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method. The first metabolite database(called METLIN) for searching m/z values from mass spectrometry data was developed by scientists at The Scripps Research Institute in 2005. In January 2007, scientists at the University of Alberta and the University of Calgary completed the first draft of the human metabolome. They catalogued approximately 2500 metabolites, 1200 drugs and 3500 food components that can be found in the human body, as reported in the literature. This information, available at the Human Metabolome Database (www.hmdb.ca) and based on analysis of information available in the current scientific literature, is far from complete.
Each type of cell and tissue has a unique metabolic ‘fingerprint’ that can elucidate organ or tissue-specific information, while the study of biofluids can give more generalized though less specialized information. Commonly used biofluids are urine and plasma, as they can be obtained non-invasively or relatively non-invasively, respectively. The ease of collection facilitates high temporal resolution, and because they are always at dynamic equilibrium with the body, they can describe the host as a whole.
Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size.
A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes. By contrast, in human-based metabolomics, it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. Metabolites of foreign substances such as drugs are termed xenometabolites. The metabolome forms a large network of metabolic reactions, where outputs from one enzymaticchemical reaction are inputs to other chemical reactions.
Metabonomics is defined as “the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification”. The word origin is from the Greek μεταβολή meaning change and nomos meaning a rule set or set of laws. This approach was pioneered by Jeremy Nicholson at Imperial College London and has been used in toxicology, disease diagnosis and a number of other fields. Historically, the metabonomics approach was one of the first methods to apply the scope of systems biology to studies of metabolism.
There is a growing consensus that ‘metabolomics’ places a greater emphasis on metabolic profiling at a cellular or organ level and is primarily concerned with normal endogenous metabolism. ‘Metabonomics’ extends metabolic profiling to include information about perturbations of metabolism caused by environmental factors (including diet and toxins), disease processes, and the involvement of extragenomic influences, such as gut microflora. This is not a trivial difference; metabolomic studies should, by definition, exclude metabolic contributions from extragenomic sources, because these are external to the system being studied.
Toxicity assessment/toxicology. Metabolic profiling (especially of urine or blood plasma samples) detects the physiological changes caused by toxic insult of a chemical (or mixture of chemicals).
Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes.
Nutrigenomics is a generalised term which links genomics, transcriptomics, proteomics and metabolomics to human nutrition. In general a metabolome in a given body fluid is influenced by endogenous factors such as age, sex, body composition and genetics as well as underlying pathologies. The large bowel microflora are also a very significant potential confounder of metabolic profiles and could be classified as either an endogenous or exogenous factor. The main exogenous factors are diet and drugs. Diet can then be broken down to nutrients and non- nutrients.
The problem with genomics was it was set as explanation for everything. In fact, when something is genetic in nature the genomic reasoning works fine. However, this means whenever an inborn error is found and only in this case the genomic knowledge afterwards may indicate what is wrong and not the completely way to put biology upside down by reading everything in the DNA genetic as well as non-genetic problems.
Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012
conformational changes leading to substrate efflux.img
The cellular response is defined by a network of chemogenomic response signatures.
This chapter I made to follow signaling, rather than to precede it. I had already written much of the content before reorganizing the contents. The previous chapters on carbohydrate and on lipid metabolism have already provided much material on proteins and protein function, which was persuasive of the need to introduce signaling, which entails a substantial introduction to conformational changes in proteins that direct the trafficking of metabolic pathways, but more subtly uncovers an important role for microRNAs, not divorced from transcription, but involved in a non-transcriptional role. This is where the classic model of molecular biology lacked any integration with emerging metabolic concepts concerning regulation. Consequently, the science was bereft of understanding the ties between the multiple convergence of transcripts, the selective inhibition of transcriptions, and the relative balance of aerobic and anaerobic metabolism, the weight of the pentose phosphate shunt, and the utilization of available energy source for synthetic and catabolic adaptive responses.
The first subchapter serves to introduce the importance of transcription in translational science. The several subtitles that follow are intended to lay out the scope of the transcriptional activity, and also to direct attention toward the huge role of proteomics in the cell construct. As we have already seen, proteins engage with carbohydrates and with lipids in important structural and signaling processes. They are integrasl to the composition of the cytoskeleton, and also to the extracellular matrix. Many proteins are actually enzymes, carrying out the transformation of some substrate, a derivative of the food we ingest. They have a catalytic site, and they function with a cofactor – either a multivalent metal or a nucleotide.
The amino acids that go into protein synthesis include “indispensable” nutrients that are not made for use, but must be derived from animal protein, although the need is partially satisfied by plant sources. The essential amino acids are classified into well established groups. There are 20 amino acids commonly found in proteins. They are classified into the following groups based on the chemical and/or structural properties of their side chains :
Aliphatic Amino Acids
Cyclic Amino Acid
AAs with Hydroxyl or Sulfur-containing side chains
Inosine triphosphate pyrophosphatase – Pyrophosphatase that hydrolyzes the non-canonical purine nucleotides inosine triphosphate (ITP), deoxyinosine triphosphate (dITP) as well as 2′-deoxy-N-6-hydroxylaminopurine triposphate (dHAPTP) and xanthosine 5′-triphosphate (XTP) to their respective monophosphate derivatives. The enzyme does not distinguish between the deoxy- and ribose forms. Probably excludes non-canonical purines from RNA and DNA precursor pools, thus preventing their incorporation into RNA and DNA and avoiding chromosomal lesions.
Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown.
Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR).
ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes.
Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.
Citterio-Quentin A1, Salvi JP, Boulieu R.
Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.
The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.
The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.
System wide analyses have underestimated protein abundances and the importance of transcription in mammals
Jingyi Jessica Li1, 2, Peter J Bickel1 and Mark D Biggin3
Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher.We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression.We show that the variance in translation rates directly measured by ribosome profiling is only 12% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 D 0.13). Based on this, our second strategy suggests that mRNA levels explain 81% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates only apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimat that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the ab-sence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the 40% of genes that are non-expressed.
Related studies that reveal issues that are not part of this chapter:
Ubiquitylation in relationship to tissue remodeling
Post-translational modification of proteins
Glycosylation
Phosphorylation
Methylation
Nitrosylation
Sulfation – sulfotransferases
cell-matrix communication
Acetylation and histone deacetylation (HDAC)
Connecting Protein Phosphatase to 1α (PP1α)
Acetylation complexes (such as CBP/p300 and PCAF)
Sirtuins
Rel/NF-kB Signal Transduction
Homologous Recombination Pathway of Double-Strand DNA Repair
Glycination
cyclin dependent kinases (CDKs)
lyase
transferase
This year, the Lasker award for basic medical research went to Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) for their “discoveries concerning the unfolded protein response (UPR) — an intracellular quality control system that
detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.”
About UPR: Approximately a third of cellular proteins pass through the Endoplasmic Reticulum (ER) which performs stringent quality control of these proteins. All proteins need to assume the proper 3-dimensional shape in order to function properly in the harsh cellular environment. Related to this is the fact that cells are under constant stress and have to make rapid, real time decisions about survival or death.
A major indicator of stress is the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), which triggers a transcriptional cascade in order to increase the folding capacity of the ER. If the metabolic burden is too great and homeostasis cannot be achieved, the response shifts from
damage control to the induction of pro-apoptotic pathways that would ultimately cause cell death.
This response to unfolded proteins or the UPR is conserved among all eukaryotes, and dysfunction in this pathway underlies many human diseases, including Alzheimer’s, Parkinson’s, Diabetes and Cancer.
The discovery of a new class of human proteins with previously unidentified activities
In a landmark study conducted by scientists at the Scripps Research Institute, The Hong Kong University of Science and Technology, aTyr Pharma and their collaborators, a new class of human proteins has been discovered. These proteins [nearly 250], called Physiocrines belong to the aminoacyl tRNA synthetase gene family and carry out novel, diverse and distinct biological functions.
The aminoacyl tRNA synthetase gene family codes for a group of 20 ubiquitous enzymes almost all of which are part of the protein synthesis machinery. Using recombinant protein purification, deep sequencing technique, mass spectroscopy and cell based assays, the team made this discovery. The finding is significant, also because it highlights the alternate use of a gene family whose protein product normally performs catalytic activities for non-catalytic regulation of basic and complex physiological processes spanning metabolism, vascularization, stem cell biology and immunology
Muscle maintenance and regeneration – key player identified
Muscle tissue suffers from atrophy with age and its regenerative capacity also declines over time. Most molecules discovered thus far to boost tissue regeneration are also implicated in cancers. During a quest to find safer alternatives that can regenerate tissue, scientists reported that the hormone Oxytocin is required for proper muscle tissue regeneration and homeostasis and that its levels decline with age.
Oxytocin could be an alternative to hormone replacement therapy as a way to combat aging and other organ related degeneration.
Oxytocin is an age-specific circulating hormone that is necessary for muscle maintenance and regeneration (June 2014)
Role of forkhead box protein A3 in age-associated metabolic decline.
Ma X1, Xu L1, Gavrilova O2, Mueller E3.
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 significantly and 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.
Asymmetric mRNA localization contributes to fidelity and sensitivity of spatially localized systems
Although many proteins are localized after translation, asymmetric protein distribution is also achieved by translation after mRNA localization. Why are certain mRNA transported to a distal location and translated on-site? Here we undertake a systematic, genome-scale study of asymmetrically distributed protein and mRNA in mammalian cells. Our findings suggest that asymmetric protein distribution by mRNA localization enhances interaction fidelity and signaling sensitivity. Proteins synthesized at distal locations frequently contain intrinsically disordered segments. These regions are generally rich in assembly-promoting modules and are often regulated by post-translational modifications. Such proteins are tightly regulated but display distinct temporal dynamics upon stimulation with growth factors. Thus, proteins synthesized on-site may rapidly alter proteome composition and act as dynamically regulated scaffolds to promote the formation of reversible cellular assemblies. Our observations are consistent across multiple mammalian species, cell types and developmental stages, suggesting that localized translation is a recurring feature of cell signaling and regulation.
An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis.
An overview of the potential advantages conferred by distal-site protein synthesis
Turquoise and red filled circle represents off-target and correct interaction partners, respectively. Wavy lines represent a disordered region within a distal site synthesis protein. Grey and red line in graphs represents profiles of t…
Tweaking transcriptional programming for high quality recombinant protein production
Since overexpression of recombinant proteins in E. coli often leads to the formation of inclusion bodies, producing properly folded, soluble proteins is undoubtedly the most important end goal in a protein expression campaign. Various approaches have been devised to bypass the insolubility issues during E. coli expression and in a recent report a group of researchers discuss reprogramming the E. coli proteostasis [protein homeostasis] network to achieve high yields of soluble, functional protein. The premise of their studies is that the basal E. coli proteostasis network is insufficient, and often unable, to fold overexpressed proteins, thus clogging the folding machinery.
By overexpressing a mutant, negative-feedback deficient heat shock transcription factor [σ32 I54N] before and during overexpression of the protein of interest, reprogramming can be achieved, resulting in high yields of soluble and functional recombinant target protein. The authors explain that this method is better than simply co-expressing/over-expressing chaperones, co-chaperones, foldases or other components of the proteostasis network because reprogramming readies the folding machinery and up regulates the essential folding components beforehand thus maintaining system capability of the folding machinery.
The Heat-Shock Response Transcriptional Program Enables High-Yield and High-Quality Recombinant Protein Production in Escherichia coli (July 2014)
Unfolded proteins collapse when exposed to heat and crowded environments
Proteins are important molecules in our body and they fulfil a broad range of functions. For instance as enzymes they help to release energy from food and as muscle proteins they assist with motion. As antibodies they are involved in immune defence and as hormone receptors in signal transduction in cells. Until only recently it was assumed that all proteins take on a clearly defined three-dimensional structure – i.e. they fold in order to be able to assume these functions. Surprisingly, it has been shown that many important proteins occur as unfolded coils. Researchers seek to establish how these disordered proteins are capable at all of assuming highly complex functions.
Ben Schuler’s research group from the Institute of Biochemistry of the University of Zurich has now established that an increase in temperature leads to folded proteins collapsing and becoming smaller. Other environmental factors can trigger the same effect.
Measurements using the “molecular ruler”
“The fact that unfolded proteins shrink at higher temperatures is an indication that cell water does indeed play an important role as to the spatial organisation eventually adopted by the molecules”, comments Schuler with regard to the impact of temperature on protein structure. For their studies the biophysicists use what is known as single-molecule spectroscopy. Small colour probes in the protein enable the observation of changes with an accuracy of more than one millionth of a millimetre. With this “molecular yardstick” it is possible to measure how molecular forces impact protein structure.
With computer simulations the researchers have mimicked the behaviour of disordered proteins.
(Courtesy of Jose EDS Roselino, PhD.
MLKL compromises plasma membrane integrity
Necroptosis is implicated in many diseases and understanding this process is essential in the search for new therapies. While mixed lineage kinase domain-like (MLKL) protein has been known to be a critical component of necroptosis induction, how MLKL transduces the death signal was not clear. In a recent finding, scientists demonstrated that the full four-helical bundle domain (4HBD) in the N-terminal region of MLKL is required and sufficient to induce its oligomerization and trigger cell death.
They also found a patch of positively charged amino acids on the surface of the 4HBD that bound to phosphatidylinositol phosphates (PIPs) and allowed the recruitment of MLKL to the plasma membrane that resulted in the formation of pores consisting of MLKL proteins, due to which cells absorbed excess water causing them to explode. Detailed knowledge about how MLKL proteins create pores offers possibilities for the development of new therapeutic interventions for tolerating or preventing cell death.
MLKL compromises plasma membrane integrity by binding to phosphatidylinositol phosphates (May 2014)
Mitochondrial and ER proteins implicated in dementia
Mitochondria and the endoplasmic reticulum (ER) form tight structural associations that facilitate a number of cellular functions. However, the molecular mechanisms of these interactions aren’t properly understood.
A group of researchers showed that the ER protein VAPB interacted with mitochondrial protein PTPIP51 to regulate ER-mitochondria associations and that TDP-43, a protein implicated in dementia, disturbs this interaction to regulate cellular Ca2+ homeostasis. These studies point to a new pathogenic mechanism for TDP-43 and may also provide a potential new target for the development of new treatments for devastating neurological conditions like dementia.
ER-mitochondria associations are regulated by the VAPB-PTPIP51 interaction and are disrupted by ALS/FTD-associated TDP-43. Nature (June 2014)
A novel strategy to improve membrane protein expression in Yeast
Membrane proteins play indispensable roles in the physiology of an organism. However, recombinant production of membrane proteins is one of the biggest hurdles facing protein biochemists today. A group of scientists in Belgium showed that,
by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis,
enhanced expression of recombinant membrane proteins in yeast is achieved.
Specifically, they engineered the oleotrophic yeast, Yarrowia lipolytica, by
deleting the phosphatidic acid phosphatase, PAH1 gene,
which led to massive proliferation of endoplasmic reticulum (ER) membranes.
For all 8 tested representatives of different integral membrane protein families, they obtained enhanced protein accumulation.
An unconventional method to boost recombinant protein levels
MazF is an mRNA interferase enzyme in E.coli that functions as and degrades cellular mRNA in a targeted fashion, at the “ACA” sequence. This degradation of cellular mRNA causes a precipitous drop in cellular protein synthesis. A group of scientists at the Robert Wood Johnson Medical School in New Jersey, exploited the degeneracy of the genetic code to modify all “ACA” triplets within their gene of interest in a way that the corresponding amino acid (Threonine) remained unchanged. Consequently, induction of MazF toxin caused degradation of E.coli cellular mRNA but the recombinant gene transcription and protein synthesis continued, causing significant accumulation of high quality target protein. This expression system enables unparalleled signal to noise ratios that could dramatically simplify structural and functional studies of difficult-to-purify, biologically important proteins.
Tandem fusions and bacterial strain evolution for enhanced functional membrane protein production
Membrane protein production remains a significant challenge in its characterization and structure determination. Despite the fact that there are a variety of host cell types, E.coli remains the popular choice for producing recombinant membrane proteins. A group of scientists in Netherlands devised a robust strategy to increase the probability of functional membrane protein overexpression in E.coli.
By fusing Green Fluorescent Protein (GFP) and the Erythromycin Resistance protein (ErmC) to the C-terminus of a target membrane protein they wer e able to track the folding state of their target protein while using Erythromycin to select for increased expression. By increasing erythromycin concentration in the growth media and testing different membrane targets, they were able to identify four evolved E.coli strains, all of which carried a mutation in the hns gene, whose product is implicated in genome organization and transcriptional silencing. Through their experiments the group showed that partial removal of the transcriptional silencing mechanism was related to production of proteins that were essential for functional overexpression of membrane proteins.
The role of an anti-apoptotic factor in recombinant protein production
In a recent study, scientists at the Johns Hopkins University and Frederick National Laboratory for Cancer Research examined an alternative method of utilizing the benefits of anti-apoptotic gene expression to enhance the transient expression of biotherapeutics, specifically, through the co-transfection of Bcl-xL along with the product-coding target gene.
Chinese Hamster Ovary(CHO) cells were co-transfected with the product-coding gene and a vector containing Bcl-xL, using Polyethylenimine (PEI) reagent. They found that the cells co-transfected with Bcl-xL demonstrated reduced apoptosis, increased specific productivity, and an overall increase in product yield.
B-cell lymphoma-extra-large (Bcl-xL) is a mitochondrial transmembrane protein and a member of the Bcl-2 family of proteins which are known to act as either pro- or anti-apoptotic proteins. Bcl-xL itself acts as an anti-apoptotic molecule by preventing the release of mitochondrial contents such as cytochrome c, which would lead to caspase activation. Higher levels of Bcl-xL push a cell toward survival mode by making the membranes pores less permeable and leaky.
Introduction to Protein Synthesis and DegradationUpdated 8/31/2019
N-Terminal Degradation of Proteins: The N-End Rule and N-degrons
In both prokaryotes and eukaryotes mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the addition of the N-formyl methionine residue. However in eukaryotic cytosolic ribosomes, the N terminal was assumed to be devoid of the N-formyl group. The unformylated N-terminal methionine residues of eukaryotes is then often N-acetylated (Ac) and creates specific degradation signals, the Ac N-end rule. These N-end rule pathways are proteolytic systems which recognize these N-degrons resulting in proteosomal degradation or autophagy. In prokaryotes this system is stimulated by certain amino acid deficiencies and in eukaryotes is dependent on the Psh1 E3 ligase.
Two papers in the journal Science describe this N-degron in more detail.
In both bacteria and eukaryotic mitochondria and chloroplasts, the ribosomal synthesis of proteins is initiated with the N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet is produced pretranslationally by formyltransferases, which use 10-formyltetrahydrofolate as a cosubstrate. By contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were always presumed to bear unformylated N-terminal Met (Nt-Met). The unformylated Nt-Met residue of eukaryotic proteins is often cotranslationally Nt-acetylated, a modification that creates specific degradation signals, Ac/N-degrons, which are targeted by the Ac/N-end rule pathway. The N-end rule pathways are a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria. The main determinant of an N‑degron is a destabilizing Nt-residue of a protein. Studies over the past three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing Nt‑residues. The previously known eukaryotic N-end rule pathways are the Arg/N-end rule pathway, the Ac/N-end rule pathway, and the Pro/N-end rule pathway. Regulated degradation of proteins and their natural fragments by the N-end rule pathways has been shown to mediate a broad range of biological processes.
RATIONALE
The chemical similarity of the formyl and acetyl groups and their identical locations in, respectively, Nt‑formylated and Nt-acetylated proteins led us to suggest, and later to show, that the Nt-fMet residues of nascent bacterial proteins can act as bacterial N-degrons, termed fMet/N-degrons. Here we wished to determine whether Nt-formylated proteins might also form in the cytosol of a eukaryote such as the yeast Saccharomyces cerevisiae and to determine the metabolic fates of Nt-formylated proteins if they could be produced outside mitochondria. Our approaches included molecular genetic techniques, mass spectrometric analyses of proteins’ N termini, and affinity-purified antibodies that selectively recognized Nt-formylated reporter proteins.
RESULTS
We discovered that the yeast formyltransferase Fmt1, which is imported from the cytosol into the mitochondria inner matrix, can generate Nt-formylated proteins in the cytosol, because the translocation of Fmt1 into mitochondria is not as efficacious, even under unstressful conditions, as had previously been assumed. We also found that Nt‑formylated proteins are greatly up-regulated in stationary phase or upon starvation for specific amino acids. The massive increase of Nt-formylated proteins strictly requires the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. Notably, the ability of Gcn2 to retain a large fraction of Fmt1 in the cytosol of nutritionally stressed cells is confined to Fmt1, inasmuch as the Gcn2 kinase does not have such an effect, under the same conditions, on other examined nuclear DNA–encoded mitochondrial matrix proteins. The Gcn2-Fmt1 protein localization circuit is a previously unknown signal transduction pathway. A down-regulation of cytosolic Nt‑formylation was found to increase the sensitivity of cells to undernutrition stresses, to a prolonged cold stress, and to a toxic compound. We also discovered that the Nt-fMet residues of Nt‑formylated cytosolic proteins act as eukaryotic fMet/N-degrons and identified the Psh1 E3 ubiquitin ligase as the recognition component (fMet/N-recognin) of the previously unknown eukaryotic fMet/N-end rule pathway, which destroys Nt‑formylated proteins.
CONCLUSION
The Nt-formylation of proteins, a long-known pretranslational protein modification, is mediated by formyltransferases. Nt-formylation was thought to be confined to bacteria and bacteria-descended eukaryotic organelles but was found here to also occur at the start of translation by the cytosolic ribosomes of a eukaryote. The levels of Nt‑formylated eukaryotic proteins are greatly increased upon specific stresses, including undernutrition, and appear to be important for adaptation to these stresses. We also discovered that Nt-formylated cytosolic proteins are selectively destroyed by the eukaryotic fMet/N-end rule pathway, mediated by the Psh1 E3 ubiquitin ligase. This previously unknown proteolytic system is likely to be universal among eukaryotes, given strongly conserved mechanisms that mediate Nt‑formylation and degron recognition.
(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.
(Top) Under undernutrition conditions, the Gcn2 kinase augments the cytosolic localization of the Fmt1 formyltransferase, and possibly also its enzymatic activity. Consequently, Fmt1 up-regulates the cytosolic fMet–tRNAi (initiator transfer RNA), and thereby increases the levels of cytosolic Nt-formylated proteins, which are required for the adaptation of cells to specific stressors. (Bottom) The Psh1 E3 ubiquitin ligase targets the N-terminal fMet-residues of eukaryotic cytosolic proteins, such as Cse4, Pgd1, and Rps22a, for the polyubiquitylation-mediated, proteasome-dependent degradation.
The second paper describes a glycine specific N-degron pathway in humans. Specifically the authors set up a screen to identify specific N-terminal degron motifs in the human. Findings included an expanded repertoire for the UBR E3 ligases to include substrates with arginine and lysine following an intact initiator methionine and a glycine at the extreme N-terminus, which is a potent degron.
Glycine N-degron regulation revealed
For more than 30 years, N-terminal sequences have been known to influence protein stability, but additional features of these N-end rule, or N-degron, pathways continue to be uncovered. Timms et al. used a global protein stability (GPS) technology to take a broader look at these pathways in human cells. Unexpectedly, glycine exposed at the N terminus could act as a potent degron; proteins bearing N-terminal glycine were targeted for proteasomal degradation by two Cullin-RING E3 ubiquitin ligases through the substrate adaptors ZYG11B and ZER1. This pathway may be important, for example, to degrade proteins that fail to localize properly to cellular membranes and to destroy protein fragments generated during cell death.
The ubiquitin-proteasome system is the major route through which the cell achieves selective protein degradation. The E3 ubiquitin ligases are the major determinants of specificity in this system, which is thought to be achieved through their selective recognition of specific degron motifs in substrate proteins. However, our ability to identify these degrons and match them to their cognate E3 ligase remains a major challenge.
RATIONALE
It has long been known that the stability of proteins is influenced by their N-terminal residue, and a large body of work over the past three decades has characterized a collection of N-end rule pathways that target proteins for degradation through N-terminal degron motifs. Recently, we developed Global Protein Stability (GPS)–peptidome technology and used it to delineate a suite of degrons that lie at the extreme C terminus of proteins. We adapted this approach to examine the stability of the human N terminome, allowing us to reevaluate our understanding of N-degron pathways in an unbiased manner.
RESULTS
Stability profiling of the human N terminome identified two major findings: an expanded repertoire for UBR family E3 ligases to include substrates that begin with arginine and lysine following an intact initiator methionine and, more notably, that glycine positioned at the extreme N terminus can act as a potent degron. We established human embryonic kidney 293T reporter cell lines in which unstable peptides that bear N-terminal glycine degrons were fused to green fluorescent protein, and we performed CRISPR screens to identify the degradative machinery involved. These screens identified two Cul2 Cullin-RING E3 ligase complexes, defined by the related substrate adaptors ZYG11B and ZER1, that act redundantly to target substrates bearing N-terminal glycine degrons for proteasomal degradation. Moreover, through the saturation mutagenesis of example substrates, we defined the composition of preferred N-terminal glycine degrons specifically recognized by ZYG11B and ZER1.
We found that preferred glycine degrons are depleted from the native N termini of metazoan proteomes, suggesting that proteins have evolved to avoid degradation through this pathway, but are strongly enriched at annotated caspase cleavage sites. Stability profiling of N-terminal peptides lying downstream of all known caspase cleavages sites confirmed that Cul2ZYG11Band Cul2ZER1 could make a substantial contribution to the removal of proteolytic cleavage products during apoptosis. Last, we identified a role for ZYG11B and ZER1 in the quality control of N-myristoylated proteins. N-myristoylation is an important posttranslational modification that occurs exclusively on N-terminal glycine. By profiling the stability of the human N-terminome in the absence of the N-myristoyltransferases NMT1 and NMT2, we found that a failure to undergo N-myristoylation exposes N-terminal glycine degrons that are otherwise obscured. Thus, conditional exposure of glycine degrons to ZYG11B and ZER1 permits the selective proteasomal degradation of aberrant proteins that have escaped N-terminal myristoylation.
CONCLUSION
These data demonstrate that an additional N-degron pathway centered on N-terminal glycine regulates the stability of metazoan proteomes. Cul2ZYG11B– and Cul2ZER1-mediated protein degradation through N-terminal glycine degrons may be particularly important in the clearance of proteolytic fragments generated by caspase cleavage during apoptosis and in the quality control of protein N-myristoylation.
Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.
Stability profiling of the human N-terminome revealed that N-terminal glycine acts as a potent degron. CRISPR screening revealed two Cul2 complexes, defined by the related substrate adaptors ZYG11B and ZER1, that recognize N-terminal glycine degrons. This pathway may be particularly important for the degradation of caspase cleavage products during apoptosis and the removal of proteins that fail to undergo N-myristoylation.
In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it. Furthermore, he observes – Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.
acetylCoA
In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view. The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics. The inherent difficulty lies in two places:
Interactions between differently weighted determinants
A large part of the genome is concerned with regulatory function, not expression of the code
The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell. Completion of the tutorial would provide
a basic understanding signal transduction and
the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
In addition – detailed knowledge of –
the role of Tyrosine kinases and
G protein-coupled receptors in cell signaling.
serine
threonine
protein kinase
We are constantly receiving and interpreting signals from our environment, which can come
in the form of light, heat, odors, touch or sound.
The cells of our bodies are also
constantly receiving signals from other cells.
These signals are important to
keep cells alive and functioning as well as
to stimulate important events such as
cell division and differentiation.
Signals are most often chemicals that can be found
in the extracellular fluid around cells.
These chemicals can come
from distant locations in the body (endocrine signaling by hormones), from
nearby cells (paracrine signaling) or can even
be secreted by the same cell (autocrine signaling).
Signaling molecules may trigger any number of cellular responses, including
changing the metabolism of the cell receiving the signal or
result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.
To which I would now add..
result in either an inhibitory or a stimulatory effect
The three stages of cell signaling are:
Cell signaling can be divided into 3 stages:
Reception: A cell detects a signaling molecule from the outside of the cell.
Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.
Response: Finally, the signal triggers a specific cellular response.
Signal Transduction – ligand binds to surface receptor
Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.
by changing shape or
by joining with another protein
once a specific ligand binds to it.
Examples of membrane receptors include
G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.
Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).
Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation. That does not have to be the case in the normal homeostatic case.
This point is the differentiating case between what JEDS Roselino has referred as
a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
a one-to-one transcription of the genetic code.
The rate of transcription can be controlled, or it can be blocked. This is in large part in response to the metabolites in the immediate interstitium.
This might only be
a change in the rate of a transcription or a suppression of expression through RNA.
Or through a conformational change in an enzyme
Swinging domains in HECT E3 enzymes
Since signaling systems need to be
responsive to small concentrations of chemical signals and act quickly,
cells often use a multi-step pathway that transmits the signal quickly,
while amplifying the signal to numerous molecules at each step.
Signal transduction pathways are shown (simplified):
Signal Transduction
Signal transduction occurs when an
extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.
In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.
Guanosine monophosphate structure
In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.
Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.
This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.
Integrin
Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.
steroid hormone receptor
Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.
Signal transduction cascades amplify the signal output
Signal transduction cascades amplify the signal output
G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
signal transduction pathways
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling
Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.
The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.
Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.
insulin receptor and and insulin receptor signaling pathway (IRS)
To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.
RTKs
The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.
Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.
Signal-Transduction-Pathway
As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are
members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.
They act as molecular switches usually
tethered to membranes by isoprenyl groups linked to their carboxyl ends.
Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate
small G proteins that activate guanine nucleotide exchange factors such as SOS1.
Once activated, these exchange factors can activate more small G proteins, thus
amplifying the receptor’s initial signal.
The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.
Integrin
Integrin
Integrin-mediated signal transduction
An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).
Integrins are produced by a wide variety of cells; they play a role in
cell attachment to other cells and the extracellular matrix and
in the transduction of signals from extracellular matrix components such as fibronectin and collagen.
Ligand binding to the extracellular domain of integrins
changes the protein’s conformation,
clustering it at the cell membrane to
initiate signal transduction.
Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.
As shown in the picture, cooperative integrin-RTK signaling determines the
timing of cellular survival,
apoptosis,
proliferation, and
differentiation.
integrin-mediated signal transduction
Integrin signaling
ion channel
A ligand-gated ion channel, upon binding with a ligand, changes conformation
to open a channel in the cell membrane
through which ions relaying signals can pass.
An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels
induces action potentials, such as those that travel along nerves,
by depolarizing the membrane of post-synaptic cells,
resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR
An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;
it acts as a second messenger
initiating signal transduction cascades and
altering the physiology of the responding cell.
This results in amplification of the synapse response between synaptic cells
by remodelling the dendritic spines involved in the synapse.
In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,
cAMP
Inositol_1,4,5-trisphosphate.svg
the latter controlling the release of intracellular calcium stores into the cytoplasm.
Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,
calcium ions bind to the EF hand domains of calmodulin,
allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor
PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.
Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.
Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.
Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,
DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.
DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.
On the other hand,
zinc is the most abundant trace metal in the human brain.
There are many scientific studies linking zinc, especially
excess amounts of free zinc, to cellular death.
Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between
omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.
Many other studies have also suggested a possible
omega-3 and zinc effect on neurodegeneration and cellular death.
Therefore, in this review, we will examine
the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.
Moreover, we will evaluate the collective understanding of
mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.
Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to
reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in
Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as
cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.
Despite the significance of these
two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both
omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].
the reverse process can be seen with increased omega-3 fatty acids in the body.
Many other factors, such as
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,
which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.
Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in
immune responses,
gene expression and
intercellular communications.
omega-3 fatty acids are known to be vital in
the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels
.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.
The main
In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that
only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline
In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown
a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.
A trial of subjects with mild memory complaints demonstrated
an improvement with 900 mg of DHA.
We review key findings on
the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.
DHA is the most abundant fatty acid in neural membranes, imparting appropriate
fluidity and other properties,
and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated
in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.
In adult rats’ brain, DHA comprises approximately
17% of the total fatty acid weight, and
in the retina it is as high as 33%.
DHA is believed to have played a major role in the evolution of the modern human –
in particular the well-developed brain.
Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.
Analysis of human cadaver brains have shown that
people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals
Furthermore, studies in mice have increased support for the
protective role of omega-3 fatty acids.
Mice administrated with a dietary intake of DHA showed
an increase in DHA levels in the hippocampus.
Errors in memory were decreased in these mice and they demonstrated
reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.
Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary
DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.
Zinc
Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to
growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process
and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as
a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.
Approximately 3% of all proteins contain
zinc binding motifs .
The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;
catalytic,
coactive and
Indeed, it is the only metal found in all six different subclasses
of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.
Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated
through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.
Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and
its release is dependent on neural activity.
Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.
The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to
form insoluble amyloid in the presence of
high physiological levels of zinc.
Insoluble amyloid is thought to
aggregate to form plaques,
which is a main pathological feature of AD. Further studies have shown that
chelation of zinc ions can deform and disaggregate plaques.
In AD, the most prominent injuries are found in
hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.
All of these neurons are known to favor
rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.
This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of
inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).
High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.
If the human diet is deficient in zinc, the body
efficiently conserves zinc at the tissue level by compensating other cellular mechanisms
to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and
redistribution of available zinc to more zinc dependent cells or organs.
A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the
assessment of the zinc status of individuals and populations.
In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting
as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters
Zinc Transporters
Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur
via intermembrane proteins and zinc transporter (ZnT) proteins
These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).
Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).
The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where
it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,
Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg
Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].
omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).
In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between
increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels
DHA and Zinc Homeostasis
Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in
increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.
Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,
a significant reduction in plaque load
and the presence of insoluble amyloid were observed.
In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant
reduction in free zinc availability in the hippocampus
and cerebral cortex.
Collectively, the findings from this study are very interesting and indicate a clear connection between
zinc availability and amyloid plaque formation,
thus indicating a possible link to AD.
DHA supplementation has also been reported to limit the following:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.
In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.
In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.
When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible
neuroprotective mechanism of DHA,
which we believe is exerted through
a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.
DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and
free zinc levels in the cytosol were significantly low compared to the control
This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had
low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.
These findings are consistent with previous published data and further strengthen the possible
correlation between zinc, DHA and neurodegeneration.
On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of
ZnT3 in memory and AD pathology.
For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of
ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory
Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.
Complex Models of Signaling: Therapeutic Implications
Curator: Larry H. Bernstein, MD, FCAP
Updated 6/24/2019
Fishy Business: Effect of Omega-3 Fatty Acids on Zinc Transporters and Free Zinc Availability in Human Neuronal Cells
Damitha De Mel and Cenk Suphioglu *
NeuroAllergy Research Laboratory (NARL), School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Waurn Ponds, Victoria, Australia.
Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).
Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,
DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.
DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.
On the other hand,
zinc is the most abundant trace metal in the human brain.
There are many scientific studies linking zinc, especially
excess amounts of free zinc, to cellular death.
Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between
omega-3 fatty acids, zinc transporter levels and
free zinc availability at cellular levels.
Many other studies have also suggested a possible
omega-3 and zinc effect on neurodegeneration and cellular death.
Therefore, in this review, we will examine
the effect of omega-3 fatty acids on zinc transporters and
the importance of free zinc for human neuronal cells.
Moreover, we will evaluate the collective understanding of
mechanism(s) for the interaction of these elements in neuronal research and their
significance for the diagnosis and treatment of neurodegeneration.
Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to
reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in
Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as
cellular differentiation,
photoreceptor membrane biogenesis and
active synaptogenesis.
Despite the significance of these
two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both
omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
opposing physiological effects. In the human body,
high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
thereby increase inflammatory processes [10].
the reverse process can be seen with increased omega-3 fatty acids in the body.
Many other factors, such as
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
C-reactive protein,
which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.
Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in
immune responses,
gene expression and
intercellular communications.
omega-3 fatty acids are known to be vital in
the prevention of fatal ventricular arrhythmias, and
are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels
.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.
The main omega-3 fatty acids in the mammalian body are
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
eicosapentaenoic acid (EPA).
In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between
increased fish consumption and reduced risk of AD
and eight out of ten studies have reported a link between higher blood omega-3 levels
Table 1. Total percentage of omega-3 fatty acids in common foods and supplements.
Food/Supplement
EPA
DHA
ALA
Total %
Fish
SalmonSardine
Anchovy
Halibut
Herring
Mackerel
Tuna
Fresh Bluefin
XX
X
X
X
X
X
X
XX
X
X
X
X
X
X
>50%>50%
>50%
>50%
>50%
>50%
>50%
>50%
Oils/Supplements
Fish oil capsulesCod liver oils
Salmon oil
Sardine oil
XX
X
X
XX
X
X
>50%>50%
>50%
>50%
Black currant oilCanola oil Mustard seed oils
Soybean oil
Walnut oil
Wheat germ oil
XX
X
X
X
X
10%–50%10%–50%
10%–50%
10%–50%
10%–50%
10%–50%
Seeds and other foods
Flaxseeds/LinseedsSpinach
Wheat germ Human milk
Peanut butter
Soybeans
Olive oil
Walnuts
XX
X
X
X
X
X
X
>50%>50%
10%–50%
10%–50%
<10%
<10%
<10%
<10%
Table adopted from Maclean C.H. et al. [18].
In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that
only high fish consumption, but
not dietary omega-3 acid intake,
had a protective effect on cognitive decline
In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown
a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.
A trial of subjects with mild memory complaints demonstrated
an improvement with 900 mg of DHA.
We review key findings on
the effect of the omega-3 fatty acid DHA on zinc transporters and the
importance of free zinc to human neuronal cells.
DHA is the most abundant fatty acid in neural membranes, imparting appropriate
fluidity and other properties,
and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated
in membrane phospholipids at synapses and
in retinal photoreceptors and
also in the testis and sperm.
In adult rats’ brain, DHA comprises approximately
17% of the total fatty acid weight, and
in the retina it is as high as 33%.
DHA is believed to have played a major role in the evolution of the modern human –
in particular the well-developed brain.
Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.
Analysis of human cadaver brains have shown that
people with AD have less DHA in their frontal lobe
and hippocampus compared with unaffected individuals
Furthermore, studies in mice have increased support for the
protective role of omega-3 fatty acids.
Mice administrated with a dietary intake of DHA showed
an increase in DHA levels in the hippocampus.
Errors in memory were decreased in these mice and they demonstrated
reduced peroxide and free radical levels,
suggesting a role in antioxidant defense.
Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary
DHA supplementation had a protective effect against reduction in
drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
decreased caspase activity.
Zinc
Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to
growth,
development,
differentiation,
immune response,
receptor activity,
DNA synthesis,
gene expression,
neuro-transmission,
enzymatic catalysis,
hormonal storage and release,
tissue repair,
memory,
the visual process
and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects, as
a component of over 300 different enzymes
an integral component of a metallothioneins
a gene regulatory protein.
Approximately 3% of all proteins contain
zinc binding motifs .
The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;
catalytic,
coactive and
Indeed, it is the only metal found in all six different subclasses
of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.
Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated
through glutamate receptors, and
it inhibits excitatory and inhibitory receptors.
Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and
its release is dependent on neural activity.
Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.
The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to
form insoluble amyloid in the presence of
high physiological levels of zinc.
Insoluble amyloid is thought to
aggregate to form plaques,
which is a main pathological feature of AD. Further studies have shown that
chelation of zinc ions can deform and disaggregate plaques.
In AD, the most prominent injuries are found in
hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
somatostatin-containing neurons in the forebrain.
All of these neurons are known to favor
rapid and direct entry of zinc in high concentration
leaving neurons frequently exposed to high dosages of zinc.
This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of
inhibiting Ca2+ and Na+ voltage gated channels
and up-regulating the cellular levels of reactive oxygen species (ROS).
High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.
If the human diet is deficient in zinc, the body
efficiently conserves zinc at the tissue level by compensating other cellular mechanisms
to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and
redistribution of available zinc to more zinc dependent cells or organs.
A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the
assessment of the zinc status of individuals and populations.
In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting
as a target for zinc ion binding and thus
assisting in the trafficking of zinc ions through the cell,
which may be similar to that of zinc transporters
Zinc Transporters
Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur
via intermembrane proteins and zinc transporter (ZnT) proteins
These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).
Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).
The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where
it is responsible for the transport of zinc into the synaptic vesicles of
glutamatergic neurons in the hippocampus and neocortex,
Figure 1. Putative cellular localization of some of the different human zinc transporters (i.e., Zip1- Zip4 and ZnT1- ZnT7). Arrows indicate the direction of zinc passage by the appropriate putative zinc transporters in a generalized human cell. Although there are fourteen Zips and eight ZnTs known so far, only the main zinc transporters are illustrated in this figure for clarity and brevity.
Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/. http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg
zinc transporters
Early zinc signaling (EZS) and late zinc signaling (LZS)
Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].
DHA and Zinc Homeostasis
Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in
increased zinc levels in the hippocampus and
elevated expression of the putative zinc transporter, ZnT3, in the rat brain.
Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,
a significant reduction in plaque load
and the presence of insoluble amyloid were observed.
In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant
reduction in free zinc availability in the hippocampus
and cerebral cortex.
Collectively, the findings from this study are very interesting and indicate a clear connection between
zinc availability and amyloid plaque formation,
thus indicating a possible link to AD.
DHA supplementation has also been reported to limit the following:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
cognitive deficits in transgenic mouse model of AD.
In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.
In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.
When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible
neuroprotective mechanism of DHA,
which we believe is exerted through
a reduction in cellular zinc levels (through altering zinc transporter expression levels)
that in turn inhibits apoptosis.
DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and
free zinc levels in the cytosol were significantly low compared to the control
This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had
low levels of active caspase-3 and
high Bcl-2 levels compared to the control treatment.
These findings are consistent with previous published data and further strengthen the possible
correlation between zinc, DHA and neurodegeneration.
On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of
ZnT3 in memory and AD pathology.
For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of
ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
required for hippocampus-dependent memory
Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.
Collectively from these studies, the following possible mechanism can be proposed (Figure 2).
possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter
Figure 2. Proposed neuroprotection mechanism of docosahexaenoic acid (DHA) in reference to synaptic zinc. Schematic diagram showing possible benefits of DHA in neuroprotection through reduction of ZnT3 transporter expression levels in human neuronal cells, which results in a reduction of zinc flux and thus lowering zinc concentrations in neuronal synaptic vesicles, and therefore contributing to a lower incidence of neurodegenerative diseases (ND), such as Alzheimer’s disease (AD).
More recent data from our research group have also shown a link between the expression levels of histone H3 and H4 proteins in human neuronal cells in relation to DHA and zinc. Following DHA treatment, both H3 and H4 levels were up-regulated. In contrast, zinc treatment resulted in a down-regulation of histone levels. Both zinc and DHA have shown opposing effects on histone post-translational modifications, indicating a possible distinctive epigenetic pattern. Upon treatment with zinc, M17 cells displayed an increase in histone deacetylase (HDACs) and a reduction in histone acetylation. Conversely, with DHA treatment, HDAC levels were significantly reduced and the acetylation of histones was up-regulated. These findings also support a possible interaction between DHA and zinc availability.
Conclusions
It is possible to safely claim that there is more than one potential pathway by which DHA and zinc interact at a cellular level, at least in cultured human neuronal cells. Significance and importance of both DHA and zinc in neuronal survival is attested by the presence of these multiple mechanisms.
Most of these reported studies were conducted using human neuroblastoma cells, or similar cell types, due to the lack of live mature human neuronal cells. Thus, the results may differ from results achieved under actual human physiological conditions due to the structural and functional differences between these cells and mature human neurons. Therefore, an alternative approach that can mimic the human neuronal cells more effectively would be advantageous.
Sphingosine-1-phosphate signaling as a therapeutic target
E Giannoudaki, DJ Swan, JA Kirby, S Ali
Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
Cell Health and Cytoskeleton 2012; 4: 63–72
S1P is a 379Da member of the lysophospholipid family. It is the direct metabolite of sphingosine through the action of two sphingosine kinases, SphK1 and SphK2. The main metabolic pathway starts with the hydrolysis of sphingomyelin, a membrane sphingolipid, into ceramide by the enzyme sphingomyelinase and the subsequent production of sphingosine by ceramidase (Figure 1). Ceramide can also be produced de novo in the endoplasmic reticulum (ER) from serine and palmitoyl coenzyme A through multiple intermediates. S1P production is regulated by various S1P-specific and general lipid phosphatases, as well as S1P lyase, which irreversibly degrades S1P into phosphoethanolamine and hexadecanal. The balance between intracellular S1P and its metabolite ceramide can determine cellular fate. Ceramide promotes apoptosis, while S1P suppresses cell death and promotes cell survival. This creates an S1P ceramide “rheostat” inside the cells. S1P lyase expression in tissue is higher than it is in erythrocytes and platelets, the main “suppliers” of S1P in blood. This causes a tissue–blood gradient of S1P, which is important in many S1P-mediated responses, like the lymphocyte egress from lymphoid organs.
S1P signaling overview
S1P is produced inside cells; however, it can also be found extracellularly, in a variety of different tissues. It is abundant in the blood, at concentrations of 0.4–1.5 μM, where it is mainly secreted by erythrocytes and platelets. Blood S1P can be found separately, but mainly it exists in complexes with high-density lipoprotein (HDL) (∼60%). Many of the cardioprotective effects of HDL are hypothesized to involve S1P. Before 1996, S1P was thought to act mainly intracellularly as a second messenger. However, the identification of several GPCRs that bind S1P led to the initiation of many studies on
extracellular S1P signaling through those receptors.
There are five receptors that have been identified currently. These can be coupled with different G-proteins. Assuming that each receptor coupling with a G protein has a slightly different function, one can recognize the complexity of S1P receptor signaling.
S1P as a second messenger
S1P is involved in many cellular processes through its GPCR signaling; studies demonstrate that S1P also acts at an intracellular level. Intracellular S1P plays a role in maintaining the balance of cell survival signal toward apoptotic signals, creating a
cell “rheostat” between S1P and its precursor ceramide.
Important evidence that S1P can act intracellularly as a second messenger came from yeast (Saccharomyces cerevisiae) and plant (Arabidopsis thaliana) cells. Yeast cells do not express any S1P receptors, although they can be affected by S1P during heat-shock responses. Similarly, Arabidopsis has only one GPCR-like protein, termed “GCR1,” which does not bind S1P, although S1P regulates stomata closure during drought.
Sphingosine-1-phosphate
In mammals, the sphingosine kinases have been found to localize in different cell compartments, being responsible for the accumulation of S1P in those compartments to give intracellular signals. In mitochondria, for instance,
S1P was recently found to interact with prohibitin 2,
a conserved protein that maintains mitochondria assembly and function. According to the same study,
SphK2 is the major producer of S1P in mitochondria and the knockout of its gene can cause
disruption of mitochondrial respiration and cytochrome c oxidase function.
SphK2 is also present in the nucleus of many cells and has been implicated to cause cell cycle arrest, and it causes S1P accumulation in the nucleus. It seems that nuclear S1P is affiliated with the histone deacetylases HDAC1 and HDAC2,
inhibiting their activity, thus having an indirect effect in epigenetic regulation of gene expression.
In the ER, SphK2 has been identified to translocate during stress, and promote apoptosis. It seems that S1P has specific targets in the ER that cause apoptosis, probably through calcium mobilization signals.
Sphingosine 1-phosphate (S1P) is a small bioactive lipid molecule that is involved in several processes both intracellularly and extracellularly. It acts intracellularly
to promote the survival and growth of the cell,
through its interaction with molecules in different compartments of the cell.
It can also exist at high concentrations extracellularly, in the blood plasma and lymph. This causes an S1P gradient important for cell migration. S1P signals through five G protein-coupled receptors, S1PR1–S1PR5, whose expression varies in different types of cells and tissue. S1P signaling can be involved in physiological and pathophysiological conditions of the cardiovascular, nervous, and immune systems and diseases such as ischemia/reperfusion injury, autoimmunity, and cancer. In this review, we discuss how it can be used to discover novel therapeutic targets.
The involvement of S1P signaling in disease
In a mouse model of myocardial ischemia-reperfusion injury (IRI), S1P and its carrier, HDL, can help protect myocardial tissue and decrease the infarct size. It seems they reduce cardiomyocyte apoptosis and neutrophil recruitment to the ischemic tissue and may decrease leukocyte adhesion to the endothelium. This effect appears to be S1PR3 mediated, since in S1PR3 knockout mice it is alleviated.
Ischemia activates SphK1, which is then translocated to the plasma membrane. This leads to an increase of intracellular S1P, helping to promote cardiomyocyte survival against apoptosis, induced by ceramide. SphK1 knockout mice cannot be preconditioned against IRI, whereas SphK1 gene induction in the heart protects it from IRI. Interestingly, a recent study shows SphK2 may also play a role, since its knockout reduces the cardioprotective effects of preconditioning. Further, administration of S1P or sphingosine during reperfusion results in better recovery and attenuation of damage to cardiomyocytes. As with preconditioning, SphK1 deficiency also affects post-conditioning of mouse hearts after ischemia reperfusion (IR).
S1P does not only protect the heart from IRI. During intestinal IR, multiple organs can be damaged, including the lungs. S1P treatment of mice during intestinal IR seems to have a protective effect on lung injury, probably due to suppression of iNOS-induced nitric oxide generation. In renal IRI, SphK1 seems to be important, since its deficiency increased the damage in kidney tissue, whereas the lentiviral overexpression of the SphK1 gene protected from injury. Another study suggests that, after IRI, apoptotic renal cells release S1P, which recruits macrophages through S1PR3 activation and might contribute to kidney regeneration and restoration of renal epithelium. However, SphK2 is negatively implicated in hepatic IRI, its inhibition helping protect hepatocytes and restoring mitochondrial function.
Further studies are implicating S1P signaling or sphingosine kinases in several kinds of cancer as well as autoimmune diseases.
Figure 2 FTY720-P causes retention of T cells in the lymph nodes.
Notes: C57BL/6 mice were injected with BALB/c splenocytes in the footpad to create an allogenic response then treated with FTY720-P or vehicle every day on days 2 to 5. On day 6, the popliteal lymph nodes were removed. Popliteal node-derived cells were mixed with BALB/c splenocytes in interferon gamma (IFN-γ) cultured enzyme-linked immunosorbent spot reactions. Bars represent the mean number of IFN-γ spot-forming cells per 1000 popliteal node-derived cells, from six mice treated with vehicle and seven with FTY720-P. **P , 0.01. (not shown)
Fingolimod (INN, trade name Gilenya, Novartis) is an immunomodulating drug, approved for treating multiple sclerosis. It has reduced the rate of relapses in relapsing-remitting multiple sclerosis by over half. Fingolimod is a sphingosine-1-phosphate receptor modulator, which sequesters lymphocytes in lymph nodes, preventing them from contributing to an autoimmune reaction.
The S1P antagonist FTY720 has been approved by the US Food and Drug Administration to be used as a drug against multiple sclerosis (MS). FTY720 is in fact a prodrug, since it is phosphorylated in vivo by SphK2 into FTY720-P, an S1P structural analog, which can activate S1PR1, 3, 4, and 5. FTY720-P binding to S1PR1 causes internalization of the receptor, as does S1P – but instead of recycling it back to the cell surface, it promotes its ubiquitination and degradation at the proteasome. This has a direct effect on lymphocyte trafficking through the lymph nodes, since it relies on S1PR1 signaling and S1P gradient (Figure 2). In MS, it stops migrating lymphocytes into the brain, but it may also have direct effects on the CNS through neuroprotection. FTY720 can pass the blood–brain barrier and it could be phosphorylated by local sphingosine kinases to act through S1PR1 and S1PR3 receptors that are mainly expressed in the CNS. In MS lesions, astrocytes upregulate those two receptors and it has been shown that FTY720-P treatment in vitro inhibits astrocyte production of inflammatory cytokines. A recent study confirms the importance of S1PR3 signaling on activated astrocytes, as well as SphK1, that are upregulated and promote the secretion of the potentially neuroprotective cytokine CXCL-1.
There are several studies implicating the intracellular S1P ceramide rheostat to cancer cell survival or apoptosis and resistance to chemotherapy or irradiation in vitro. Studies with SphK1 inhibition in pancreatic, prostate cancers, and leukemia, show increased ceramide/S1P ratio and induction of apoptosis. However, S1P receptor signaling plays conflicting roles in cancer cell migration and metastasis.
Modulation of S1P signaling: therapeutic potential
S1P signaling can be involved in many pathophysiological conditions. This means that we could look for therapeutic targets in all the molecules taking part in S1P signaling and production, most importantly the S1P receptors and the sphingosine kinases. S1P agonists and antagonists could also be used to modulate S1P signaling during pathological conditions.
S1P can have direct effects on the cardiovascular system. During IRI, intracellular S1P can protect the cardiomyocytes and promote their survival. Pre- or post-conditioning of the heart with S1P could be used as a treatment, but upregulation of sphingosine kinases could also increase intracellular S1P bioavailability. S1P could also have effects on endothelial cells and neutrophil trafficking. Vascular endothelial cells mainly express S1PR1 and S1PR3; only a few types express S1PR2. S1PR1 and S1PR3 activation on these cells has been shown to enhance their chemotactic migration, probably through direct phosphorylation of S1PR1 by Akt, in a phosphatidylinositol 3-kinase and Rac1-dependent signaling pathway. Moreover, it stimulates endothelial cell proliferation through an ERK pathway. S1PR2 activation, however, inhibits endothelial cell migration, morphogenesis, and angiogenesis, most likely through Rho-dependent inhibition of Rac signaling pathway, as Inoki et al showed in mouse cells with the use of S1PR1 and S1PR3 specific antagonists.
Regarding permeability of the vascular endothelium and endothelial barrier integrity, S1P receptors can have different effects. S1PR1 activation enhances endothelial barrier integrity by stimulation of cellular adhesion and upregulation of adhesion molecules. However, S1PR2 and S1PR3 have been shown to have barrier-disrupting effects in vitro, and vascular permeability increasing effects in vivo. All the effects S1P can have on vascular endothelium and smooth muscle cells suggest that activation of S1PR2, not S1PR1 and S1PR3, signaling, perhaps with the use of S1PR2 specific agonists, could be used therapeutically to inhibit angiogenesis and disrupt vasculature, suppressing tumor growth and progression.
An important aspect of S1P signaling that is being already therapeutically targeted, but could be further investigated, is immune cell trafficking. Attempts have already been made to regulate lymphocyte cell migration with the use of the drug FTY720, whose phosphorylated form can inhibit the cells S1PR1-dependent egress from the lymph nodes, causing lymphopenia. FTY720 is used as an immunosuppressant for MS but is also being investigated for other autoimmune conditions and for transplantation. Unfortunately, Phase II and III clinical trials for the prevention of kidney graft rejection have not shown an advantage over standard therapies. Moreover, FTY720 can have some adverse cardiac effects, such as bradycardia. However, there are other S1PR1 antagonists that could be considered instead, including KRP-203, AUY954, and SEW2871. KRP-203 in particular has been shown to prolong rat skin and heart allograft survival and attenuate chronic rejection without causing bradycardia, especially when combined with other immunomodulators.
There are studies that argue S1P pretreatment has a negative effect on neutrophil chemotaxis toward the chemokine CXCL-8 (interleukin-8) or the potent chemoattractant formyl-methionyl-leucyl-phenylalanine. S1P pretreatment might also inhibit trans-endothelial migration of neutrophils, without affecting their adhesion to the endothelium. S1P effects on neutrophil migration toward CXCL-8 might be the result of S1PRs cross-linking with the CXCL-8 receptors in neutrophils, CXCR-1 and CXCR-2. Indeed, there is evidence suggesting S1PR4 and S1PR3 form heterodimers with CXCR-1 in neutrophils. Another indication that S1P plays a role in neutrophil trafficking is a recent paper on S1P lyase deficiency, a deficiency that impairs neutrophil migration from blood to tissue in knockout mice.
S1P lyase and S1PRs in neutrophils may be new therapeutic targets against IRI and inflammatory conditions in general. Consistent with these results, another study has shown that inhibition of S1P lyase can have a protective effect on the heart after IRI and this effect is alleviated when pretreated with an S1PR1 and S1PR3 antagonist. Inhibition was achieved with a US Food and Drug Administration-approved food additive, 2-acetyl-4-tetrahydroxybutylimidazole, providing a possible new drug perspective. Another S1P lyase inhibitor, LX2931, a synthetic analog of 2-acetyl-4-tetrahydroxybutylimidazole, has been shown to cause peripheral lymphopenia when administered in mice, providing a potential treatment for autoimmune diseases and prevention of graft rejection in transplantation. This molecule is currently under Phase II clinical trials in rheumatoid arthritis patients.
S1P signaling research has the potential to discover novel therapeutic targets. S1P signaling is involved in many physiological and pathological processes. However, the complexity of S1P signaling makes it necessary to consider every possible pathway, either through its GPCRs, or intracellularly, with S1P as a second messenger. Where the activation of one S1P receptor may lead to the desired outcome, the simultaneous activation of another S1P receptor may lead to the opposite outcome. Thus, if we are to target a specific signaling pathway, we might need specific agonists for S1P receptors to activate one S1P receptor pathway, while, at the same time, we might need to inhibit another through S1P receptor antagonists.
Evidence of sphingolipid signaling in cancer
Biologically active lipids are important cellular signaling molecules and play a role in cell communication and cancer cell proliferation, and cancer stem cell biology. A recent study in ovarian cancer cell lines shows that exogenous sphingosine 1 phosphate (SIP1) or overexpression of the sphingosine kinase (SPHK1) increases ovarian cancer cell proliferation, invasion and contributes to cancer stem cell like phenotype. The diabetes drug metformin was shown to be an inhibitor of SPHK1 and reduce ovarian cancer tumor growth.
Mol Cancer Res. 2019 Apr;17(4):870-881. doi: 10.1158/1541-7786.MCR-18-0409. Epub 2019 Jan 17.
SPHK1 Is a Novel Target of Metformin in Ovarian Cancer.
The role of phospholipid signaling in ovarian cancer is poorly understood. Sphingosine-1-phosphate (S1P) is a bioactive metabolite of sphingosine that has been associated with tumor progression through enhanced cell proliferation and motility. Similarly, sphingosine kinases (SPHK), which catalyze the formation of S1P and thus regulate the sphingolipid rheostat, have been reported to promote tumor growth in a variety of cancers. The findings reported here show that exogenous S1P or overexpression of SPHK1 increased proliferation, migration, invasion, and stem-like phenotypes in ovarian cancer cell lines. Likewise, overexpression of SPHK1 markedly enhanced tumor growth in a xenograft model of ovarian cancer, which was associated with elevation of key markers of proliferation and stemness. The diabetes drug, metformin, has been shown to have anticancer effects. Here, we found that ovarian cancer patients taking metformin had significantly reduced serum S1P levels, a finding that was recapitulated when ovarian cancer cells were treated with metformin and analyzed by lipidomics. These findings suggested that in cancer the sphingolipid rheostat may be a novel metabolic target of metformin. In support of this, metformin blocked hypoxia-induced SPHK1, which was associated with inhibited nuclear translocation and transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Further, ovarian cancer cells with high SPHK1 were found to be highly sensitive to the cytotoxic effects of metformin, whereas ovarian cancer cells with low SPHK1 were resistant. Together, the findings reported here show that hypoxia-induced SPHK1 expression and downstream S1P signaling promote ovarian cancer progression and that tumors with high expression of SPHK1 or S1P levels might have increased sensitivity to the cytotoxic effects of metformin. IMPLICATIONS: Metformin targets sphingolipid metabolism through inhibiting SPHK1, thereby impeding ovarian cancer cell migration, proliferation, and self-renewal.
Nrf2:INrf2(Keap1) Signaling in Oxidative Stress
James W. Kaspar, Suresh K. Niture, and Anil K. Jaiswal*
Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD
Nrf2:INrf2(Keap1) are cellular sensors of chemical and radiation induced oxidative and electrophilic stress. Nrf2 is a nuclear transcription factor that
controls the expression and coordinated induction of a battery of defensive genes encoding detoxifying enzymes and antioxidant proteins.
This is a mechanism of critical importance for cellular protection and cell survival. Nrf2 is retained in the cytoplasm by an inhibitor INrf2. INrf2 functions as an adapter for
Cul3/Rbx1 mediated degradation of Nrf2.
In response to oxidative/electrophilic stress,
Nrf2 is switched on and then off by distinct
early and delayed mechanisms.
Oxidative/electrophilic modification of INrf2cysteine151 and/or PKC phosphorylation of Nrf2serine40 results in the escape or release of Nrf2 from INrf2. Nrf2 is stabilized and translocates to the nucleus, forms heterodimers with unknown proteins, and binds antioxidant response element (ARE) that leads to coordinated activation of gene expression. It takes less than fifteen minutes from the time of exposure
to switch on nuclear import of Nrf2.
This is followed by activation of a delayed mechanism that controls
switching off of Nrf2 activation of gene expression.
GSK3β phosphorylates Fyn at unknown threonine residue(s) leading to
nuclear localization of Fyn.
Fyn phosphorylates Nrf2tyrosine568 resulting in
nuclear export of Nrf2,
binding with INrf2 and
degradation of Nrf2.
The switching on and off of Nrf2 protects cells against free radical damage, prevents apoptosis and promotes cell survival.
NPRA-mediated suppression of AngII-induced ROS production contributes to the antiproliferative effects of B-type natriuretic peptide in VSMC
Pan Gao, De-Hui Qian, Wei Li, Lan Huang
Mol Cell Biochem (2009) 324:165–172
Excessive proliferation of vascular smooth cells (VSMCs) plays a critical role in the pathogenesis of diverse vascular disorders, and inhibition of VSMCs proliferation has been proved to be beneficial to these diseases.
In this study, we investigated the antiproliferative effect of
B-type natriuretic peptide (BNP), a natriuretic peptide with potent antioxidant capacity,
on rat aortic VSMCs, and the possible mechanisms involved. The results indicate that
BNP potently inhibited Angiotensin II (AngII)-induced VSMCs proliferation,
as evaluated by [3H]-thymidine incorporation assay. Consistently, BNP significantly decreased
AngII-induced intracellular reactive oxygen species (ROS)
and NAD(P)H oxidase activity.
8-Br-cGMP, a cGMP analog,
mimicked these effects.
To confirm its mechanism, siRNA of natriuretic peptide receptor-A(NRPA) strategy technology was used
to block cGMP production in VSMCs, and
siNPRA attenuated the inhibitory effects of BNP in VSMCs.
Taken together, these results indicate that
BNP was capable of inhibiting VSMCs proliferation by
NPRA/cGMP pathway,
which might be associated with
the suppression of ROS production.
These results might be related, at least partly, to the anti-oxidant property of BNP.
Cellular prion protein is required for neuritogenesis: fine-tuning of multiple signaling pathways involved in focal adhesions and actin cytoskeleton dynamics
A Alleaume-Butaux, C Dakowski, M Pietri, S Mouillet-Richard, Jean-Marie Launay, O Kellermann, B Schneider
1INSERM, UMR-S 747, 2Paris Descartes University, Sorbonne Paris, 3Public Hospital of Paris, Department of Biochemistry, Paris, France; 4Pharma Research Department, Hoffmann La Roche Ltd, Basel, Switzerland
Cell Health and Cytoskeleton 2013; 5: 1–12
Neuritogenesis is a complex morphological phenomena accompanying neuronal differentiation. Neuritogenesis relies on the initial breakage of the rather spherical symmetry of neuroblasts and the formation of buds emerging from the postmitotic neuronal soma. Buds then evolve into neurites, which later convert into an axon or dendrites. At the distal tip of neurites, the growth cone integrates extracellular signals and guides the neurite to its target. The acquisition of neuronal polarity depends on deep modifications of the neuroblast cytoskeleton characterized by the remodeling and activation of focal adhesions (FAs) and localized destabilization of the actin network in the neuronal sphere.Actin instability in unpolarized neurons allows neurite sprouting, ie, the protrusion of microtubules, and subsequent neurite outgrowth. Once the neurite is formed, actin microfilaments recover their stability and exert a sheathed action on neurites, a dynamic process necessary for the maintenance and integrity of neurites.
A combination of extrinsic and intrinsic cues pilots the architectural and functional changes in FAs and the actin network along neuritogenesis. This process includes neurotrophic factors (nerve growth factor, brain derived neurotrophic factor, neurotrophin, ciliary neurotrophic factor, glial derived neurotrophic factor) and their receptors, protein components of the extracellular matrix (ECM) (laminin, vitronectin, fibronectin), plasma membrane integrins and neural cell adhesion molecules (NCAM), and intracellular molecular protagonists such as small G proteins (RhoA, Rac, Cdc42) and their downstream targets.
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
Percent Composition: C 64.95%, H 4.49%, O 20.36%, S 10.20%
LitRef: Selective cyclooxygenase-2 (COX-2) inhibitor. Prepn: Y. Ducharme et al.,WO9500501; eidem,US5474995 (both 1995 to Merck Frosst).
Therap-Cat: Anti-inflammatory; analgesic.
Rofecoxib/ˌrɒfɨˈkɒksɪb/ is a nonsteroidal anti-inflammatory drug (NSAID) that has now been withdrawn over safety concerns. It was marketed by Merck & Co. to treat osteoarthritis, acute pain conditions, and dysmenorrhoea. Rofecoxib was approved by the Food and Drug Administration (FDA) on May 20, 1999, and was marketed under the brand namesVioxx, Ceoxx, and Ceeoxx.
Rofecoxib
Rofecoxib gained widespread acceptance among physicians treating patients with arthritis and other conditions causing chronic or acute pain. Worldwide, over 80 million people were prescribed rofecoxib at some time.[1]
On September 30, 2004, Merck withdrew rofecoxib from the market because of concerns about increased risk of heart attack and stroke associated with long-term, high-dosage use. Merck withdrew the drug after disclosures that it withheld information about rofecoxib’s risks from doctors and patients for over five years, resulting in between 88,000 and 140,000 cases of serious heart disease.[2] Rofecoxib was one of the most widely used drugs ever to be withdrawn from the market. In the year before withdrawal, Merck had sales revenue of US$2.5 billion from Vioxx.[3] Merck reserved $970 million to pay for its Vioxx-related legal expenses through 2007, and have set aside $4.85bn for legal claims from US citizens.
Rofecoxib was available on prescription in both tablet-form and as an oral suspension. It was available by injection for hospital use.
Mode of action
Cyclooxygenase (COX) has two well-studied isoforms, called COX-1 and COX-2.
COX-1 mediates the synthesis of prostaglandins responsible for protection of the stomach lining, while
COX-2 mediates the synthesis of prostaglandins responsible for pain and inflammation.
prostaglandin PGE2
By creating “selective” NSAIDs that inhibit COX-2, but not COX-1, the same pain relief as traditional NSAIDs is offered, but with greatly reduced risk of fatal or debilitating peptic ulcers. Rofecoxib is a selective COX-2 inhibitor, or “coxib”.
Others include Merck’s etoricoxib (Arcoxia), Pfizer’s celecoxib (Celebrex) and valdecoxib (Bextra). Interestingly, at the time of its withdrawal, rofecoxib was the only coxib with clinical evidence of its superior gastrointestinal adverse effect profile over conventional NSAIDs. This was largely based on the VIGOR (Vioxx GI Outcomes Research) study, which compared the efficacy and adverse effect profiles of rofecoxib and naproxen.[4]
Pharmacokinetics
The therapeutic recommended dosages were 12.5, 25, and 50 mg with an approximate bioavailability of 93%.[5][6][7] Rofecoxib crossed the placenta and blood–brain barrier,[5][6][8]and took 1–3 hours to reach peak plasma concentration with an effective half-life (based on steady-state levels) of approximately 17 hours.[5][7][9] The metabolic products are cis-dihydro and trans-dihydro derivatives of rofecoxib[5][9] which are primarily excreted through urine.
Fabricated efficacy studies
On March 11, 2009, Scott S. Reuben, former chief of acute pain at Baystate Medical Center, Springfield, Mass., revealed that data for 21 studies he had authored for the efficacy of the drug (along with others such as celecoxib) had been fabricated in order to augment the analgesic effects of the drugs. There is no evidence that Reuben colluded with Merck in falsifying his data. Reuben was also a former paid spokesperson for the drug company Pfizer (which owns the intellectual property rights for marketing celecoxib in the United States). The retracted studies were not submitted to either the FDA or the European Union’s regulatory agencies prior to the drug’s approval. Drug manufacturer Merckhad no comment on the disclosure.[10]
Aside from the reduced incidence of gastric ulceration, rofecoxib exhibits a similar adverse effect profile to other NSAIDs.
Prostaglandin is a large family of lipids. Prostaglandin I2/PGI2/prostacyclin is just one member of it. Prostaglandins other than PGI2 (such as PGE2) also play important roles in vascular tone regulation. Prostacyclin/thromboxane are produced by both COX-1 and COX-2, and rofecoxib suppresses just COX-2 enzyme, so there is no reason to believe that prostacyclin levels are significantly reduced by the drug. And there is no reason to believe that only the balance between quantities of prostacyclin and thromboxane is the determinant factor for vascular tone.[11] Indeed Merck has stated that there was no effect on prostacyclin production in blood vessels in animal testing.[12] Other researchers have speculated that the cardiotoxicity may be associated with maleic anhydride metabolites formed when rofecoxib becomes ionized under physiological conditions. (Reddy & Corey, 2005)
Adverse cardiovascular events
VIGOR study and publishing controversy
The VIGOR (Vioxx GI Outcomes Research) study, conducted by Bombardier, et al., which compared the efficacy and adverse effect profiles of rofecoxib and naproxen, had indicated a significant 4-fold increased risk of acute myocardial infarction (heart attack) in rofecoxib patients when compared with naproxen patients (0.4% vs 0.1%, RR 0.25) over the 12 month span of the study. The elevated risk began during the second month on rofecoxib. There was no significant difference in the mortality from cardiovascular events between the two groups, nor was there any significant difference in the rate of myocardial infarction between the rofecoxib and naproxen treatment groups in patients without high cardiovascular risk. The difference in overall risk was by the patients at higher risk of heart attack, i.e. those meeting the criteria for low-dose aspirin prophylaxis of secondary cardiovascular events (previous myocardial infarction, angina, cerebrovascular accident, transient ischemic attack, or coronary artery bypass).
Merck’s scientists interpreted the finding as a protective effect of naproxen, telling the FDA that the difference in heart attacks “is primarily due to” this protective effect (Targum, 2001). Some commentators have noted that naproxen would have to be three times as effective as aspirin to account for all of the difference (Michaels 2005), and some outside scientists warned Merck that this claim was implausible before VIGOR was published.[13] No evidence has since emerged for such a large cardioprotective effect of naproxen, although a number of studies have found protective effects similar in size to those of aspirin.[14][15] Though Dr. Topol’s 2004 paper criticized Merck’s naproxen hypothesis, he himself co-authored a 2001 JAMA article stating “because of the evidence for an antiplatelet effect of naproxen, it is difficult to assess whether the difference in cardiovascular event rates in VIGOR was due to a benefit from naproxen or to a prothrombotic effect from rofecoxib.” (Mukherjee, Nissen and Topol, 2001.)
The results of the VIGOR study were submitted to the United States Food and Drug Administration (FDA) in February 2001. In September 2001, the FDA sent a warning letter to the CEO of Merck, stating, “Your promotional campaign discounts the fact that in the VIGOR study, patients on Vioxx were observed to have a four to five fold increase in myocardial infarctions (MIs) compared to patients on the comparator non-steroidal anti-inflammatory drug (NSAID), Naprosyn (naproxen).”[16] This led to the introduction, in April 2002, of warnings on Vioxx labeling concerning the increased risk of cardiovascular events (heart attack and stroke).
Months after the preliminary version of VIGOR was published in the New England Journal of Medicine, the journal editors learned that certain data reported to the FDA were not included in the NEJM article. Several years later, when they were shown a Merck memo during the depositions for the first federal Vioxx trial, they realized that these data had been available to the authors months before publication. The editors wrote an editorial accusing the authors of deliberately withholding the data.[17] They released the editorial to the media on December 8, 2005, before giving the authors a chance to respond. NEJM editor Gregory Curfman explained that the quick release was due to the imminent presentation of his deposition testimony, which he feared would be misinterpreted in the media. He had earlier denied any relationship between the timing of the editorial and the trial. Although his testimony was not actually used in the December trial, Curfman had testified well before the publication of the editorial.[18]
The editors charged that “more than four months before the article was published, at least two of its authors were aware of critical data on an array of adverse cardiovascular events that were not included in the VIGOR article.” These additional data included three additional heart attacks, and raised the relative risk of Vioxx from 4.25-fold to 5-fold. All the additional heart attacks occurred in the group at low risk of heart attack (the “aspirin not indicated” group) and the editors noted that the omission “resulted in the misleading conclusion that there was a difference in the risk of myocardial infarction between the aspirin indicated and aspirin not indicated groups.” The relative risk for myocardial infarctions among the aspirin not indicated patients increased from 2.25 to 3 (although it remained statitistically insignificant). The editors also noted a statistically significant (2-fold) increase in risk for serious thromboembolic events for this group, an outcome that Merck had not reported in the NEJM, though it had disclosed that information publicly in March 2000, eight months before publication.[19]
The authors of the study, including the non-Merck authors, responded by claiming that the three additional heart attacks had occurred after the prespecified cutoff date for data collection and thus were appropriately not included. (Utilizing the prespecified cutoff date also meant that an additional stroke in the naproxen population was not reported.) Furthermore, they said that the additional data did not qualitatively change any of the conclusions of the study, and the results of the full analyses were disclosed to the FDA and reflected on the Vioxx warning label. They further noted that all of the data in the “omitted” table were printed in the text of the article. The authors stood by the original article.[20]
NEJM stood by its editorial, noting that the cutoff date was never mentioned in the article, nor did the authors report that the cutoff for cardiovascular adverse events was before that for gastrointestinal adverse events. The different cutoffs increased the reported benefits of Vioxx (reduced stomach problems) relative to the risks (increased heart attacks).[19]
Some scientists have accused the NEJM editorial board of making unfounded accusations.[21][22] Others have applauded the editorial. Renowned research cardiologist Eric Topol,[23] a prominent Merck critic, accused Merck of “manipulation of data” and said “I think now the scientific misconduct trial is really fully backed up”.[24] Phil Fontanarosa, executive editor of the prestigious Journal of the American Medical Association, welcomed the editorial, saying “this is another in the long list of recent examples that have generated real concerns about trust and confidence in industry-sponsored studies”.[25]
On May 15, 2006, the Wall Street Journal reported that a late night email, written by an outside public relations specialist and sent to Journal staffers hours before the Expression of Concern was released, predicted that “the rebuke would divert attention to Merck and induce the media to ignore the New England Journal of Medicine‘s own role in aiding Vioxx sales.”[26]
“Internal emails show the New England Journal’s expression of concern was timed to divert attention from a deposition in which Executive Editor Gregory Curfman made potentially damaging admissions about the journal’s handling of the Vioxx study. In the deposition, part of the Vioxx litigation, Dr. Curfman acknowledged that lax editing might have helped the authors make misleading claims in the article.” The Journal stated that NEJM‘s “ambiguous” language misled reporters into incorrectly believing that Merck had deleted data regarding the three additional heart attacks, rather than a blank table that contained no statistical information; “the New England Journal says it didn’t attempt to have these mistakes corrected.”[26]
APPROVe study
In 2001, Merck commenced the APPROVe (Adenomatous Polyp PRevention On Vioxx) study, a three-year trial with the primary aim of evaluating the efficacy of rofecoxib for theprophylaxis of colorectal polyps. Celecoxib had already been approved for this indication, and it was hoped to add this to the indications for rofecoxib as well. An additional aim of the study was to further evaluate the cardiovascular safety of rofecoxib.
The APPROVe study was terminated early when the preliminary data from the study showed an increased relative risk of adverse thrombotic cardiovascular events (includingheart attack and stroke), beginning after 18 months of rofecoxib therapy. In patients taking rofecoxib, versus placebo, the relative risk of these events was 1.92 (rofecoxib 1.50 events vs placebo 0.78 events per 100 patient years). The results from the first 18 months of the APPROVe study did not show an increased relative risk of adverse cardiovascular events. Moreover, overall and cardiovascular mortality rates were similar between the rofecoxib and placebo populations.[28]
In summary, the APPROVe study suggested that long-term use of rofecoxib resulted in nearly twice the risk of suffering a heart attack or stroke compared to patients receiving a placebo.
Other studies
Several very large observational studies have also found elevated risk of heart attack from rofecoxib. For example, a recent retrospective study of 113,000 elderly Canadians suggested a borderline statistically significant increased relative risk of heart attacks of 1.24 from Vioxx usage, with a relative risk of 1.73 for higher-dose Vioxx usage. (Levesque, 2005). Another study, using Kaiser Permanente data, found a 1.47 relative risk for low-dose Vioxx usage and 3.58 for high-dose Vioxx usage compared to current use of celecoxib, though the smaller number was not statistically significant, and relative risk compared to other populations was not statistically significant. (Graham, 2005).
Furthermore, a more recent meta-study of 114 randomized trials with a total of 116,000+ participants, published in JAMA, showed that Vioxx uniquely increased risk of renal (kidney) disease, and heart arrhythmia.[31]
Other COX-2 inhibitors
Any increased risk of renal and arrhythmia pathologies associated with the class of COX-2 inhibitors, e.g. celecoxib (Celebrex), valdecoxib (Bextra), parecoxib (Dynastat),lumiracoxib, and etoricoxib is not evident,[31] although smaller studies[32][33] had demonstrated such effects earlier with the use of celecoxib, valdecoxib and parecoxib.
Nevertheless, it is likely that trials of newer drugs in the category will be extended in order to supply additional evidence of cardiovascular safety. Examples are some more specific COX-2 inhibitors, including etoricoxib (Arcoxia) and lumiracoxib (Prexige), which are currently (circa 2005) undergoing Phase III/IV clinical trials.
Besides, regulatory authorities worldwide now require warnings about cardiovascular risk of COX-2 inhibitors still on the market. For example, in 2005, EU regulators required the following changes to the product information and/or packaging of all COX-2 inhibitors:[34]
Contraindications stating that COX-2 inhibitors must not be used in patients with established ischaemic heart disease and/or cerebrovascular disease (stroke), and also in patients with peripheral arterial disease
Reinforced warnings to healthcare professionals to exercise caution when prescribing COX-2 inhibitors to patients with risk factors for heart disease, such as hypertension, hyperlipidaemia (high cholesterol levels), diabetes and smoking
Given the association between cardiovascular risk and exposure to COX-2 inhibitors, doctors are advised to use the lowest effective dose for the shortest possible duration of treatment
Other NSAIDs
Since the withdrawal of Vioxx it has come to light that there may be negative cardiovascular effects with not only other COX-2 inhibitiors, but even the majority of other NSAIDs. It is only with the recent development of drugs like Vioxx that drug companies have carried out the kind of well executed trials that could establish such effects and these sort of trials have never been carried out in older “trusted” NSAIDs such as ibuprofen, diclofenac and others. The possible exceptions may be aspirin and naproxen due to their anti-platelet aggregation properties.
Withdrawal
Due to the findings of its own APPROVe study, Merck publicly announced its voluntary withdrawal of the drug from the market worldwide on September 30, 2004.[35]
In addition to its own studies, on September 23, 2004 Merck apparently received information about new research by the FDA that supported previous findings of increased risk of heart attack among rofecoxib users (Grassley, 2004). FDA analysts estimated that Vioxx caused between 88,000 and 139,000 heart attacks, 30 to 40 percent of which were probably fatal, in the five years the drug was on the market.[36]
On November 5, the medical journal The Lancet published a meta-analysis of the available studies on the safety of rofecoxib (Jüni et al., 2004). The authors concluded that, owing to the known cardiovascular risk, rofecoxib should have been withdrawn several years earlier. The Lancet published an editorial which condemned both Merck and the FDA for the continued availability of rofecoxib from 2000 until the recall. Merck responded by issuing a rebuttal of the Jüni et al. meta-analysis that noted that Jüni omitted several studies that showed no increased cardiovascular risk. (Merck & Co., 2004).
In 2005, advisory panels in both the U.S. and Canada encouraged the return of rofecoxib to the market, stating that rofecoxib’s benefits outweighed the risks for some patients. The FDA advisory panel voted 17-15 to allow the drug to return to the market despite being found to increase heart risk. The vote in Canada was 12-1, and the Canadian panel noted that the cardiovascular risks from rofecoxib seemed to be no worse than those from ibuprofen—though the panel recommended that further study was needed for all NSAIDs to fully understand their risk profiles. Notwithstanding these recommendations, Merck has not returned rofecoxib to the market.[37]
In 2005, Merck retained Debevoise & Plimpton LLP to investigate Vioxx study results and communications conducted by Merck. Through the report, it was found that Merck’s senior management acted in good faith, and that the confusion over the clinical safety of Vioxx was due to the sales team’s overzealous behavior. The report that was filed gave a timeline of the events surrounding Vioxx and showed that Merck intended to operate honestly throughout the process. Any mistakes that were made regarding the mishandling of clinical trial results and withholding of information was the result of oversight, not malicious behavior….The report was published in February 2006, and Merck was satisfied with the findings of the report and promised to consider the recommendations contained in the Martin Report. Advisers to the US Food and Drug Administration (FDA) have voted, by a narrow margin, that it should not ban Vioxx — the painkiller withdrawn by drug-maker Merck.
They also said that Pfizer’s Celebrex and Bextra, two other members of the family of painkillers known as COX-2 inhibitors, should remain available, despite the fact that they too boost patients’ risk of heart attack and stroke. url = http://www.nature.com/drugdisc/news/articles/433790b.html The recommendations of the arthritis and drug safety advisory panel offer some measure of relief to the pharmaceutical industry, which has faced a barrage of criticism for its promotion of the painkillers. But the advice of the panel, which met near Washington DC over 16–18 February, comes with several strings attached.
For example, most panel members said that manufacturers should be required to add a prominent warning about the drugs’ risks to their labels; to stop direct-to-consumer advertising of the drugs; and to include detailed, written risk information with each prescription. The panel also unanimously stated that all three painkillers “significantly increase the risk of cardiovascular events”.
This is the final article in a robust series on metabolism, metabolomics, and the “-OMICS-“ biological synthesis that is creating a more holistic and interoperable view of natural sciences, including the biological disciplines, climate science, physics, chemistry, toxicology, pharmacology, and pathophysiology with as yet unforeseen consequences.
There have been impressive advances already in the research into developmental biology, plant sciences, microbiology, mycology, and human diseases, most notably, cancer, metabolic , and infectious, as well as neurodegenerative diseases.
Acknowledgements:
I write this article in honor of my first mentor, Harry Maisel, Professor and Emeritus Chairman of Anatomy, Wayne State University, Detroit, MI and to my stimulating mentors, students, fellows, and associates over many years:
Masahiro Chiga, MD, PhD, Averill A Liebow, MD, Nathan O Kaplan, PhD, Johannes Everse, PhD, Norio Shioura, PhD, Abraham Braude, MD, Percy J Russell, PhD, Debby Peters, Walter D Foster, PhD, Herschel Sidransky, MD, Sherman Bloom, MD, Matthew Grisham, PhD, Christos Tsokos, PhD, IJ Good, PhD, Distinguished Professor, Raool Banagale, MD, Gustavo Reynoso, MD,Gustave Davis, MD, Marguerite M Pinto, MD, Walter Pleban, MD, Marion Feietelson-Winkler, RD, PhD, John Adan,MD, Joseph Babb, MD, Stuart Zarich, MD, Inder Mayall, MD, A Qamar, MD, Yves Ingenbleek, MD, PhD, Emeritus Professor, Bette Seamonds, PhD, Larry Kaplan, PhD, Pauline Y Lau, PhD, Gil David, PhD, Ronald Coifman, PhD, Emeritus Professor, Linda Brugler, RD, MBA, James Rucinski, MD, Gitta Pancer, Ester Engelman, Farhana Hoque, Mohammed Alam, Michael Zions, William Fleischman, MD, Salman Haq, MD, Jerard Kneifati-Hayek, Madeleine Schleffer, John F Heitner, MD, Arun Devakonda,MD, Liziamma George,MD, Suhail Raoof, MD, Charles Oribabor,MD, Anthony Tortolani, MD, Prof and Chairman, JRDS Rosalino, PhD, Aviva Lev Ari, PhD, RN, Rosser Rudolph, MD, PhD, Eugene Rypka, PhD, Jay Magidson, PhD, Izaak Mayzlin, PhD, Maurice Bernstein, PhD, Richard Bing, Eli Kaplan, PhD, Maurice Bernstein, PhD.
This article has EIGHT parts, as follows:
Part 1
Metabolomics Continues Auspicious Climb
Part 2
Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells
Part 3
Neuroscience
Part 4
Cancer Research
Part 5
Metabolic Syndrome
Part 6
Biomarkers
Part 7
Epigenetics and Drug Metabolism
Part 8
Pictorial
genome cartoon
iron metabolism
personalized reference range within population range
Part 1. MetabolomicsSurge
metagraph _OMICS
Metabolomics Continues Auspicious Climb
Jeffery Herman, Ph.D.
GEN May 1, 2012 (Vol. 32, No. 9)
Aberrant biochemical and metabolite signaling plays an important role in
the development and progression of diseased tissue.
This concept has been studied by the science community for decades. However, with relatively
recent advances in analytical technology and bioinformatics as well as
the development of the Human Metabolome Database (HMDB),
metabolomics has become an invaluable field of research.
At the “International Conference and Exhibition on Metabolomics & Systems Biology” held recently in San Francisco, researchers and industry leaders discussed how
the underlying cellular biochemical/metabolite fingerprint in response to
a specific disease state,
toxin exposure, or
pharmaceutical compound
is useful in clinical diagnosis and biomarker discovery and
in understanding disease development and progression.
Developed by BASF, MetaMap® Tox is
a database that helps identify in vivo systemic effects of a tested compound, including
targeted organs,
mechanism of action, and
adverse events.
Based on 28-day systemic rat toxicity studies, MetaMap Tox is composed of
differential plasma metabolite profiles of rats
after exposure to a large variety of chemical toxins and pharmaceutical compounds.
“Using the reference data,
we have developed more than 110 patterns of metabolite changes, which are
specific and predictive for certain toxicological modes of action,”
said Hennicke Kamp, Ph.D., group leader, department of experimental toxicology and ecology at BASF.
With MetaMap Tox, a potential drug candidate
can be compared to a similar reference compound
using statistical correlation algorithms,
which allow for the creation of a toxicity and mechanism of action profile.
“MetaMap Tox, in the context of early pre-clinical safety enablement in pharmaceutical development,” continued Dr. Kamp,
has been independently validated “
by an industry consortium (Drug Safety Executive Council) of 12 leading biopharmaceutical companies.”
Dr. Kamp added that this technology may prove invaluable
allowing for quick and accurate decisions and
for high-throughput drug candidate screening, in evaluation
on the safety and efficacy of compounds
during early and preclinical toxicological studies,
by comparing a lead compound to a variety of molecular derivatives, and
the rapid identification of the most optimal molecular structure
with the best efficacy and safety profiles might be streamlined.
Dynamic Construct of the –Omics
Targeted Tandem Mass Spectrometry
Biocrates Life Sciences focuses on targeted metabolomics, an important approach for
the accurate quantification of known metabolites within a biological sample.
Originally used for the clinical screening of inherent metabolic disorders from dried blood-spots of newborn children, Biocrates has developed
a tandem mass spectrometry (MS/MS) platform, which allows for
the identification,
quantification, and
mapping of more than 800 metabolites to specific cellular pathways.
It is based on flow injection analysis and high-performance liquid chromatography MS/MS.
common drug targets
The MetaDisIDQ® Kit is a
“multiparamatic” diagnostic assay designed for the “comprehensive assessment of a person’s metabolic state” and
the early determination of pathophysiological events with regards to a specific disease.
MetaDisIDQ is designed to quantify
a diverse range of 181 metabolites involved in major metabolic pathways
from a small amount of human serum (10 µL) using isotopically labeled internal standards,
This kit has been demonstrated to detect changes in metabolites that are commonly associated with the development of
metabolic syndrome, type 2 diabetes, and diabetic nephropathy,
Dr. Dallman reports that data generated with the MetaDisIDQ kit correlates strongly with
routine chemical analyses of common metabolites including glucose and creatinine
Biocrates has also developed the MS/MS-based AbsoluteIDQ® kits, which are
an “easy-to-use” biomarker analysis tool for laboratory research.
The kit functions on MS machines from a variety of vendors, and allows for the quantification of 150-180 metabolites.
The SteroIDQ® kit is a high-throughput standardized MS/MS diagnostic assay,
validated in human serum, for the rapid and accurate clinical determination of 16 known steroids.
Initially focusing on the analysis of steroid ranges for use in hormone replacement therapy, the SteroIDQ Kit is expected to have a wide clinical application.
Hormone-Resistant Breast Cancer
Scientists at Georgetown University have shown that
breast cancer cells can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.
To grow, cells need energy, and energy is a product of cellular metabolism. For nearly a century, it was thought that
the uncoupling of glycolysis from the mitochondria,
leading to the inefficient but rapid metabolism of glucose and
the formation of lactic acid (the Warburg effect), was
the major and only metabolism driving force for unchecked proliferation and tumorigenesis of cancer cells.
Other aspects of metabolism were often overlooked.
“.. we understand now that
cellular metabolism is a lot more than just metabolizing glucose,”
said Robert Clarke, Ph.D., professor of oncology and physiology and biophysics at Georgetown University. Dr. Clarke, in collaboration with the Waters Center for Innovation at Georgetown University (led by Albert J. Fornace, Jr., M.D.), obtained
the metabolomic profile of hormone-sensitive and -resistant breast cancer cells through the use of UPLC-MS.
They demonstrated that breast cancer cells, through a rather complex and not yet completely understood process,
can functionally coordinate cell-survival and cell-proliferation mechanisms,
while maintaining a certain degree of cellular metabolism.
This is at least partly accomplished through the upregulation of important pro-survival mechanisms; including
the unfolded protein response;
a regulator of endoplasmic reticulum stress and
initiator of autophagy.
Normally, during a stressful situation, a cell may
enter a state of quiescence and undergo autophagy,
a process by which a cell can recycle organelles
in order to maintain enough energy to survive during a stressful situation or,
if the stress is too great,
undergo apoptosis.
By integrating cell-survival mechanisms and cellular metabolism
advanced ER+ hormone-resistant breast cancer cells
can maintain a low level of autophagy
to adapt and resist hormone/chemotherapy treatment.
This adaptation allows cells
to reallocate important metabolites recovered from organelle degradation and
provide enough energy to also promote proliferation.
With further research, we can gain a better understanding of the underlying causes of hormone-resistant breast cancer, with
the overall goal of developing effective diagnostic, prognostic, and therapeutic tools.
NMR
Over the last two decades, NMR has established itself as a major tool for metabolomics analysis. It is especially adept at testing biological fluids. [Bruker BioSpin]
Historically, nuclear magnetic resonance spectroscopy (NMR) has been used for structural elucidation of pure molecular compounds. However, in the last two decades, NMR has established itself as a major tool for metabolomics analysis. Since
the integral of an NMR signal is directly proportional to
the molar concentration throughout the dynamic range of a sample,
“the simultaneous quantification of compounds is possible
without the need for specific reference standards or calibration curves,” according to Lea Heintz of Bruker BioSpin.
NMR is adept at testing biological fluids because of
high reproducibility,
standardized protocols,
low sample manipulation, and
the production of a large subset of data,
Bruker BioSpin is presently involved in a project for the screening of inborn errors of metabolism in newborn children from Turkey, based on their urine NMR profiles. More than 20 clinics are participating to the project that is coordinated by INFAI, a specialist in the transfer of advanced analytical technology into medical diagnostics. The construction of statistical models are being developed
for the detection of deviations from normality, as well as
automatic quantification methods for indicative metabolites
Bruker BioSpin recently installed high-resolution magic angle spinning NMR (HRMAS-NMR) systems that can rapidly analyze tissue biopsies. The main objective for HRMAS-NMR is to establish a rapid and effective clinical method to assess tumor grade and other important aspects of cancer during surgery.
Combined NMR and Mass Spec
There is increasing interest in combining NMR and MS, two of the main analytical assays in metabolomic research, as a means
to improve data sensitivity and to
fully elucidate the complex metabolome within a given biological sample.
to realize a potential for cancer biomarker discovery in the realms of diagnosis, prognosis, and treatment.
.
Using combined NMR and MS to measure the levels of nearly 250 separate metabolites in the patient’s blood, Dr. Weljie and other researchers at the University of Calgary were able to rapidly determine the malignancy of a pancreatic lesion (in 10–15% of the cases, it is difficult to discern between benign and malignant), while avoiding unnecessary surgery in patients with benign lesions.
When performing NMR and MS on a single biological fluid, ultimately “we are,” noted Dr. Weljie,
“splitting up information content, processing, and introducing a lot of background noise and error and
then trying to reintegrate the data…
It’s like taking a complex item, with multiple pieces, out of an IKEA box and trying to repackage it perfectly into another box.”
By improving the workflow between the initial splitting of the sample, they improved endpoint data integration, proving that
a streamlined approach to combined NMR/MS can be achieved,
leading to a very strong, robust and precise metabolomics toolset.
Metabolomics Research Picks Up Speed
Field Advances in Quest to Improve Disease Diagnosis and Predict Drug Response
John Morrow Jr., Ph.D.
GEN May 1, 2011 (Vol. 31, No. 9)
As an important discipline within systems biology, metabolomics is being explored by a number of laboratories for
its potential in pharmaceutical development.
Studying metabolites can offer insights into the relationships between genotype and phenotype, as well as between genotype and environment. In addition, there is plenty to work with—there are estimated to be some 2,900 detectable metabolites in the human body, of which
309 have been identified in cerebrospinal fluid,
1,122 in serum,
458 in urine, and
roughly 300 in other compartments.
Guowang Xu, Ph.D., a researcher at the Dalian Institute of Chemical Physics. is investigating the causes of death in China,
and how they have been changing over the years as the country has become a more industrialized nation.
the increase in the incidence of metabolic disorders such as diabetes has grown to affect 9.7% of the Chinese population.
Dr. Xu, collaborating with Rainer Lehman, Ph.D., of the University of Tübingen, Germany, compared urinary metabolites in samples from healthy individuals with samples taken from prediabetic, insulin-resistant subjects. Using mass spectrometry coupled with electrospray ionization in the positive mode, they observed striking dissimilarities in levels of various metabolites in the two groups.
“When we performed a comprehensive two-dimensional gas chromatography, time-of-flight mass spectrometry analysis of our samples, we observed several metabolites, including
2-hydroxybutyric acid in plasma,
as potential diabetes biomarkers,” Dr. Xu explains.
In other, unrelated studies, Dr. Xu and the German researchers used a metabolomics approach to investigate the changes in plasma metabolite profiles immediately after exercise and following a 3-hour and 24-hour period of recovery. They found that
medium-chain acylcarnitines were the most distinctive exercise biomarkers, and
they are released as intermediates of partial beta oxidation in human myotubes and mouse muscle tissue.
Dr. Xu says. “The traditional approach of assessment based on a singular biomarker is being superseded by the introduction of multiple marker profiles.”
Typical of the studies under way by Dr. Kaddurah-Daouk and her colleaguesat Duke University
is a recently published investigation highlighting the role of an SNP variant in
the glycine dehydrogenase gene on individual response to antidepressants.
patients who do not respond to the selective serotonin uptake inhibitors citalopram and escitalopram
carried a particular single nucleotide polymorphism in the GD gene.
“These results allow us to pinpoint a possible
role for glycine in selective serotonin reuptake inhibitor response and
illustrate the use of pharmacometabolomics to inform pharmacogenomics.
These discoveries give us the tools for prognostics and diagnostics so that
we can predict what conditions will respond to treatment.
“This approach to defining health or disease in terms of metabolic states opens a whole new paradigm.
By screening hundreds of thousands of molecules, we can understand
the relationship between human genetic variability and the metabolome.”
Dr. Kaddurah-Daouk talks about statins as a current
model of metabolomics investigations.
It is now known that the statins have widespread effects, altering a range of metabolites. To sort out these changes and develop recommendations for which individuals should be receiving statins will require substantial investments of energy and resources into defining the complex web of biochemical changes that these drugs initiate.
Furthermore, Dr. Kaddurah-Daouk asserts that,
“genetics only encodes part of the phenotypic response.
One needs to take into account the
net environment contribution in order to determine
how both factors guide the changes in our metabolic state that determine the phenotype.”
Interactive Metabolomics
Researchers at the University of Nottingham use diffusion-edited nuclear magnetic resonance spectroscopy to assess the effects of a biological matrix on metabolites. Diffusion-edited NMR experiments provide a way to
separate the different compounds in a mixture
based on the differing translational diffusion coefficients (which reflect the size and shape of the molecule).
The measurements are carried out by observing
the attenuation of the NMR signals during a pulsed field gradient experiment.
Clare Daykin, Ph.D., is a lecturer at the University of Nottingham, U.K. Her field of investigation encompasses “interactive metabolomics,”which she defines as
“the study of the interactions between low molecular weight biochemicals and macromolecules in biological samples ..
without preselection of the components of interest.
“Blood plasma is a heterogeneous mixture of molecules that
undergo a variety of interactions including metal complexation,
chemical exchange processes,
micellar compartmentation,
enzyme-mediated biotransformations, and
small molecule–macromolecular binding.”
Many low molecular weight compounds can exist
freely in solution,
bound to proteins, or
within organized aggregates such as lipoprotein complexes.
Therefore, quantitative comparison of plasma composition from
diseased individuals compared to matched controls provides an incomplete insight to plasma metabolism.
“It is not simply the concentrations of metabolites that must be investigated,
but their interactions with the proteins and lipoproteins within this complex web.
Rather than targeting specific metabolites of interest, Dr. Daykin’s metabolite–protein binding studies aim to study
the interactions of all detectable metabolites within the macromolecular sample.
Such activities can be studied through the use of diffusion-edited nuclear magnetic resonance (NMR) spectroscopy, in which one can assess
the effects of the biological matrix on the metabolites.
“This can lead to a more relevant and exact interpretation
for systems where metabolite–macromolecule interactions occur.”
Diffusion-edited NMR experiments provide a way to separate the different compounds in a mixture based on
