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George A. Miller, a Pioneer in Cognitive Psychology, Is Dead at 92

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 5.10

5.10 George A. Miller, a Pioneer in Cognitive Psychology, Is Dead at 92

By PAUL VITELLOAUG. 1, 2012

http://www.nytimes.com/2012/08/02/us/george-a-miller-cognitive-psychology-pioneer-dies-at-92.html?_r=0

Miller started his education focusing on speech and language and published papers on these topics, focusing on mathematicalcomputational 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 UniversityMIT 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.

http://www.psychologicalscience.org/index.php/publications/observer/2012/october-12/remembering-george-a-miller.html

Working memory

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]

Language and Communication, 1951[edit]

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]

Plans and the Structure of Behavior, 1960[edit]

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]

The Psychology of Communication, 1967[edit]

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]

https://en.wikipedia.org/wiki/George_Armitage_Miller

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Neonatal Pathophysiology

Neonatal Pathophysiology

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

 

Introduction

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

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

  1. Genetic and metabolic diseases
  2. Nervous system
  3. Cardiovascular
  4. Pulmonary
  5. Skeletal – bone and muscle
  6. Hematological
  7. Liver
  8. Esophagus, stomach, and intestines
  9. Kidneys
  10. Immune system

Fetal Development

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

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

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

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

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

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

fetal-development

fetal-development

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

Inherited Metabolic Disorders

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

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

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

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

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

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

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

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

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

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

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

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

Organic acidemias: methylmalonic acidemia and propionic acidemia.

Urea cycle disorders: ornithine transcarbamylase deficiency and citrullinemia

Hemoglobinopathies – thalassemias, sickle cell disease

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

This list is by no means complete.

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

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

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

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

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

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

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

Diagnosis and Management of Hereditary Hemochromatosis

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

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

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

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

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

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

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

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

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

Neurological and Behavioral Disorders

Estrogen receptor signaling during vertebrate development

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

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

 

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

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

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

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

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

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

representations of the HPA axis

representations of the HPA axis

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

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

Fetal programming of schizophrenia: Select mechanisms

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

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

NMDA receptor dysfunction in autism spectrum disorders

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

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

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

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

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

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

Chemokines roles within the hippocampus

Chemokines roles within the hippocampus

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

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

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

Three Classes of Glutamate Receptors

Three Classes of Glutamate Receptors

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

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

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

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

Fig not shown.

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

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

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

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

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

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

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

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

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

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

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

Impact of bilirubin-induced neurologic dysfunction on neurodevelopmental outcomes

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

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

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

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

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

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

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

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

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

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

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

  • Cerebellar vulnerability to bilirubin-induced injury
  • Cerebellar injury phenotypes and BIND
  • Cerebellar projections
Transverse section of cerebellum and brainstem

Transverse section of cerebellum and 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

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

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

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

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

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

Audiologic impairment associated with bilirubin-induced neurologic damage

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

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

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

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

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

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

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

Bilirubin-induced neurologic dysfunction (BIND)

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

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

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

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

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

Sensitive time-windows for susceptibility in neurodevelopmental disorders

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

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

Fig (not shown)

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

Fig (not shown)

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

Outstanding questions

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

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

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

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

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

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

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

Cardiovascular

Cardiac output monitoring in newborns

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

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

Fick principle

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

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

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

Cardiovascular adaptation to extra uterine life

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

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

Fetal vascular structures that exist to direct blood flow

Fetal structure Function
Arterial duct Connects pulmonary artery to the aorta and shunts blood right to left; diverting flow away from fetal lungs
Foramen ovale Opening between the two atria thatdirects blood flow returning to right

atrium through the septal wall into the left atrium bypassing lungs

Ductus venosus Receives oxygenated blood fromumbilical vein and directs it to the

inferior vena cava and right atrium

Umbilical arteries Carrying deoxygenated blood fromthe fetus to the placenta
Umbilical vein Carrying oxygenated blood from theplacenta to the fetus

Maternal causes of congenital heart disease

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

 

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

Clinical monitoring of systemic hemodynamics in critically ill newborns

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

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

Key guidelines

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

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

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

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

Monitoring oxygen saturation and heart rate in the early neonatal period

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

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

Practice points

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

Using pulse oximetry assists clinicians:

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

Research directions

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

Peripheral haemodynamics in newborns: Best practice guidelines

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

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

graphic

NIRS with partial venous occlusion to measure venous oxygen saturation

NIRS with partial venous occlusion to measure venous oxygen saturation

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

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

The oxygen-dissociation curve

graphic

The oxygen-dissociation curve

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

Peripheral oxygenation and management in the perinatal period

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

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

Fig (not shown)

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

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

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

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

Peripheral fractional oxygen extraction in babies

Peripheral fractional oxygen extraction in babies

graphic

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

Practice points

  • Peripheral tissue DO2 is complex: cardiac function, blood gases, Hb concentration and the proportion of HbF, and peripheral temperature all play a part in determining blood flow and oxygen extraction in the sick, preterm infant. Blood pressure appears to be less important.
  • Other factors likely to play a role are the administration of vasoactive medications and ventilation strategies, which affect blood gases and cardiac output by changing intrathoracic pressure.
  • Central blood pressure is a poor surrogate measurement for the adequacy of DO2 to the periphery. Direct measurement, using NIRS, laser Doppler flowmetry or other means, may give more useful information.
  • Reasons for total hemoglobin concentration (Hb) being a relatively poor indicator of the adequacy of the provision of oxygen to the tissues:
  1. Hb is only indirectly related to red blood cell volume, which may be a better indicator of the body’s oxygen delivering capacity.
  2. Hb-dependent oxygen availability depends on the position of the oxygen-hemoglobin dissociation curve.
  3. An individual’s oxygen requirements vary with time and from organ to organ. This means that DO2 also needs to vary.
  4. 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.
  5. The normal decrease of Hb during the first few weeks of life in both full-term and preterm babies usually occurs without symptoms or signs of anemia or clinical consequences.

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

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

Pulmonary

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

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

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

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

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

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

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

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

Neonatal ventilation strategies and long-term respiratory outcomes

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

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

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

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

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

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

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

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

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

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

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

Anemia, Apnea of Prematurity, and Blood Transfusions

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Long-term pulmonary outcomes of patients with bronchopulmonary dysplasia

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

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

Neonatal ventilation strategies and long-term respiratory outcomes

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

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

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

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

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

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

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

Pulmonary hypertension in bronchopulmonary dysplasia

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

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

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

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

Methylxanthines
Diuretics and bronchodilators
Corticosteroids
Macrolide antibiotics
Recombinant human Clara cell 10-kilodalton protein(rhCC10)
Vitamin A
Surfactant
Leukotriene receptor antagonist
Pulmonary vasodilators

Skeletal and Muscle

Skeletal Stem Cells in Space and Time

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Identification and Specification of the Mouse Skeletal Stem Cell

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

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

Bone mesenchymal development

Bone mesenchymal development

Bone mesenchymal development

The bone-remodeling cycle

The bone-remodeling cycle

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

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

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

Hematopoietic

Hematopoietic Stem Cell Arrival Triggers Dynamic Remodeling of the Perivascular Niche

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

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

Neonatal anemia

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

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

Management of anemia in the newborn

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

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

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

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

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

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

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

Clinical and hematological presentation among Indian patients with common hemoglobin variants

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

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

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

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

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

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

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

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

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

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

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

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

KEY POINTS

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

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

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

Hemolytic conditions in the neonate

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

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

  c. ABO blood group: Anti-A, -B

2. Red blood cell (RBC) enzyme defects

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

  b. Pyruvate kinase deficiency

  c. Others

3. RBC membrane defects

  a. Hereditary spherocytosis

  b. Elliptocytosis

  c. Stomatocytosis

  d. Pyknocytosis

  e. Others

4. Hemoglobinopathies

  a. alpha-thalassemia

  b. gamma-thalassemia

Standard maternal antibody screeningAlloantibody                                 Blood Group

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

K, k, Kpa, Jsa                                  Kell

Fya, Fyb                                          Duffy

Jka, Jkb                                           Kidd

Xga                                                  Xg

Lea, Leb                                          Lewis

S, s, M, N                                        MNS

P1                                                    P

Lub                                                  Lutheran

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

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

 

Red Blood Cell Enzymopathies

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

Red Blood Cell Membrane Defects

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

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

Risk factors for bilirubin neurotoxicityIsoimmune hemolytic disease

G6PD deficiency

Asphyxia

Sepsis

Acidosis

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

Polycythemia

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

 

 

Liver

Metabolic disorders presenting as liver disease

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

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

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

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

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

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

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

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

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

Alpha1-antitrypsin deficiency

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

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

Citrin deficiency

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

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

HFE-associated hemochromatosis

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

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

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

 

The liver in haemochromatosis

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

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

Gastrointestinal

Decoding the enigma of necrotizing enterocolitis in premature infants

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

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

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

Human gut microbiome

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

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

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

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

Current Issues in the Management of Necrotizing Enterocolitis

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

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

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

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

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

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

Premature newborns born prior to the 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

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

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

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

Intestinal Microbiota Development in Preterm Neonates and Effect of Perinatal Antibiotics

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

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

Human gut microbiome

Human gut microbiome

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

Ischemia and necrotizing enterocolitis: where, when, and how

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

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

Schematic representation of the intestinal microcirculation

Schematic representation of the intestinal microcirculation

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

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

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

Necrotizing enterocolitis: An update

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

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

Immune Deficiencies

Primary immunodeficiencies: A rapidly evolving story

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

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

Combined Immunodeficiencies

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

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

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

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

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

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

TCR signaling

TCR signaling

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

  • Uncoordinated 119 deficiency: Idiopathic CD41 lymphopenia

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

Well-Defined Syndromes with Immunodeficiency

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

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

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

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

Predominantly Antibody Defects

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

early colitis

Defects Of Immune Dysregulation

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

Congenital Defects Of Phagocyte Number, Function, Or Both

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

Defects In Innate Immunity

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

Autoinflammatory Disorders

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

 

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

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

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

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

Primary immunodeficiencies

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

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

 

 

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Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer

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

Article ID #160: Summary and Perspectives: Impairments in Pathological States: Endocrine Disorders, Stress Hypermetabolism and Cancer. Published on 11/9/2014

WordCloud Image Produced by Adam Tubman

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.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41282/title/The-Ever-Transcendent-Cell/

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.

http://www.the-scientist.com/Nov2014/TE_21.jpg

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.”

J.K. Goodrich et al., “Human genetics shape the gut microbiome,” Cell,  http://dx.doi.org:/10.1016/j.cell.2014.09.053, 2014.

Light-Operated Drugs

Scientists create a photosensitive pharmaceutical to target a glutamate receptor.
By Ruth Williams | The Scentist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41279/title/Light-Operated-Drugs/

light operated drugs MO1

light operated drugs MO1

http://www.the-scientist.com/Nov2014/MO1.jpg

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)

Enhanced Enhancers

The recent discovery of super-enhancers may offer new drug targets for a range of diseases.
By Eric Olson | The Scientist Nov 1, 2014
http://www.the-scientist.com/?articles.view/articleNo/41281/title/Enhanced-Enhancers/

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).

Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization 
Andrea Sánchez-Vallet, et al.   eLife 2013;2:e00790 http://elifesciences.org/content/2/e00790#sthash.LnqVMJ9p.dpuf

LysM effector

LysM effector

http://img.scoop.it/ZniCRKQSvJOG18fHbb4p0Tl72eJkfbmt4t8yenImKBVvK0kTmF0xjctABnaLJIm9

While host immune receptors

  • 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.”

Darnell Reflections - James_Darnell

Darnell Reflections – James_Darnell

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Recent findings on presenilins and signal peptide peptidase

By Dinu-Valantin Bălănescu

γ-secretase and SPP

γ-secretase and SPP

Fig. 1 from the minireview shows a schematic depiction of γ-secretase and SPP

http://www.asbmb.org/assets/0/366/418/428/85528/85529/85530/c2de032a-daad-41e5-ba19-87a17bd26362.png

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:

  1. nicastrin,
  2. anterior pharynx defective 1 and
  3. 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

From www.pnas.org –  Sep 3, 2013 4:24 PM

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

  • IFN-γ enzyme-linked immunosorbent spot responses (P = 0.042, multiple regression).

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.

Metabolomics in drug target discovery

J D Rabinowitz et al.

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

Metabolic Pathways as Targets for Drug Screening, Metabolomics, Dr Ute Roessner (Ed.), ISBN: 978-953-51-0046-1, InTech, Available from: http://www.intechopen.com/books/metabolomics/metabolic-pathways-as-targets-for-drug-screening

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

Drug Discov. Today 19 (2014), 171–182     http://dx.doi.org:/10.1016/j.drudis.2013.07.014

Highlights

  • We now have metabolic network models; the metabolome is represented by their nodes.
  • Metabolite levels are sensitive to changes in enzyme activities.
  • Drugs hitchhike on metabolite transporters to get into and out of cells.
  • The consensus network Recon2 represents the present state of the art, and has predictive power.
  • Constraint-based modelling relates network structure to metabolic fluxes.

Metabolism represents the ‘sharp end’ of systems biology, because changes in metabolite concentrations are

  • necessarily amplified relative to changes in the transcriptome, proteome and enzyme activities, which can be modulated by drugs.

To understand such behaviour, we therefore need (and increasingly have) reliable consensus (community) models of

  • the human metabolic network that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters, because

  • drugs bear structural similarities to metabolites known from the network reconstructions and
  • from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic network reconstruction; it can predict inter alia:

(i) the effects of inborn errors of metabolism;

(ii) which metabolites are exometabolites, and

(iii) how metabolism varies between tissues and cellular compartments.

However, even these qualitative network models are not yet complete. As our understanding improves

  • so do we recognise more clearly the need for a systems (poly)pharmacology.

Introduction – a systems biology approach to drug discovery

It is clearly not news that the productivity of the pharmaceutical industry has declined significantly during recent years

  • following an ‘inverse Moore’s Law’, Eroom’s Law, or
  • that many commentators, consider that the main cause of this is
  • because of an excessive focus on individual molecular target discovery rather than a more sensible strategy
  • based on a systems-level approach (Fig. 1).
drug discovery science

drug discovery science

Figure 1.

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.

metabolomics and systems pharmacology

metabolomics and systems pharmacology

http://ars.els-cdn.com/content/image/1-s2.0-S1359644613002481-gr2.jpg

Metabolic Disease Drug Discovery— “Hitting the Target” Is Easier Said Than Done

David E. Moller, et al.   http://dx.doi.org:/10.1016/j.cmet.2011.10.012

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—

  1. glucagon-like peptide-1 receptor agonists and
  2. 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

  • predict or evaluate the metabolism of pharmaceutical compounds, and
  • to better understand the pharmacokinetic profile of a drug.

Alternatively, pharmacometabolomics can be applied to measure metabolite levels

  • following the administration of a pharmaceutical compound, in order to
  • monitor the effects of the compound on certain metabolic pathways(pharmacodynamics).

This provides detailed mapping of drug effects on metabolism and

  • the pathways that are implicated in mechanism of variation of response to treatment.

In addition, the metabolic profile of an individual at baseline (metabotype) provides information about

  • how individuals respond to treatment and highlights heterogeneity within a disease state.

All three approaches require the quantification of metabolites found

relationship between -OMICS

relationship between -OMICS

http://upload.wikimedia.org/wikipedia/commons/thumb/e/eb/OMICS.png/350px-OMICS.png

Pharmacometabolomics is thought to provide information that

Looking at the characteristics of an individual down through these different levels of detail, there is an

  • increasingly more accurate prediction of a person’s ability to respond to a pharmaceutical compound.
  1. the genome, made up of 25 000 genes, can indicate possible errors in drug metabolism;
  2. the transcriptome, made up of 85,000 transcripts, can provide information about which genes important in metabolism are being actively transcribed;
  3. and the proteome, >10,000,000 members, depicts which proteins are active in the body to carry out these functions.

Pharmacometabolomics complements the omics with

  • direct measurement of the products of all of these reactions, but with perhaps a relatively
  • smaller number of members: that was initially projected to be approximately 2200 metabolites,

but could be a larger number when gut derived metabolites and xenobiotics are added to the list. Overall, the goal of pharmacometabolomics is

  • to more closely predict or assess the response of an individual to a pharmaceutical compound,
  • permitting continued treatment with the right drug or dosage
  • depending on the variations in their metabolism and ability to respond to treatment.

Pharmacometabolomic analyses, through the use of a metabolomics approach,

  • can provide a comprehensive and detailed metabolic profile or “metabolic fingerprint” for an individual patient.

Such metabolic profiles can provide a complete overview of individual metabolite or pathway alterations,

This approach can then be applied to the prediction of response to a pharmaceutical compound

  • by patients with a particular metabolic profile.

Pharmacometabolomic analyses of drug response are

Pharmacogenetics focuses on the identification of genetic variations (e.g. single-nucleotide polymorphisms)

  • within patients that may contribute to altered drug responses and overall outcome of a certain treatment.

The results of pharmacometabolomics analyses can act to “inform” or “direct”

  • pharmacogenetic analyses by correlating aberrant metabolite concentrations or metabolic pathways to potential alterations at the genetic level.

This concept has been established with two seminal publications from studies of antidepressants serotonin reuptake inhibitors

  • where metabolic signatures were able to define a pathway implicated in response to the antidepressant and
  • that lead to identification of genetic variants within a key gene
  • within the highlighted pathway as being implicated in variation in response.

These genetic variants were not identified through genetic analysis alone and hence

  • illustrated how metabolomics can guide and inform genetic data.

en.wikipedia.org/wiki/Pharmacometabolomics

Benznidazole Biotransformation and Multiple Targets in Trypanosoma cruzi Revealed by Metabolomics

Andrea Trochine, Darren J. Creek, Paula Faral-Tello, Michael P. Barrett, Carlos Robello
Published: May 22, 2014   http://dx.doi.org:/10.1371/journal.pntd.0002844

The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi,

  • involves administration of benznidazole (Bzn).

Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active. We used a

  • non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.

Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols

  1. trypanothione,
  2. homotrypanothione and
  3. cysteine

were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment.

These metabolites included reduction products, fragments and covalent adducts of reduced Bzn

  • linked to each of the major low molecular weight thiols:
  1. trypanothione,
  2. glutathione,
  3. g-glutamylcysteine,
  4. glutathionylspermidine,
  5. cysteine and
  6. ovothiol A.

Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI,

  • were found within the parasites,
  • but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.

Our data is indicative of a major role of the

  • thiol binding capacity of Bzn reduction products
  • in the mechanism of Bzn toxicity against T. cruzi.

 

 

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Summary to Metabolomics

Summary to Metabolomics

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

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?

http://masspec.scripps.edu/metabo_science/recommended_readings.php
 Recommended Readings and Historical Perspectives

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] mRNA gene 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 enzymatic chemical 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.

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

Jose Eduardo des Salles Roselino

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

Coordination of the transcriptome and metabolome by the circadian clock PNAS 2012

analysis of metabolomic data and differential metabolic regulation for fetal lungs, and maternal blood plasma

conformational changes leading to substrate efflux.img

conformational changes leading to substrate efflux.img

The cellular response is defined by a network of chemogenomic response signatures.

The cellular response is defined by a network of chemogenomic response signatures.

Dynamic Construct of the –Omics

Dynamic Construct of the –Omics

 genome cartoon

genome cartoon

central dogma phenotype

central dogma phenotype

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Introduction to Protein Synthesis and Degradation

Curator: Larry H. Bernstein, MD, FCAP

Updated 8/31/2019

 

Introduction to Protein Synthesis and Degradation

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 :

  1. Aliphatic Amino Acids
  2. Cyclic Amino Acid
  3. AAs with Hydroxyl or Sulfur-containing side chains
  4. Aromatic Amino Acids
  5. Basic Amino Acids
  6. Acidic Amino Acids and their Amides

Examples include:

Alanine                  aliphatic hydrophobic neutral
Arginine                 polar hydrophilic charged (+)
Cysteine                polar hydrophobic neutral
Glutamine             polar hydrophilic neutral
Histidine                aromatic polar hydrophilic charged (+)
Lysine                   polar hydrophilic charged (+)
Methionine            hydrophobic neutral
Serine                   polar hydrophilic neutral
Tyrosine                aromatic polar hydrophobic

Transcribe and Translate a Gene

  1. For each RNA base there is a corresponding DNA base
  2. Cells use the two-step process of transcription and translation to read each gene and produce the string of amino acids that makes up a protein.
  3. mRNA is produced in the nucleus, and is transferred to the ribosome
  4. mRNA uses uracil instead of thymine
  5. the ribosome reads the RNA sequence and makes protein
  6. There is a sequence combination to fit each amino acid to a three letter RNA code
  7. The ribosome starts at AUG (start), and it reads each codon three letters at a time
  8. Stop codons are UAA, UAG and UGA

 

protein synthesis

protein synthesis

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mcell-transcription-translation

mcell-transcription-translation

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transcription_translation

transcription_translation

 

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What about the purine inosine?

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.

Gastroenterology. 2011 Apr;140(4):1314-21.  http://dx.doi.org:/10.1053/j.gastro.2010.12.038. Epub 2011 Jan 1.

Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function.

Hitomi Y1, Cirulli ET, Fellay J, McHutchison JG, Thompson AJ, Gumbs CE, Shianna KV, Urban TJ, Goldstein DB.

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.

Ther Drug Monit. 2012 Aug;34(4):477-80.  http://dx.doi.org:/10.1097/FTD.0b013e31825c2703.

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

PeerJ 2:e270; http://dx.doi.org:/10.7717/peerj.270

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:

  1. Ubiquitylation in relationship to tissue remodeling
  2. Post-translational modification of proteins
    1. Glycosylation
    2. Phosphorylation
    3. Methylation
    4. Nitrosylation
    5. Sulfation – sulfotransferases
      cell-matrix communication
    6. 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
    7. Glycination
    8. cyclin dependent kinases (CDKs)
    9. lyase
    10. 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)

 

Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14289-94.   http://dx.doi.org:/10.1073/pnas.1407640111. Epub 2014 Sep 15.

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

RJ Weatheritt, TJ Gibson & MM Babu
Nature Structural & Molecular Biology 24 Aug, 2014; 21: 833–839 http://dx.do.orgi:/10.1038/nsmb.2876

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

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…

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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 Degradation Updated 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.

Structured Abstract
INTRODUCTION

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.

The eukaryotic fMet/N-end rule pathway.

(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.

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The eukaryotic fMet/N-end rule pathway.

(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.

 

A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Richard T. Timms1,2Zhiqian Zhang1,2David Y. Rhee3J. Wade Harper3Itay Koren1,2,*,Stephen J. Elledge1,2

Science  05 Jul 2019: Vol. 365, Issue 6448

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.

Science, this issue p. eaaw4912

Structured Abstract

INTRODUCTION

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.

The glycine N-degron pathway.

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.

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The glycine N-degron pathway.

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.

 

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Summary of Signaling and Signaling Pathways

Summary of Signaling and Signaling Pathways

Author and Curator: Larry H Bernstein, MD, FCAP

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

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:

  1. Interactions between differently weighted determinants
  2. 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

  1. a basic understanding signal transduction and
  2. the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

In addition – detailed knowledge of –

  1. the role of Tyrosine kinases and
  2. G protein-coupled receptors in cell signaling.
serine

serine

threonine

threonine

protein kinase

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).

Notch-mediated juxtacrine signal between adjacent cells. 220px-Notchccr

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.

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

signal transduction

http://www.hartnell.edu/tutorials/biology/images/signaltransduction_simple.jpg

The initiation is depicted as follows:

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.

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

  • Receptor Tyrosine Kinases.
intracellular signaling

intracellular signaling

http://www.hartnell.edu/tutorials/biology/images/membrane_receptor_tk.jpg

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

  1. a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
  2. 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

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

Signal transduction occurs when an

  1. extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
  2. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
  3. Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
  4. 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

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

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

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

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

signal transduction pathways

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

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)

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

RTKs

The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.

Allosteric_Regulation.svg

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

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

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

  1. timing of cellular survival,
  2. apoptosis,
  3. proliferation, and
  4. differentiation.
integrin-mediated signal transduction

integrin-mediated signal transduction

Integrin signaling

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

  1. induces action potentials, such as those that travel along nerves,
  2. by depolarizing the membrane of post-synaptic cells,
  3. resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR

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

cAMP

Inositol_1,4,5-trisphosphate.svg

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

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.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_via_metabolomics_and_systems_biology_driven_approaches

Related articles

  1. Systems Biology Approach Reveals Genome to Phenome Correlation in Type 2 Diabetes (plosone.org)
  2. Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease – Novel Clues to Drug Targets and Disease Mechanisms? (plosone.org)
  3. Activation of the Jasmonic Acid Plant Defence Pathway Alters the Composition of Rhizosphere

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

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

  1. thromboxane A2 (TXA2),
  2. leukotriene
  3. B4 (LTB4),
  4. IL-1,
  5. IL-6,
  6. tumor necrosis factor (TNF) and
  7. 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;

  1. catalytic,
  2. 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

  1. α-linolenic acid (ALA),
  2. docosahexenoic acid (DHA) and
  3. 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:

  1. amyloid presence,
  2. synaptic marker loss,
  3. hyper-phosphorylation of Tau,
  4. oxidative damage and
  5. 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.

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Complex Models of Signaling: Therapeutic Implications

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.

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

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

  1. thromboxane A2 (TXA2),
  2. leukotriene
  3. B4 (LTB4),
  4. IL-1,
  5. IL-6,
  6. tumor necrosis factor (TNF) and
  7. 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

  1. α-linolenic acid (ALA),
  2. docosahexenoic acid (DHA) and
  3. 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;

  1. catalytic,
  2. 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

zinc transporters

 

 

Early zinc signaling (EZS) and late zinc signaling (LZS)

Early zinc signaling (EZS) and late zinc signaling (LZS)

http://www.hindawi.com/journals/jnme/2012/floats/173712/thumbnails/173712.fig.002_th.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].

 

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:

  1. amyloid presence,
  2. synaptic marker loss,
  3. hyper-phosphorylation of Tau,
  4. oxidative damage and
  5. 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

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

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.

Fingolimod3Dan

Fingolimod3Dan

 

http://upload.wikimedia.org/wikipedia/commons/thumb/4/48/Fingolimod3Dan.gif/200px-Fingolimod3Dan.gif

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.

 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.

Abstract

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

Free Radic Biol Med. 2009 Nov 1; 47(9): 1304–1309. http://dx.doi.org:/10.1016/j.freeradbiomed.2009.07.035

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

http://dx.doi.org/10.1007/s11010-008-9995-y

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

  1. elements of the extracellular matrix,
  2. plasma membrane receptors,
  3. adhesion molecules, and
  4. soluble factors that control actin cytoskeleton turnover through Rho-GTPase signaling.

Once neurons have reached their terminal stage of differentiation and acquired their polarized morphology, PrPC also

  • takes part in the maintenance of neurites.

By acting on tissue nonspecific alkaline phosphatase, or

  • matrix metalloproteinase type 9,

PrPC stabilizes interactions between

  • neurites and the extracellular matrix.

Keywords: prion, neuronal differentiation

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Larry H. Bernstein, MD, FCAP, Reporter, Reposted

Leaders in Pharmaceutical Intelligence

DR ANTHONY MELVIN CRASTO …..FOR BLOG HOME CLICK HERE

http://pharmaceuticalintelligence.com/10/29/2010/larryhbern/Rofecoxib

ROFECOXIB

MK-966, MK-0966, Vioxx

162011-90-7

C17-H14-O4-S
314.3596
\
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., WO 9500501; 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 osteoarthritisacute pain conditions, and dysmenorrhoea. Rofecoxib was approved by the Food and Drug Administration (FDA) on May 20, 1999, and was marketed under the brand names VioxxCeoxx, and Ceeoxx.

Rofecoxib

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

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]

Adverse drug reactions

VIOXX sample blister pack.jpg

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 accidenttransient 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 polypsCelecoxib 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 ibuprofendiclofenac 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”.

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Metabolomics Summary and Perspective

Metabolomics Summary and Perspective

Author and Curator: Larry H Bernstein, MD, FCAP 

 

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

genome cartoon

 iron metabolism

iron metabolism

personalized reference range within population range

personalized reference range within population range

Part 1.  MetabolomicsSurge

metagraph  _OMICS

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

  1. recent advances in analytical technology and bioinformatics as well as
  2. 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
  1. a specific disease state,
  2. toxin exposure, or
  3. 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
  1. targeted organs,
  2. mechanism of action, and
  3. 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
  1. on the safety and efficacy of compounds
  2. during early and preclinical toxicological studies,
  3. 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

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
  1. the identification,
  2. quantification, and
  3. mapping of more than 800 metabolites to specific cellular pathways.

It is based on flow injection analysis and high-performance liquid chromatography MS/MS.

Clarification of Pathway-Specific Inhibition by Fourier Transform Ion Cyclotron Resonance.Mass Spectrometry-Based Metabolic Phenotyping Studies F5.large

common drug targets

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

  1. the uncoupling of glycolysis from the mitochondria,
  2. leading to the inefficient but rapid metabolism of glucose and
  3. 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,

  1. can functionally coordinate cell-survival and cell-proliferation mechanisms,
  2. 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

  1.  high reproducibility,
  2. standardized protocols,
  3. low sample manipulation, and
  4. 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,

  1. “splitting up information content, processing, and introducing a lot of background noise and error and
  2. 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

  1. 309 have been identified in cerebrospinal fluid,
  2. 1,122 in serum,
  3. 458 in urine, and
  4. 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

  1. undergo a variety of interactions including metal complexation,
  2. chemical exchange processes,
  3. micellar compartmentation,
  4. enzyme-mediated biotransformations, and
  5. 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

  • 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.

Pushing the Limits

It is widely recognized that many drug candidates fail during development due to ancillary toxicity. Uwe Sauer, Ph.D., professor, and Nicola Zamboni, Ph.D., researcher, both at the Eidgenössische Technische Hochschule, Zürich (ETH Zürich), are applying

  • high-throughput intracellular metabolomics to understand
  • the basis of these unfortunate events and
  • head them off early in the course of drug discovery.

“Since metabolism is at the core of drug toxicity, we developed a platform for

  • measurement of 50–100 targeted metabolites by
  • a high-throughput system consisting of flow injection
  • coupled to tandem mass spectrometry.”

Using this approach, Dr. Sauer’s team focused on

  • the central metabolism of the yeast Saccharomyces cerevisiae, reasoning that
  • this core network would be most susceptible to potential drug toxicity.

Screening approximately 41 drugs that were administered at seven concentrations over three orders of magnitude, they observed changes in metabolome patterns at much lower drug concentrations without attendant physiological toxicity.

The group carried out statistical modeling of about

  • 60 metabolite profiles for each drug they evaluated.

This data allowed the construction of a “profile effect map” in which

  • the influence of each drug on metabolite levels can be followed, including off-target effects, which
  • provide an indirect measure of the possible side effects of the various drugs.

Dr. Sauer says.“We have found that this approach is

  • at least 100 times as fast as other omics screening platforms,”

“Some drugs, including many anticancer agents,

  • disrupt metabolism long before affecting growth.”
killing cancer cells

killing cancer cells

Furthermore, they used the principle of 13C-based flux analysis, in which

  • metabolites labeled with 13C are used to follow the utilization of metabolic pathways in the cell.

These 13C-determined intracellular responses of metabolic fluxes to drug treatment demonstrate

  • the functional performance of the network to be rather robust,
conformational changes leading to substrate efflux.

conformational changes leading to substrate efflux.

leading Dr. Sauer to the conclusion that

  • the phenotypic vigor he observes to drug challenges
  • is achieved by a flexible make up of the metabolome.

Dr. Sauer is confident that it will be possible to expand the scope of these investigations to hundreds of thousands of samples per study. This will allow answers to the questions of

  • how cells establish a stable functioning network in the face of inevitable concentration fluctuations.

Is Now the Hour?

There is great enthusiasm and agitation within the biotech community for

  • metabolomics approaches as a means of reversing the dismal record of drug discovery

that has accumulated in the last decade.

While the concept clearly makes sense and is being widely applied today, there are many reasons why drugs fail in development, and metabolomics will not be a panacea for resolving all of these questions. It is too early at this point to recognize a trend or a track record, and it will take some time to see how this approach can aid in drug discovery and shorten the timeline for the introduction of new pharmaceutical agents.

Degree of binding correlated with function

Degree of binding correlated with function

Diagram_of_a_two-photon_excitation_microscope_

Diagram_of_a_two-photon_excitation_microscope_

Part 2.  Biologists Find ‘Missing Link’ in the Production of Protein Factories in Cells

Biologists at UC San Diego have found

  • the “missing link” in the chemical system that
  • enables animal cells to produce ribosomes

—the thousands of protein “factories” contained within each cell that

  • manufacture all of the proteins needed to build tissue and sustain life.
‘Missing Link’

‘Missing Link’

Their discovery, detailed in the June 23 issue of the journal Genes & Development, will not only force

  • a revision of basic textbooks on molecular biology, but also
  • provide scientists with a better understanding of
  • how to limit uncontrolled cell growth, such as cancer,
  • that might be regulated by controlling the output of ribosomes.

Ribosomes are responsible for the production of the wide variety of proteins that include

  1. enzymes;
  2. structural molecules, such as hair,
  3. skin and bones;
  4. hormones like insulin; and
  5. components of our immune system such as antibodies.

Regarded as life’s most important molecular machine, ribosomes have been intensively studied by scientists (the 2009 Nobel Prize in Chemistry, for example, was awarded for studies of its structure and function). But until now researchers had not uncovered all of the details of how the proteins that are used to construct ribosomes are themselves produced.

In multicellular animals such as humans,

  • ribosomes are made up of about 80 different proteins
    (humans have 79 while some other animals have a slightly different number) as well as
  • four different kinds of RNA molecules.

In 1969, scientists discovered that

  • the synthesis of the ribosomal RNAs is carried out by specialized systems using two key enzymes:
  • RNA polymerase I and RNA polymerase III.

But until now, scientists were unsure if a complementary system was also responsible for

  • the production of the 80 proteins that make up the ribosome.

That’s essentially what the UC San Diego researchers headed by Jim Kadonaga, a professor of biology, set out to examine. What they found was the missing link—the specialized

  • system that allows ribosomal proteins themselves to be synthesized by the cell.

Kadonaga says that he and coworkers found that ribosomal proteins are synthesized via

  • a novel regulatory system with the enzyme RNA polymerase II and
  • a factor termed TRF2,”

“For the production of most proteins,

  1. RNA polymerase II functions with
  2. a factor termed TBP,
  3. but for the synthesis of ribosomal proteins, it uses TRF2.”
  •  this specialized TRF2-based system for ribosome biogenesis
  • provides a new avenue for the study of ribosomes and
  • its control of cell growth, and

“it should lead to a better understanding and potential treatment of diseases such as cancer.”

Coordination of the transcriptome and metabolome

Coordination of the transcriptome and metabolome

the potential advantages conferred by distal-site protein synthesis

the potential advantages conferred by distal-site protein synthesis

Other authors of the paper were UC San Diego biologists Yuan-Liang Wang, Sascha Duttke and George Kassavetis, and Kai Chen, Jeff Johnston, and Julia Zeitlinger of the Stowers Institute for Medical Research in Kansas City, Missouri. Their research was supported by two grants from the National Institutes of Health (1DP2OD004561-01 and R01 GM041249).

Turning Off a Powerful Cancer Protein

Scientists have discovered how to shut down a master regulatory transcription factor that is

  • key to the survival of a majority of aggressive lymphomas,
  • which arise from the B cells of the immune system.

The protein, Bcl6, has long been considered too complex to target with a drug since it is also crucial

  • to the healthy functioning of many immune cells in the body, not just B cells gone bad.

The researchers at Weill Cornell Medical College report that it is possible

  • to shut down Bcl6 in diffuse large B-cell lymphoma (DLBCL)
  • while not affecting its vital function in T cells and macrophages
  • that are needed to support a healthy immune system.

If Bcl6 is completely inhibited, patients might suffer from systemic inflammation and atherosclerosis. The team conducted this new study to help clarify possible risks, as well as to understand

  • how Bcl6 controls the various aspects of the immune system.

The findings in this study were inspired from

  • preclinical testing of two Bcl6-targeting agents that Dr. Melnick and his Weill Cornell colleagues have developed
  • to treat DLBCLs.

These experimental drugs are

  • RI-BPI, a peptide mimic, and
  • the small molecule agent 79-6.

“This means the drugs we have developed against Bcl6 are more likely to be

  • significantly less toxic and safer for patients with this cancer than we realized,”

says Ari Melnick, M.D., professor of hematology/oncology and a hematologist-oncologist at NewYork-Presbyterian Hospital/Weill Cornell Medical Center.

Dr. Melnick says the discovery that

  • a master regulatory transcription factor can be targeted
  • offers implications beyond just treating DLBCL.

Recent studies from Dr. Melnick and others have revealed that

  • Bcl6 plays a key role in the most aggressive forms of acute leukemia, as well as certain solid tumors.

Bcl6 can control the type of immune cell that develops in the bone marrow—playing many roles

  • in the development of B cells, T cells, macrophages, and other cells—including a primary and essential role in
  • enabling B-cells to generate specific antibodies against pathogens.

According to Dr. Melnick, “When cells lose control of Bcl6,

  • lymphomas develop in the immune system.

Lymphomas are ‘addicted’ to Bcl6, and therefore

  • Bcl6 inhibitors powerfully and quickly destroy lymphoma cells,” .

The big surprise in the current study is that rather than functioning as a single molecular machine,

  • Bcl6 functions like a Swiss Army knife,
  • using different tools to control different cell types.

This multifunction paradigm could represent a general model for the functioning of other master regulatory transcription factors.

“In this analogy, the Swiss Army knife, or transcription factor, keeps most of its tools folded,

  • opening only the one it needs in any given cell type,”

He makes the following analogy:

  • “For B cells, it might open and use the knife tool;
  • for T cells, the cork screw;
  • for macrophages, the scissors.”

“this means that you only need to prevent the master regulator from using certain tools to treat cancer. You don’t need to eliminate the whole knife,” . “In fact, we show that taking out the whole knife is harmful since

  • the transcription factor has many other vital functions that other cells in the body need.”

Prior to these study results, it was not known that a master regulator could separate its functions so precisely. Researchers hope this will be a major benefit to the treatment of DLBCL and perhaps other disorders that are influenced by Bcl6 and other master regulatory transcription factors.

The study is published in the journal Nature Immunology, in a paper titled “Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms”.

Part 3. Neuroscience

Vesicles influence function of nerve cells 
Oct, 06 2014        source: http://feeds.sciencedaily.com

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Neurons (blue) which have absorbed exosomes (green) have increased levels of the enzyme catalase (red), which helps protect them against peroxides.

Tiny vesicles containing protective substances

  • which they transmit to nerve cells apparently
  • play an important role in the functioning of neurons.

As cell biologists at Johannes Gutenberg University Mainz (JGU) have discovered,

  • nerve cells can enlist the aid of mini-vesicles of neighboring glial cells
  • to defend themselves against stress and other potentially detrimental factors.

These vesicles, called exosomes, appear to stimulate the neurons on various levels:

  • they influence electrical stimulus conduction,
  • biochemical signal transfer, and
  • gene regulation.

Exosomes are thus multifunctional signal emitters

  • that can have a significant effect in the brain.
Exosome

Exosome

The researchers in Mainz already observed in a previous study that

  • oligodendrocytes release exosomes on exposure to neuronal stimuli.
  • these are absorbed by the neurons and improve neuronal stress tolerance.

Oligodendrocytes, a type of glial cell, form an

  • insulating myelin sheath around the axons of neurons.

The exosomes transport protective proteins such as

  • heat shock proteins,
  • glycolytic enzymes, and
  • enzymes that reduce oxidative stress from one cell type to another,
  • but also transmit genetic information in the form of ribonucleic acids.

“As we have now discovered in cell cultures, exosomes seem to have a whole range of functions,” explained Dr. Eva-Maria Krmer-Albers. By means of their transmission activity, the small bubbles that are the vesicles

  • not only promote electrical activity in the nerve cells, but also
  • influence them on the biochemical and gene regulatory level.

“The extent of activities of the exosomes is impressive,” added Krmer-Albers. The researchers hope that the understanding of these processes will contribute to the development of new strategies for the treatment of neuronal diseases. Their next aim is to uncover how vesicles actually function in the brains of living organisms.

http://labroots.com/user/news/article/id/217438/title/vesicles-influence-function-of-nerve-cells

The above story is based on materials provided by Universitt Mainz.

Universitt Mainz. “Vesicles influence function of nerve cells.” ScienceDaily. ScienceDaily, 6 October 2014. www.sciencedaily.com/releases/2014/10/141006174214.htm

Neuroscientists use snail research to help explain “chemo brain”

10/08/2014
It is estimated that as many as half of patients taking cancer drugs experience a decrease in mental sharpness. While there have been many theories, what causes “chemo brain” has eluded scientists.

In an effort to solve this mystery, neuroscientists at The University of Texas Health Science Center at Houston (UTHealth) conducted an experiment in an animal memory model and their results point to a possible explanation. Findings appeared in The Journal of Neuroscience.

In the study involving a sea snail that shares many of the same memory mechanisms as humans and a drug used to treat a variety of cancers, the scientists identified

  • memory mechanisms blocked by the drug.

Then, they were able to counteract or

  • unblock the mechanisms by administering another agent.

“Our research has implications in the care of people given to cognitive deficits following drug treatment for cancer,” said John H. “Jack” Byrne, Ph.D., senior author, holder of the June and Virgil Waggoner Chair and Chairman of the Department of Neurobiology and Anatomy at the UTHealth Medical School. “There is no satisfactory treatment at this time.”

Byrne’s laboratory is known for its use of a large snail called Aplysia californica to further the understanding of the biochemical signaling among nerve cells (neurons).  The snails have large neurons that relay information much like those in humans.

When Byrne’s team compared cell cultures taken from normal snails to

  • those administered a dose of a cancer drug called doxorubicin,

the investigators pinpointed a neuronal pathway

  • that was no longer passing along information properly.

With the aid of an experimental drug,

  • the scientists were able to reopen the pathway.

Unfortunately, this drug would not be appropriate for humans, Byrne said. “We want to identify other drugs that can rescue these memory mechanisms,” he added.

According the American Cancer Society, some of the distressing mental changes cancer patients experience may last a short time or go on for years.

Byrne’s UT Health research team includes co-lead authors Rong-Yu Liu, Ph.D., and Yili Zhang, Ph.D., as well as Brittany Coughlin and Leonard J. Cleary, Ph.D. All are affiliated with the W.M. Keck Center for the Neurobiology of Learning and Memory.

Byrne and Cleary also are on the faculty of The University of Texas Graduate School of Biomedical Sciences at Houston. Coughlin is a student at the school, which is jointly operated by UT Health and The University of Texas MD Anderson Cancer Center.

The study titled “Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase” received support from National Institutes of Health grant (NS019895) and the Zilkha Family Discovery Fellowship.

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Source: Univ. of Texas Health Science Center at Houston

http://www.rdmag.com/news/2014/10/neuroscientists-use-snail-research-help-explain-E2_9_Cchemo-brain

Doxorubicin Attenuates Serotonin-Induced Long-Term Synaptic Facilitation by Phosphorylation of p38 Mitogen-Activated Protein Kinase

Rong-Yu Liu*,  Yili Zhang*,  Brittany L. Coughlin,  Leonard J. Cleary, and  John H. Byrne   +Show Affiliations
The Journal of Neuroscience, 1 Oct 2014, 34(40): 13289-13300;
http://dx.doi.org:/10.1523/JNEUROSCI.0538-14.2014

Doxorubicin (DOX) is an anthracycline used widely for cancer chemotherapy. Its primary mode of action appears to be

  • topoisomerase II inhibition, DNA cleavage, and free radical generation.

However, in non-neuronal cells, DOX also inhibits the expression of

  • dual-specificity phosphatases (also referred to as MAPK phosphatases) and thereby
  1. inhibits the dephosphorylation of extracellular signal-regulated kinase (ERK) and
  2. p38 mitogen-activated protein kinase (p38 MAPK),
  3. two MAPK isoforms important for long-term memory (LTM) formation.

Activation of these kinases by DOX in neurons, if present,

  • could have secondary effects on cognitive functions, such as learning and memory.

The present study used cultures of rat cortical neurons and sensory neurons (SNs) of Aplysia

  • to examine the effects of DOX on levels of phosphorylated ERK (pERK) and
  • phosphorylated p38 (p-p38) MAPK.

In addition, Aplysia neurons were used to examine the effects of DOX on

  • long-term enhanced excitability, long-term synaptic facilitation (LTF), and
  • long-term synaptic depression (LTD).

DOX treatment led to elevated levels of

  • pERK and p-p38 MAPK in SNs and cortical neurons.

In addition, it increased phosphorylation of

  • the downstream transcriptional repressor cAMP response element-binding protein 2 in SNs.

DOX treatment blocked serotonin-induced LTF and enhanced LTD induced by the neuropeptide Phe-Met-Arg-Phe-NH2. The block of LTF appeared to be attributable to

  • overriding inhibitory effects of p-p38 MAPK, because
  • LTF was rescued in the presence of an inhibitor of p38 MAPK
    (SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole]) .

These results suggest that acute application of DOX might impair the formation of LTM via the p38 MAPK pathway.
Terms: Aplysia chemotherapy ERK  p38 MAPK serotonin synaptic plasticity

Technology that controls brain cells with radio waves earns early BRAIN grant

10/08/2014

bright spots = cells with increased calcium after treatment with radio waves,  allows neurons to fire

bright spots = cells with increased calcium after treatment with radio waves, allows neurons to fire

BRAIN control: The new technology uses radio waves to activate or silence cells remotely. The bright spots above represent cells with increased calcium after treatment with radio waves, a change that would allow neurons to fire.

A proposal to develop a new way to

  • remotely control brain cells

from Sarah Stanley, a research associate in Rockefeller University’s Laboratory of Molecular Genetics, headed by Jeffrey M. Friedman, is

  • among the first to receive funding from U.S. President Barack Obama’s BRAIN initiative.

The project will make use of a technique called

  • radiogenetics that combines the use of radio waves or magnetic fields with
  • nanoparticles to turn neurons on or off.

The National Institutes of Health is one of four federal agencies involved in the BRAIN (Brain Research through Advancing Innovative Neurotechnologies) initiative. Following in the ambitious footsteps of the Human Genome Project, the BRAIN initiative seeks

  • to create a dynamic map of the brain in action,

a goal that requires the development of new technologies. The BRAIN initiative working group, which outlined the broad scope of the ambitious project, was co-chaired by Rockefeller’s Cori Bargmann, head of the Laboratory of Neural Circuits and Behavior.

Stanley’s grant, for $1.26 million over three years, is one of 58 projects to get BRAIN grants, the NIH announced. The NIH’s plan for its part of this national project, which has been pitched as “America’s next moonshot,” calls for $4.5 billion in federal funds over 12 years.

The technology Stanley is developing would

  • enable researchers to manipulate the activity of neurons, as well as other cell types,
  • in freely moving animals in order to better understand what these cells do.

Other techniques for controlling selected groups of neurons exist, but her new nanoparticle-based technique has a

  • unique combination of features that may enable new types of experimentation.
  • it would allow researchers to rapidly activate or silence neurons within a small area of the brain or
  • dispersed across a larger region, including those in difficult-to-access locations.

Stanley also plans to explore the potential this method has for use treating patients.

“Francis Collins, director of the NIH, has discussed

  • the need for studying the circuitry of the brain,
  • which is formed by interconnected neurons.

Our remote-control technology may provide a tool with which researchers can ask new questions about the roles of complex circuits in regulating behavior,” Stanley says.
Rockefeller University’s Laboratory of Molecular Genetics
Source: Rockefeller Univ.

Part 4.  Cancer

Two Proteins Found to Block Cancer Metastasis

Why do some cancers spread while others don’t? Scientists have now demonstrated that

  • metastatic incompetent cancers actually “poison the soil”
  • by generating a micro-environment that blocks cancer cells
  • from settling and growing in distant organs.

The “seed and the soil” hypothesis proposed by Stephen Paget in 1889 is now widely accepted to explain how

  • cancer cells (seeds) are able to generate fertile soil (the micro-environment)
  • in distant organs that promotes cancer’s spread.

However, this concept had not explained why some tumors do not spread or metastasize.

The researchers, from Weill Cornell Medical College, found that

  • two key proteins involved in this process work by
  • dramatically suppressing cancer’s spread.

The study offers hope that a drug based on these

  • potentially therapeutic proteins, prosaposin and Thrombospondin 1 (Tsp-1),

might help keep human cancer at bay and from metastasizing.

Scientists don’t understand why some tumors wouldn’t “want” to spread. It goes against their “job description,” says the study’s senior investigator, Vivek Mittal, Ph.D., an associate professor of cell and developmental biology in cardiothoracic surgery and director of the Neuberger Berman Foundation Lung Cancer Laboratory at Weill Cornell Medical College. He theorizes that metastasis occurs when

  • the barriers that the body throws up to protect itself against cancer fail.

But there are some tumors in which some of the barriers may still be intact. “So that suggests

  • those primary tumors will continue to grow, but that
  • an innate protective barrier still exists that prevents them from spreading and invading other organs,”

The researchers found that, like typical tumors,

  • metastasis-incompetent tumors also send out signaling molecules
  • that establish what is known as the “premetastatic niche” in distant organs.

These niches composed of bone marrow cells and various growth factors have been described previously by others including Dr. Mittal as the fertile “soil” that the disseminated cancer cell “seeds” grow in.

Weill Cornell’s Raúl Catena, Ph.D., a postdoctoral fellow in Dr. Mittal’s laboratory, found an important difference between the tumor types. Metastatic-incompetent tumors

  • systemically increased expression of Tsp-1, a molecule known to fight cancer growth.
  • increased Tsp-1 production was found specifically in the bone marrow myeloid cells
  • that comprise the metastatic niche.

These results were striking, because for the first time Dr. Mittal says

  • the bone marrow-derived myeloid cells were implicated as
  • the main producers of Tsp-1,.

In addition, Weill Cornell and Harvard researchers found that

  • prosaposin secreted predominantly by the metastatic-incompetent tumors
  • increased expression of Tsp-1 in the premetastatic lungs.

Thus, Dr. Mittal posits that prosaposin works in combination with Tsp-1

  • to convert pro-metastatic bone marrow myeloid cells in the niche
  • into cells that are not hospitable to cancer cells that spread from a primary tumor.
  • “The very same myeloid cells in the niche that we know can promote metastasis
  • can also be induced under the command of the metastatic incompetent primary tumor to inhibit metastasis,”

The research team found that

  • the Tsp-1–inducing activity of prosaposin
  • was contained in only a 5-amino acid peptide region of the protein, and
  • this peptide alone induced Tsp-1 in the bone marrow cells and
  • effectively suppressed metastatic spread in the lungs
  • in mouse models of breast and prostate cancer.

This 5-amino acid peptide with Tsp-1–inducing activity

  • has the potential to be used as a therapeutic agent against metastatic cancer,

The scientists have begun to test prosaposin in other tumor types or metastatic sites.

Dr. Mittal says that “The clinical implications of the study are:

  • “Not only is it theoretically possible to design a prosaposin-based drug or drugs
  • that induce Tsp-1 to block cancer spread, but
  • you could potentially create noninvasive prognostic tests
  • to predict whether a cancer will metastasize.”

The study was reported in the April 30 issue of Cancer Discovery, in a paper titled “Bone Marrow-Derived Gr1+ Cells Can Generate a Metastasis-Resistant Microenvironment Via Induced Secretion of Thrombospondin-1”.

Disabling Enzyme Cripples Tumors, Cancer Cells

First Step of Metastasis

First Step of Metastasis

Published: Sep 05, 2013  http://www.technologynetworks.com/Metabolomics/news.aspx?id=157138

Knocking out a single enzyme dramatically cripples the ability of aggressive cancer cells to spread and grow tumors.

The paper, published in the journal Proceedings of the National Academy of Sciences, sheds new light on the importance of lipids, a group of molecules that includes fatty acids and cholesterol, in the development of cancer.

Researchers have long known that cancer cells metabolize lipids differently than normal cells. Levels of ether lipids – a class of lipids that are harder to break down – are particularly elevated in highly malignant tumors.

“Cancer cells make and use a lot of fat and lipids, and that makes sense because cancer cells divide and proliferate at an accelerated rate, and to do that,

  • they need lipids, which make up the membranes of the cell,”

said study principal investigator Daniel Nomura, assistant professor in UC Berkeley’s Department of Nutritional Sciences and Toxicology. “Lipids have a variety of uses for cellular structure, but what we’re showing with our study is that

  • lipids can send signals that fuel cancer growth.”

In the study, Nomura and his team tested the effects of reducing ether lipids on human skin cancer cells and primary breast tumors. They targeted an enzyme,

  • alkylglycerone phosphate synthase, or AGPS,
  • known to be critical to the formation of ether lipids.

The researchers confirmed that

  1. AGPS expression increased when normal cells turned cancerous.
  2. inactivating AGPS substantially reduced the aggressiveness of the cancer cells.

“The cancer cells were less able to move and invade,” said Nomura.

The researchers also compared the impact of

  • disabling the AGPS enzyme in mice that had been injected with cancer cells.

Nomura. observes -“Among the mice that had the AGPS enzyme inactivated,

  • the tumors were nonexistent,”

“The mice that did not have this enzyme

  • disabled rapidly developed tumors.”

The researchers determined that

  • inhibiting AGPS expression depleted the cancer cells of ether lipids.
  • AGPS altered levels of other types of lipids important to the ability of the cancer cells to survive and spread, including
    • prostaglandins and acyl phospholipids.

“What makes AGPS stand out as a treatment target is that the enzyme seems to simultaneously

  • regulate multiple aspects of lipid metabolism
  • important for tumor growth and malignancy.”

Future steps include the

  • development of AGPS inhibitors for use in cancer therapy,

“This study sheds considerable light on the important role that AGPS plays in ether lipid metabolism in cancer cells, and it suggests that

  • inhibitors of this enzyme could impair tumor formation,”

said Benjamin Cravatt, Professor and Chair of Chemical Physiology at The Scripps Research Institute, who is not part of the UC.

Agilent Technologies Thought Leader Award Supports Translational Research Program
Published: Mon, March 04, 2013

The award will support Dr DePinho’s research into

  • metabolic reprogramming in the earliest stages of cancer.

Agilent Technologies Inc. announces that Dr. Ronald A. DePinho, a world-renowned oncologist and researcher, has received an Agilent Thought Leader Award.

DePinho is president of the University of Texas MD Anderson Cancer Center. DePinho and his team hope to discover and characterize

  • alterations in metabolic flux during tumor initiation and maintenance, and to identify biomarkers for early detection of pancreatic cancer together with
  • novel therapeutic targets.

Researchers on his team will work with scientists from the university’s newly formed Institute of Applied Cancer Sciences.

The Agilent Thought Leader Award provides funds to support personnel as well as a state-of-the-art Agilent 6550 iFunnel Q-TOF LC/MS system.

“I am extremely pleased to receive this award for metabolomics research, as the survival rates for pancreatic cancer have not significantly improved over the past 20 years,” DePinho said. “This technology will allow us to

  • rapidly identify new targets that drive the formation, progression and maintenance of pancreatic cancer.

Discoveries from this research will also lead to

  • the development of effective early detection biomarkers and novel therapeutic interventions.”

“We are proud to support Dr. DePinho’s exciting translational research program, which will make use of

  • metabolomics and integrated biology workflows and solutions in biomarker discovery,”

said Patrick Kaltenbach, Agilent vice president, general manager of the Liquid Phase Division, and the executive sponsor of this award.

The Agilent Thought Leader Program promotes fundamental scientific advances by support of influential thought leaders in the life sciences and chemical analysis fields.

The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis

Ping Xie, Minghua Zhang, Shan He, Kefeng Lu, Yuhan Chen, Guichun Xing, et al.
Nature Communications
  2014; 5(3733).  http://dx.doi.org:/10.1038/ncomms4733

Neddylation, the covalent attachment of ubiquitin-like protein Nedd8, of the Cullin-RING E3 ligase family

  • regulates their ubiquitylation activity.

However, regulation of HECT ligases by neddylation has not been reported to date. Here we show that

  • the C2-WW-HECT ligase Smurf1 is activated by neddylation.

Smurf1 physically interacts with

  1. Nedd8 and Ubc12,
  2. forms a Nedd8-thioester intermediate, and then
  3. catalyses its own neddylation on multiple lysine residues.

Intriguingly, this autoneddylation needs

  • an active site at C426 in the HECT N-lobe.

Neddylation of Smurf1 potently enhances

  • ubiquitin E2 recruitment and
  • augments the ubiquitin ligase activity of Smurf1.

The regulatory role of neddylation

  • is conserved in human Smurf1 and yeast Rsp5.

Furthermore, in human colorectal cancers,

  • the elevated expression of Smurf1, Nedd8, NAE1 and Ubc12
  • correlates with cancer progression and poor prognosis.

These findings provide evidence that

  • neddylation is important in HECT ubiquitin ligase activation and
  • shed new light on the tumour-promoting role of Smurf1.
 Swinging domains in HECT E3

Swinging domains in HECT E3

Subject terms: Biological sciences Cancer Cell biology

Figure 1: Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

Smurf1 expression is elevated in colorectal cancer tissues.

(a) Smurf1 expression scores are shown as box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th percentiles, respectively; and the vertical bars representing the ra

Figure 2: Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer.

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

Positive correlation of Smurf1 expression with Nedd8 and its interacting enzymes in colorectal cancer

(a) Representative images from immunohistochemical staining of Smurf1, Ubc12, NAE1 and Nedd8 in the same colorectal cancer tumour. Scale bars, 100 μm. (bd) The expression scores of Nedd8 (b, n=283 ), NAE1 (c, n=281) and Ubc12 (d, n=19…

Figure 3: Smurf1 interacts with Ubc12.

Smurf1 interacts with Ubc12

Smurf1 interacts with Ubc12

(a) GST pull-down assay of Smurf1 with Ubc12. Both input and pull-down samples were subjected to immunoblotting with anti-His and anti-GST antibodies. Smurf1 interacted with Ubc12 and UbcH5c, but not with Ubc9. (b) Mapping the regions…

Figure 4: Nedd8 is attached to Smurf1through C426-catalysed autoneddylation.

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

Nedd8 is attached to Smurf1through C426-catalysed autoneddylation

(a) Covalent neddylation of Smurf1 in vitro.Purified His-Smurf1-WT or C699A proteins were incubated with Nedd8 and Nedd8-E1/E2. Reactions were performed as described in the Methods section. Samples were analysed by western blotting wi…

Figure 5: Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

Neddylation of Smurf1 activates its ubiquitin ligase activity.

(a) In vivo Smurf1 ubiquitylation assay. Nedd8 was co-expressed with Smurf1 WT or C699A in HCT116 cells (left panels). Twenty-four hours post transfection, cells were treated with MG132 (20 μM, 8 h). HCT116 cells were transfected with…

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The deubiquitylase USP33 discriminates between RALB functions in autophagy and innate immune response

M Simicek, S Lievens, M Laga, D Guzenko, VN. Aushev, et al.
Nature Cell Biology 2013; 15, 1220–1230    http://dx.doi.org:/10.1038/ncb2847

The RAS-like GTPase RALB mediates cellular responses to nutrient availability or viral infection by respectively

  • engaging two components of the exocyst complex, EXO84 and SEC5.
  1. RALB employs SEC5 to trigger innate immunity signalling, whereas
  2. RALB–EXO84 interaction induces autophagocytosis.

How this differential interaction is achieved molecularly by the RAL GTPase remains unknown.

We found that whereas GTP binding

  • turns on RALB activity,

ubiquitylation of RALB at Lys 47

  • tunes its activity towards a particular effector.

Specifically, ubiquitylation at Lys 47

  • sterically inhibits RALB binding to EXO84, while
  • facilitating its interaction with SEC5.

Double-stranded RNA promotes

  • RALB ubiquitylation and
  • SEC5–TBK1 complex formation.

In contrast, nutrient starvation

  • induces RALB deubiquitylation
  • by accumulation and relocalization of the deubiquitylase USP33
  • to RALB-positive vesicles.

Deubiquitylated RALB

  • promotes the assembly of the RALB–EXO84–beclin-1 complexes
  • driving autophagosome formation. Thus,
  • ubiquitylation within the effector-binding domain
  • provides the switch for the dual functions of RALB in
    • autophagy and innate immune responses.

Part 5. Metabolic Syndrome

Single Enzyme is Necessary for Development of Diabetes

Published: Aug 20, 2014 http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169416

12-LO enzyme promotes the obesity-induced oxidative stress in the pancreatic cells.

An enzyme called 12-LO promotes the obesity-induced oxidative stress in the pancreatic cells that leads

  • to pre-diabetes, and diabetes.

12-LO’s enzymatic action is the last step in

  • the production of certain small molecules that harm the cell,

according to a team from Indiana University School of Medicine, Indianapolis.

The findings will enable the development of drugs that can interfere with this enzyme, preventing or even reversing diabetes. The research is published ahead of print in the journal Molecular and Cellular Biology.

In earlier studies, these researchers and their collaborators at Eastern Virginia Medical School showed that

  • 12-LO (which stands for 12-lipoxygenase) is present in these cells
  • only in people who become overweight.

The harmful small molecules resulting from 12-LO’s enzymatic action are known as HETEs, short for hydroxyeicosatetraenoic acid.

  1. HETEs harm the mitochondria, which then
  2. fail to produce sufficient energy to enable
  3. the pancreatic cells to manufacture the necessary quantities of insulin.

For the study, the investigators genetically engineered mice that

  • lacked the gene for 12-LO exclusively in their pancreas cells.

Mice were either fed a low-fat or high-fat diet.

Both the control mice and the knockout mice on the high fat diet

  • developed obesity and insulin resistance.

The investigators also examined the pancreatic beta cells of both knockout and control mice, using both microscopic studies and molecular analysis. Those from the knockout mice were intact and healthy, while

  • those from the control mice showed oxidative damage,
  • demonstrating that 12-LO and the resulting HETEs
  • caused the beta cell failure.

Mirmira notes that fatty diet used in the study was the Western Diet, which comprises mostly saturated-“bad”-fats. Based partly on a recent study of related metabolic pathways, he says that

  • the unsaturated and mono-unsaturated fats-which comprise most fats in the healthy,
  • relatively high fat Mediterranean diet-are unlikely to have the same effects.

“Our research is the first to show that 12-LO in the beta cell

  • is the culprit in the development of pre-diabetes, following high fat diets,” says Mirmira.

“Our work also lends important credence to the notion that

  • the beta cell is the primary defective cell in virtually all forms of diabetes and pre-diabetes.”

A New Player in Lipid Metabolism Discovered

Published: Aug18, 2014  http://www.technologynetworks.com/Metabolomics/news.aspx?ID=169356

Specially engineered mice gained no weight, and normal counterparts became obese

  • on the same high-fat, obesity-inducing Western diet.

Specially engineered mice that lacked a particular gene did not gain weight

  • when fed a typical high-fat, obesity-inducing Western diet.

Yet, these mice ate the same amount as their normal counterparts that became obese.

The mice were engineered with fat cells that lacked a gene called SEL1L,

  • known to be involved in the clearance of mis-folded proteins
  • in the cell’s protein making machinery called the endoplasmic reticulum (ER).

When mis-folded proteins are not cleared but accumulate,

  • they destroy the cell and contribute to such diseases as
  1. mad cow disease,
  2. Type 1 diabetes and
  3. cystic fibrosis.

“The million-dollar question is why don’t these mice gain weight? Is this related to its inability to clear mis-folded proteins in the ER?” said Ling Qi, associate professor of molecular and biochemical nutrition and senior author of the study published online July 24 in Cell Metabolism. Haibo Sha, a research associate in Qi’s lab, is the paper’s lead author.

Interestingly, the experimental mice developed a host of other problems, including

  • postprandial hypertriglyceridemia,
  • and fatty livers.

“Although we are yet to find out whether these conditions contribute to the lean phenotype, we found that

  • there was a lipid partitioning defect in the mice lacking SEL1L in fat cells,
  • where fat cells cannot store fat [lipids], and consequently
  • fat goes to the liver.

During the investigation of possible underlying mechanisms, we discovered

  • a novel function for SEL1L as a regulator of lipid metabolism,” said Qi.

Sha said “We were very excited to find that

  • SEL1L is required for the intracellular trafficking of
  • lipoprotein lipase (LPL), acting as a chaperone,” .

and added that “Using several tissue-specific knockout mouse models,

  • we showed that this is a general phenomenon,”

Without LPL, lipids remain in the circulation;

  • fat and muscle cells cannot absorb fat molecules for storage and energy combustion,

People with LPL mutations develop

  • postprandial hypertriglyceridemia similar to
  • conditions found in fat cell-specific SEL1L-deficient mice, said Qi.

Future work will investigate the

  • role of SEL1L in human patients carrying LPL mutations and
  • determine why fat cell-specific SEL1L-deficient mice remain lean under Western diets, said Sha.

Co-authors include researchers from Cedars-Sinai Medical Center in Los Angeles; Wageningen University in the Netherlands; Georgia State University; University of California, Los Angeles; and the Medical College of Soochow University in China.

The study was funded by the U.S. National Institutes of Health, the Netherlands Organization for Health Research and Development National Institutes of Health, the Cedars-Sinai Medical Center, Chinese National Science Foundation, the American Diabetes Association, Cornell’s Center for Vertebrate Genomics and the Howard Hughes Medical Institute.

Part 6. Biomarkers

Biomarkers Take Center Stage

Josh P. Roberts
GEN May 1, 2013 (Vol. 33, No. 9)  http://www.genengnews.com/

While work with biomarkers continues to grow, scientists are also grappling with research-related bottlenecks, such as

  1. affinity reagent development,
  2. platform reproducibility, and
  3. sensitivity.

Biomarkers by definition indicate some state or process that generally occurs

  • at a spatial or temporal distance from the marker itself, and

it would not be an exaggeration to say that biomedicine has become infatuated with them:

  1. where to find them,
  2. when they may appear,
  3. what form they may take, and
  4. how they can be used to diagnose a condition or
  5. predict whether a therapy may be successful.

Biomarkers are on the agenda of many if not most industry gatherings, and in cases such as Oxford Global’s recent “Biomarker Congress” and the GTC “Biomarker Summit”, they hold the naming rights. There, some basic principles were built upon, amended, and sometimes challenged.

In oncology, for example, biomarker discovery is often predicated on the premise that

  • proteins shed from a tumor will traverse to and persist in, and be detectable in, the circulation.

By quantifying these proteins—singularly or as part of a larger “signature”—the hope is

  1. to garner information about the molecular characteristics of the cancer
  2. that will help with cancer detection and
  3. personalization of the treatment strategy.

Yet this approach has not yet turned into the panacea that was hoped for. Bottlenecks exist in

  • affinity reagent development,
  • platform reproducibility, and
  • sensitivity.

There is also a dearth of understanding of some of the

  • fundamental principles of biomarker biology that we need to know the answers to,

said Parag Mallick, Ph.D., whose lab at Stanford University is “working on trying to understand where biomarkers come from.”

There are dogmas saying that

  • circulating biomarkers come solely from secreted proteins.

But Dr. Mallick’s studies indicate that fully

  • 50% of circulating proteins may come from intracellular sources or
  • proteins that are annotated as such.

“We don’t understand the processes governing

  • which tumor-derived proteins end up in the blood.”

Other questions include “how does the size of a tumor affect how much of a given protein will be in the blood?”—perhaps

  • the tumor is necrotic at the center, or
  • it’s hypervascular or hypovascular.

He points out “The problem is that these are highly nonlinear processes at work, and

  • there is a large number of factors that might affect the answer to that question,” .

Their research focuses on using

  1. mass spectrometry and
  2. computational analysis
  • to characterize the biophysical properties of the circulating proteome, and
  • relate these to measurements made of the tumor itself.

Furthermore, he said – “We’ve observed that the proteins that are likely to

  • first show up and persist in the circulation, ..
  • are more stable than proteins that don’t,”
  • “we can quantify how significant the effect is.”

The goal is ultimately to be able to

  1. build rigorous, formal mathematical models that will allow something measured in the blood
  2. to be tied back to the molecular biology taking place in the tumor.

And conversely, to use those models

  • to predict from a tumor what will be found in the circulation.

“Ultimately, the models will allow you to connect the dots between

  • what you measure in the blood and the biology of the tumor.”

Bound for Affinity Arrays

Affinity reagents are the main tools for large-scale protein biomarker discovery. And while this has tended to mean antibodies (or their derivatives), other affinity reagents are demanding a place in the toolbox.

Affimers, a type of affinity reagent being developed by Avacta, consist of

  1. a biologically inert, biophysically stable protein scaffold
  2. containing three variable regions into which
  3. distinct peptides are inserted.

The resulting three-dimensional surface formed by these peptides

  • interacts and binds to proteins and other molecules in solution,
  • much like the antigen-binding site of antibodies.

Unlike antibodies, Affimers are relatively small (13 KDa),

  • non-post-translationally modified proteins
  • that can readily be expressed in bacterial culture.

They may be made to bind surfaces through unique residues

  • engineered onto the opposite face of the Affimer,
  • allowing the binding site to be exposed to the target in solution.

“We don’t seem to see in what we’ve done so far

  • any real loss of activity or functionality of Affimers when bound to surfaces—

they’re very robust,” said CEO Alastair Smith, Ph.D.

Avacta is taking advantage of this stability and its large libraries of Affimers to develop

  • very large affinity microarrays for
  • drug and biomarker discovery.

To date they have printed arrays with around 20–25,000 features, and Dr. Smith is “sure that we can get toward about 50,000 on a slide,” he said. “There’s no real impediment to us doing that other than us expressing the proteins and getting on with it.”

Customers will be provided with these large, complex “naïve” discovery arrays, readable with standard equipment. The plan is for the company to then “support our customers by providing smaller arrays with

  • the Affimers that are binding targets of interest to them,” Dr. Smith foretold.

And since the intellectual property rights are unencumbered,

  • Affimers in those arrays can be licensed to the end users
  • to develop diagnostics that can be validated as time goes on.

Around 20,000-Affimer discovery arrays were recently tested by collaborator Professor Ann Morgan of the University of Leeds with pools of unfractionated serum from patients with symptoms of inflammatory disease. The arrays

  • “rediscovered” elevated C-reactive protein (CRP, the clinical gold standard marker)
  • as well as uncovered an additional 22 candidate biomarkers.
  • other candidates combined with CRP, appear able to distinguish between different diseases such as
  1. rheumatoid arthritis,
  2. psoriatic arthritis,
  3. SLE, or
  4. giant cell arteritis.

Epigenetic Biomarkers

Methylation of adenine

Sometimes biomarkers are used not to find disease but

  • to distinguish healthy human cell types, with
  •  examples being found in flow cytometry and immunohistochemistry.

These widespread applications, however, are difficult to standardize, being

  • subject to arbitrary or subjective gating protocols and other imprecise criteria.

Epiontis instead uses an epigenetic approach. “What we need is a unique marker that is

  • demethylated only in one cell type and
  • methylated in all the other cell types,”

Each cell of the right cell type will have

  • two demethylated copies of a certain gene locus,
  • allowing them to be enumerated by quantitative PCR.

The biggest challenge is finding that unique epigenetic marker. To do so they look through the literature for proteins and genes described as playing a role in the cell type’s biology, and then

  • look at the methylation patterns to see if one can be used as a marker,

They also “use customized Affymetrix chips to look at the

  • differential epigenetic status of different cell types on a genomewide scale.”

explained CBO and founder Ulrich Hoffmueller, Ph.D.

The company currently has a panel of 12 assays for 12 immune cell types. Among these is an assay for

  • regulatory T (Treg) cells that queries the Foxp3 gene—which is uniquely demethylated in Treg
  • even though it is transiently expressed in activated T cells of other subtypes.

Also assayed are Th17 cells, difficult to detect by flow cytometry because

  • “the cells have to be stimulated in vitro,” he pointed out.

Developing New Assays for Cancer Biomarkers

Researchers at Myriad RBM and the Cancer Prevention Research Institute of Texas are collaborating to develop

  • new assays for cancer biomarkers on the Myriad RBM Multi-Analyte Profile (MAP) platform.

The release of OncologyMAP 2.0 expanded Myriad RBM’s biomarker menu to over 250 analytes, which can be measured from a small single sample, according to the company. Using this menu, L. Stephen et al., published a poster, “Analysis of Protein Biomarkers in Prostate and Colorectal Tumor Lysates,” which showed the results of

  • a survey of proteins relevant to colorectal (CRC) and prostate (PC) tumors
  • to identify potential proteins of interest for cancer research.

The study looked at CRC and PC tumor lysates and found that 102 of the 115 proteins showed levels above the lower limit of quantification.

  • Four markers were significantly higher in PC and 10 were greater in CRC.

For most of the analytes, duplicate sections of the tumor were similar, although some analytes did show differences. In four of the CRC analytes, tumor number four showed differences for CEA and tumor number 2 for uPA.

Thirty analytes were shown to be

  • different in CRC tumor compared to its adjacent tissue.
  • Ten of the analytes were higher in adjacent tissue compared to CRC.
  • Eighteen of the markers examined demonstrated  —-

significant correlations of CRC tumor concentration to serum levels.

“This suggests.. that the Oncology MAP 2.0 platform “provides a good method for studying changes in tumor levels because many proteins can be assessed with a very small sample.”

Clinical Test Development with MALDI-ToF

While there have been many attempts to translate results from early discovery work on the serum proteome into clinical practice, few of these efforts have progressed past the discovery phase.

Matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry on unfractionated serum/plasma samples offers many practical advantages over alternative techniques, and does not require

  • a shift from discovery to development and commercialization platforms.

Biodesix claims it has been able to develop the technology into

  • a reproducible, high-throughput tool to
  • routinely measure protein abundance from serum/plasma samples.

“.. we improved data-analysis algorithms to

  • reproducibly obtain quantitative measurements of relative protein abundance from MALDI-ToF mass spectra.

Heinrich Röder, CTO points out that the MALDI-ToF measurements

  • are combined with clinical outcome data using
  • modern learning theory techniques
  • to define specific disease states
  • based on a patient’s serum protein content,”

The clinical utility of the identification of these disease states can be investigated through a retrospective analysis of differing sample sets. For example, Biodesix clinically validated its first commercialized serum proteomic test, VeriStrat®, in 85 different retrospective sample sets.

Röder adds that “It is becoming increasingly clear that

  • the patients whose serum is characterized as VeriStrat Poor show
  • consistently poor outcomes irrespective of
  1. tumor type,
  2. histology, or
  3. molecular tumor characteristics,”

MALDI-ToF mass spectrometry, in its standard implementation,

  • allows for the observation of around 100 mostly high-abundant serum proteins.

Further, “while this does not limit the usefulness of tests developed from differential expression of these proteins,

  • the discovery potential would be greatly enhanced
  • if we could probe deeper into the proteome
  • while not giving up the advantages of the MALDI-ToF approach,”

Biodesix reports that its new MALDI approach, Deep MALDI™, can perform

  • simultaneous quantitative measurement of more than 1,000 serum protein features (or peaks) from 10 µL of serum in a high-throughput manner.
  • it increases the observable signal noise ratio from a few hundred to over 50,000,
  • resulting in the observation of many lower-abundance serum proteins.

Breast cancer, a disease now considered to be a collection of many complexes of symptoms and signatures—the dominant ones are labeled Luminal A, Luminal B, Her2, and Basal— which suggests different prognose, and

  • these labels are considered too simplistic for understanding and managing a woman’s cancer.

Studies published in the past year have looked at

  1. somatic mutations,
  2. gene copy number aberrations,
  3. gene expression abnormalities,
  4. protein and miRNA expression, and
  5. DNA methylation,

coming up with a list of significantly mutated genes—hot spots—in different categories of breast cancers. Targeting these will inevitably be the focus of much coming research.

“We’ve been taking these large trials and profiling these on a variety of array or sequence platforms. We think we’ll get

  1. prognostic drivers
  2. predictive markers for taxanes and
  3. monoclonal antibodies and
  4. tamoxifen and aromatase inhibitors,”
    explained Brian Leyland-Jones, Ph.D., director of Edith Sanford Breast Cancer Research. “We will end up with 20–40 different diseases, maybe more.”

Edith Sanford Breast Cancer Research is undertaking a pilot study in collaboration with The Scripps Research Institute, using a variety of tests on 25 patients to see how the information they provide complements each other, the overall flow, and the time required to get and compile results.

Laser-captured tumor samples will be subjected to low passage whole-genome, exome, and RNA sequencing (with targeted resequencing done in parallel), and reverse-phase protein and phosphorylation arrays, with circulating nucleic acids and circulating tumor cells being queried as well. “After that we hope to do a 100- or 150-patient trial when we have some idea of the best techniques,” he said.

Dr. Leyland-Jones predicted that ultimately most tumors will be found

  • to have multiple drivers,
  • with most patients receiving a combination of two, three, or perhaps four different targeted therapies.

Reduce to Practice

According to Randox, the evidence Investigator is a sophisticated semi-automated biochip sys­tem designed for research, clinical, forensic, and veterinary applications.

Once biomarkers that may have an impact on therapy are discovered, it is not always routine to get them into clinical practice. Leaving regulatory and financial, intellectual property and cultural issues aside, developing a diagnostic based on a biomarker often requires expertise or patience that its discoverer may not possess.

Andrew Gribben is a clinical assay and development scientist at Randox Laboratories, based in Northern Ireland, U.K. The company utilizes academic and industrial collaborators together with in-house discovery platforms to identify biomarkers that are

  • augmented or diminished in a particular pathology
  • relative to appropriate control populations.

Biomarkers can be developed to be run individually or

  • combined into panels of immunoassays on its multiplex biochip array technology.

Specificity can also be gained—or lost—by the affinity of reagents in an assay. The diagnostic potential of Heart-type fatty acid binding protein (H-FABP) abundantly expressed in human myocardial cells was recognized by Jan Glatz of Maastricht University, The Netherlands, back in 1988. Levels rise quickly within 30 minutes after a myocardial infarction, peaking at 6–8 hours and return to normal within 24–30 hours. Yet at the time it was not known that H-FABP was a member of a multiprotein family, with which the polyclonal antibodies being used in development of an assay were cross-reacting, Gribben related.

Randox developed monoclonal antibodies specific to H-FABP, funded trials investigating its use alone, and multiplexed with cardiac biomarker assays, and, more than 30 years after the biomarker was identified, in 2011, released a validated assay for H-FABP as a biomarker for early detection of acute myocardial infarction.

Ultrasensitive Immunoassays for Biomarker Development

Research has shown that detection and monitoring of biomarker concentrations can provide

  • insights into disease risk and progression.

Cytokines have become attractive biomarkers and candidates

  • for targeted therapies for a number of autoimmune diseases, including rheumatoid arthritis (RA), Crohn’s disease, and psoriasis, among others.

However, due to the low-abundance of circulating cytokines, such as IL-17A, obtaining robust measurements in clinical samples has been difficult.

Singulex reports that its digital single-molecule counting technology provides

  • increased precision and detection sensitivity over traditional ELISA techniques,
  • helping to shed light on biomarker verification and validation programs.

The company’s Erenna® immunoassay system, which includes optimized immunoassays, offers LLoQ to femtogram levels per mL resolution—even in healthy populations, at an improvement of 1-3 fold over standard ELISAs or any conventional technology and with a dynamic range of up to 4-logs, according to a Singulex official, who adds that

  • this sensitivity improvement helps minimize undetectable samples that
  • could otherwise delay or derail clinical studies.

The official also explains that the Singulex solution includes an array of products and services that are being applied to a number of programs and have enabled the development of clinically relevant biomarkers, allowing translation from discovery to the clinic.

In a poster entitled “Advanced Single Molecule Detection: Accelerating Biomarker Development Utilizing Cytokines through Ultrasensitive Immunoassays,” a case study was presented of work performed by Jeff Greenberg of NYU to show how the use of the Erenna system can provide insights toward

  • improving the clinical utility of biomarkers and
  • accelerating the development of novel therapies for treating inflammatory diseases.

A panel of inflammatory biomarkers was examined in DMARD (disease modifying antirheumatic drugs)-naïve RA (rheumatoid arthritis) vs. knee OA (osteoarthritis) patient cohorts. Markers that exhibited significant differences in plasma concentrations between the two cohorts included

  • CRP, IL-6R alpha, IL-6, IL-1 RA, VEGF, TNF-RII, and IL-17A, IL-17F, and IL-17A/F.

Among the three tested isoforms of IL-17,

  • the magnitude of elevation for IL-17F in RA patients was the highest.

“Singulex provides high-resolution monitoring of baseline IL-17A concentrations that are present at low levels,” concluded the researchers. “The technology also enabled quantification of other IL-17 isoforms in RA patients, which have not been well characterized before.”

The Singulex Erenna System has also been applied to cardiovascular disease research, for which its

  • cardiac troponin I (cTnI) digital assay can be used to measure circulating
  • levels of cTnI undetectable by other commercial assays.

Recently presented data from Brigham and Women’s Hospital and the TIMI-22 study showed that

  • using the Singulex test to serially monitor cTnI helps
  • stratify risk in post-acute coronary syndrome patients and
  • can identify patients with elevated cTnI
  • who have the most to gain from intensive vs. moderate-dose statin therapy,

according to the scientists involved in the research.

The study poster, “Prognostic Performance of Serial High Sensitivity Cardiac Troponin Determination in Stable Ischemic Heart Disease: Analysis From PROVE IT-TIMI 22,” was presented at the 2013 American College of Cardiology (ACC) Annual Scientific Session & Expo by R. O’Malley et al.

Biomarkers Changing Clinical Medicine

Better Diagnosis, Prognosis, and Drug Targeting Are among Potential Benefits

  1. John Morrow Jr., Ph.D.

Researchers at EMD Chemicals are developing biomarker immunoassays

  • to monitor drug-induced toxicity including kidney damage.

The pace of biomarker development is accelerating as investigators report new studies on cancer, diabetes, Alzheimer disease, and other conditions in which the evaluation and isolation of workable markers is prominently featured.

Wei Zheng, Ph.D., leader of the R&D immunoassay group at EMD Chemicals, is overseeing a program to develop biomarker immunoassays to

  • monitor drug-induced toxicity, including kidney damage.

“One of the principle reasons for drugs failing during development is because of organ toxicity,” says Dr. Zheng.
“proteins liberated into the serum and urine can serve as biomarkers of adverse response to drugs, as well as disease states.”

Through collaborative programs with Rules-Based Medicine (RBM), the EMD group has released panels for the profiling of human renal impairment and renal toxicity. These urinary biomarker based products fit the FDA and EMEA guidelines for assessment of drug-induced kidney damage in rats.

The group recently performed a screen for potential protein biomarkers in relation to

  • kidney toxicity/damage on a set of urine and plasma samples
  • from patients with documented renal damage.

Additionally, Dr. Zheng is directing efforts to move forward with the multiplexed analysis of

  • organ and cellular toxicity.

Diseases thought to involve compromised oxidative phosphorylation include

  • diabetes, Parkinson and Alzheimer diseases, cancer, and the aging process itself.

Good biomarkers allow Dr. Zheng to follow the mantra, “fail early, fail fast.” With robust, multiplexible biomarkers, EMD can detect bad drugs early and kill them before they move into costly large animal studies and clinical trials. “Recognizing the severe liability that toxicity presents, we can modify the structure of the candidate molecule and then rapidly reassess its performance.”

Scientists at Oncogene Science a division of Siemens Healthcare Diagnostics, are also focused on biomarkers. “We are working on a number of antibody-based tests for various cancers, including a test for the Ca-9 CAIX protein, also referred to as carbonic anhydrase,” Walter Carney, Ph.D., head of the division, states.

CAIX is a transmembrane protein that is

  • overexpressed in a number of cancers, and, like Herceptin and the Her-2 gene,
  • can serve as an effective and specific marker for both diagnostic and therapeutic purposes.
  • It is liberated into the circulation in proportion to the tumor burden.

Dr. Carney and his colleagues are evaluating patients after tumor removal for the presence of the Ca-9 CAIX protein. If

  • the levels of the protein in serum increase over time,
  • this suggests that not all the tumor cells were removed and the tumor has metastasized.

Dr. Carney and his team have developed both an immuno-histochemistry and an ELISA test that could be used as companion diagnostics in clinical trials of CAIX-targeted drugs.

The ELISA for the Ca-9 CAIX protein will be used in conjunction with Wilex’ Rencarex®, which is currently in a

  • Phase III trial as an adjuvant therapy for non-metastatic clear cell renal cancer.

Additionally, Oncogene Science has in its portfolio an FDA-approved test for the Her-2 marker. Originally approved for Her-2/Neu-positive breast cancer, its indications have been expanded over time, and was approved

  • for the treatment of gastric cancer last year.

It is normally present on breast cancer epithelia but

  • overexpressed in some breast cancer tumors.

“Our products are designed to be used in conjunction with targeted therapies,” says Dr. Carney. “We are working with companies that are developing technology around proteins that are

  • overexpressed in cancerous tissues and can be both diagnostic and therapeutic targets.”

The long-term goal of these studies is to develop individualized therapies, tailored for the patient. Since the therapies are expensive, accurate diagnostics are critical to avoid wasting resources on patients who clearly will not respond (or could be harmed) by the particular drug.

“At this time the rate of response to antibody-based therapies may be very poor, as

  • they are often employed late in the course of the disease, and patients are in such a debilitated state
  • that they lack the capacity to react positively to the treatment,” Dr. Carney explains.

Nanoscale Real-Time Proteomics

Stanford University School of Medicine researchers, working with Cell BioSciences, have developed a

  • nanofluidic proteomic immunoassay that measures protein charge,
  • similar to immunoblots, mass spectrometry, or flow cytometry.
  • unlike these platforms, this approach can measure the amount of individual isoforms,
  • specifically, phosphorylated molecules.

“We have developed a nanoscale device for protein measurement, which I believe could be useful for clinical analysis,” says Dean W. Felsher, M.D., Ph.D., associate professor at Stanford University School of Medicine.

Critical oncogenic transformations involving

  • the activation of the signal-related kinases ERK-1 and ERK-2 can now be followed with ease.

“The fact that we measure nanoquantities with accuracy means that

  • we can interrogate proteomic profiles in clinical patients,

by drawing tiny needle aspirates from tumors over the course of time,” he explains.

“This allows us to observe the evolution of tumor cells and

  • their response to therapy
  • from a baseline of the normal tissue as a standard of comparison.”

According to Dr. Felsher, 20 cells is a large enough sample to obtain a detailed description. The technology is easy to automate, which allows

  • the inclusion of hundreds of assays.

Contrasting this technology platform with proteomic analysis using microarrays, Dr. Felsher notes that the latter is not yet workable for revealing reliable markers.

Dr. Felsher and his group published a description of this technology in Nature Medicine. “We demonstrated that we could take a set of human lymphomas and distinguish them from both normal tissue and other tumor types. We can

  • quantify changes in total protein, protein activation, and relative abundance of specific phospho-isoforms
  • from leukemia and lymphoma patients receiving targeted therapy.

Even with very small numbers of cells, we are able to show that the results are consistent, and

  • our sample is a random profile of the tumor.”

Splice Variant Peptides

“Aberrations in alternative splicing may generate

  • much of the variation we see in cancer cells,”

says Gilbert Omenn, Ph.D., director of the center for computational medicine and bioinformatics at the University of Michigan School of Medicine. Dr. Omenn and his colleague, Rajasree Menon, are

  • using this variability as a key to new biomarker identification.

It is becoming evident that splice variants play a significant role in the properties of cancer cells, including

  • initiation, progression, cell motility, invasiveness, and metastasis.

Alternative splicing occurs through multiple mechanisms

  • when the exons or coding regions of the DNA transcribe mRNA,
  • generating initiation sites and connecting exons in protein products.

Their translation into protein can result in numerous protein isoforms, and

  • these isoforms may reflect a diseased or cancerous state.

Regulatory elements within the DNA are responsible for selecting different alternatives; thus

  • the splice variants are tempting targets for exploitation as biomarkers.
Analyses of the splice-site mutation

Analyses of the splice-site mutation

Despite the many questions raised by these observations, splice variation in tumor material has not been widely studied. Cancer cells are known for their tremendous variability, which allows them to

  • grow rapidly, metastasize, and develop resistance to anticancer drugs.

Dr. Omenn and his collaborators used

  • mass spec data to interrogate a custom-built database of all potential mRNA sequences
  • to find alternative splice variants.

When they compared normal and malignant mammary gland tissue from a mouse model of Her2/Neu human breast cancers, they identified a vast number (608) of splice variant proteins, of which

  • peptides from 216 were found only in the tumor sample.

“These novel and known alternative splice isoforms

  • are detectable both in tumor specimens and in plasma and
  • represent potential biomarker candidates,” Dr. Omenn adds.

Dr. Omenn’s observations and those of his colleague Lewis Cantley, Ph.D., have also

  • shed light on the origins of the classic Warburg effect,
  • the shift to anaerobic glycolysis in tumor cells.

The novel splice variant M2, of muscle pyruvate kinase,

  • is observed in embryonic and tumor tissue.

It is associated with this shift, the result of

  • the expression of a peptide splice variant sequence.

It is remarkable how many different areas of the life sciences are tied into the phenomenon of splice variation. The changes in the genetic material can be much greater than point mutations, which have been traditionally considered to be the prime source of genetic variability.

“We now have powerful methods available to uncover a whole new category of variation,” Dr. Omenn says. “High-throughput RNA sequencing and proteomics will be complementary in discovery studies of splice variants.”

Splice variation may play an important role in rapid evolutionary changes, of the sort discussed by Susumu Ohno and Stephen J. Gould decades ago. They, and other evolutionary biologists, argued that

  • gene duplication, combined with rapid variability, could fuel major evolutionary jumps.

At the time, the molecular mechanisms of variation were poorly understood, but today

  • the tools are available to rigorously evaluate the role of
  • splice variation and other contributors to evolutionary change.

“Biomarkers derived from studies of splice variants, could, in the future, be exploited

  • both for diagnosis and prognosis and
  • for drug targeting of biological networks,
  • in situations such as the Her-2/Neu breast cancers,” Dr. Omenn says.

Aminopeptidase Activities

“By correlating the proteolytic patterns with disease groups and controls, we have shown that

  • exopeptidase activities contribute to the generation of not only cancer-specific
  • but also cancer type specific serum peptides.

according to Paul Tempst, Ph.D., professor and director of the Protein Center at the Memorial Sloan-Kettering Cancer Center.

So there is a direct link between peptide marker profiles of disease and differential protease activity.” For this reason Dr. Tempst argues that “the patterns we describe may have value as surrogate markers for detection and classification of cancer.”

To investigate this avenue, Dr. Tempst and his colleagues have followed

  • the relationship between exopeptidase activities and metastatic disease.

“We monitored controlled, de novo peptide breakdown in large numbers of biological samples using mass spectrometry, with relative quantitation of the metabolites,” Dr. Tempst explains. This entailed the use of magnetic, reverse-phase beads for analyte capture and a MALDI-TOF MS read-out.

“In biomarker discovery programs, functional proteomics is usually not pursued,” says Dr. Tempst. “For putative biomarkers, one may observe no difference in quantitative levels of proteins, while at the same time, there may be substantial differences in enzymatic activity.”

In a preliminary prostate cancer study, the team found a significant difference

  • in activity levels of exopeptidases in serum from patients with metastatic prostate cancer
  • as compared to primary tumor-bearing individuals and normal healthy controls.

However, there were no differences in amounts of the target protein, and this potential biomarker would have been missed if quantitative levels of protein had been the only criterion of selection.

It is frequently stated that “practical fusion energy is 30 years in the future and always will be.” The same might be said of functional, practical biomarkers that can pass muster with the FDA. But splice variation represents a new handle on this vexing problem. It appears that we are seeing the emergence of a new approach that may finally yield definitive diagnostic tests, detectable in serum and urine samples.

Part 7. Epigenetics and Drug Metabolism

DNA Methylation Rules: Studying Epigenetics with New Tools

The tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Patricia Fitzpatrick Dimond, Ph.D.

http://www.genengnews.com/media/images/AnalysisAndInsight/Feb7_2013_24454248_GreenPurpleDNA_EpigeneticsToolsII3576166141.jpg

New tools may help move the field of epigenetic analysis forward and potentially unveil novel biomarkers for cellular development, differentiation, and disease.

DNA sequencing has had the power of technology behind it as novel platforms to produce more sequencing faster and at lower cost have been introduced. But the tools to unravel the epigenetic control mechanisms that influence how cells control access of transcriptional proteins to DNA are just beginning to emerge.

Among these mechanisms, DNA methylation, or the enzymatically mediated addition of a methyl group to cytosine or adenine dinucleotides,

  • serves as an inherited epigenetic modification that
  • stably modifies gene expression in dividing cells.

The unique methylomes are largely maintained in differentiated cell types, making them critical to understanding the differentiation potential of the cell.

In the DNA methylation process, cytosine residues in the genome are enzymatically modified to 5-methylcytosine,

  • which participates in transcriptional repression of genes during development and disease progression.

5-methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. DNA methylation affects gene transcription by physically

  • interfering with the binding of proteins involved in gene transcription.

Methylated DNA may be bound by methyl-CpG-binding domain proteins (MBDs) that can

  • then recruit additional proteins. Some of these include histone deacetylases and other chromatin remodeling proteins that modify histones, thereby
  • forming compact, inactive chromatin, or heterochromatin.

While DNA methylation doesn’t change the genetic code,

  • it influences chromosomal stability and gene expression.

Epigenetics and Cancer Biomarkers

multistage chemical carcinogenesis

multistage chemical carcinogenesis

And because of the increasing recognition that DNA methylation changes are involved in human cancers, scientists have suggested that these epigenetic markers may provide biological markers for cancer cells, and eventually point toward new diagnostic and therapeutic targets. Cancer cell genomes display genome-wide abnormalities in DNA methylation patterns,

  • some of which are oncogenic and contribute to genome instability.

In particular, de novo methylation of tumor suppressor gene promoters

  • occurs frequently in cancers, thereby silencing them and promoting transformation.

Cytosine hydroxymethylation (5-hydroxymethylcytosine, or 5hmC), the aforementioned DNA modification resulting from the enzymatic conversion of 5mC into 5-hydroxymethylcytosine by the TET family of oxygenases, has been identified

  • as another key epigenetic modification marking genes important for
  • pluripotency in embryonic stem cells (ES), as well as in cancer cells.

The base 5-hydroxymethylcytosine was recently identified as an oxidation product of 5-methylcytosine in mammalian DNA. In 2011, using sensitive and quantitative methods to assess levels of 5-hydroxymethyl-2′-deoxycytidine (5hmdC) and 5-methyl-2′-deoxycytidine (5mdC) in genomic DNA, scientists at the Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California investigated

  • whether levels of 5hmC can distinguish normal tissue from tumor tissue.

They showed that in squamous cell lung cancers, levels of 5hmdC showed

  • up to five-fold reduction compared with normal lung tissue.

In brain tumors,5hmdC showed an even more drastic reduction

  • with levels up to more than 30-fold lower than in normal brain,
  • but 5hmdC levels were independent of mutations in isocitrate dehydrogenase-1, the enzyme that converts 5hmC to 5hmdC.

Immunohistochemical analysis indicated that 5hmC is “remarkably depleted” in many types of human cancer.

  • there was an inverse relationship between 5hmC levels and cell proliferation with lack of 5hmC in proliferating cells.

Their data suggest that 5hmdC is strongly depleted in human malignant tumors,

  • a finding that adds another layer of complexity to the aberrant epigenome found in cancer tissue.

In addition, a lack of 5hmC may become a useful biomarker for cancer diagnosis.

Enzymatic Mapping

But according to New England Biolabs’ Sriharsa Pradhan, Ph.D., methods for distinguishing 5mC from 5hmC and analyzing and quantitating the cell’s entire “methylome” and “hydroxymethylome” remain less than optimal.

The protocol for bisulphite conversion to detect methylation remains the “gold standard” for DNA methylation analysis. This method is generally followed by PCR analysis for single nucleotide resolution to determine methylation across the DNA molecule. According to Dr. Pradhan, “.. bisulphite conversion does not distinguish 5mC and 5hmC,”

Recently we found an enzyme, a unique DNA modification-dependent restriction endonuclease, AbaSI, which can

  • decode the hydryoxmethylome of the mammalian genome.

You easily can find out where the hydroxymethyl regions are.”

AbaSI, recognizes 5-glucosylatedmethylcytosine (5gmC) with high specificity when compared to 5mC and 5hmC, and

  • cleaves at narrow range of distances away from the recognized modified cytosine.

By mapping the cleaved ends, the exact 5hmC location can, the investigators reported, be determined.

Dr. Pradhan and his colleagues at NEB; the Department of Biochemistry, Emory University School of Medicine, Atlanta; and the New England Biolabs Shanghai R&D Center described use of this technique in a paper published in Cell Reports this month, in which they described high-resolution enzymatic mapping of genomic hydroxymethylcytosine in mouse ES cells.

In the current report, the authors used the enzyme technology for the genome-wide high-resolution hydroxymethylome, describing simple library construction even with a low amount of input DNA (50 ng) and the ability to readily detect 5hmC sites with low occupancy.

As a result of their studies, they propose that

factors affecting the local 5mC accessibility to TET enzymes play important roles in the 5hmC deposition

  • including include chromatin compaction, nucleosome positioning, or TF binding.
  •  the regularly oscillating 5hmC profile around the CTCF-binding sites, suggests 5hmC ‘‘writers’’ may be sensitive to the nucleosomal environment.
  • some transiently stable 5hmCs may indicate a poised epigenetic state or demethylation intermediate, whereas others may suggest a locally accessible chromosomal environment for the TET enzymatic apparatus.

“We were able to do complete mapping in mouse embryonic cells and are pleased about what this enzyme can do and how it works,” Dr. Pradhan said.

And the availability of novel tools that make analysis of the methylome and hypomethylome more accessible will move the field of epigenetic analysis forward and potentially novel biomarkers for cellular development, differentiation, and disease.

Patricia Fitzpatrick Dimond, Ph.D. (pdimond@genengnews.com), is technical editor at Genetic Engineering & Biotechnology News.

Epigenetic Regulation of ADME-Related Genes: Focus on Drug Metabolism and Transport

Published: Sep 23, 2013

Epigenetic regulation of gene expression refers to heritable factors that are functionally relevant genomic modifications but that do not involve changes in DNA sequence.

Examples of such modifications include

  • DNA methylation, histone modifications, noncoding RNAs, and chromatin architecture.

Epigenetic modifications are crucial for

packaging and interpreting the genome, and they have fundamental functions in regulating gene expression and activity under the influence of physiologic and environmental factors.

In this issue of Drug Metabolism and Disposition, a series of articles is presented to demonstrate the role of epigenetic factors in regulating

  • the expression of genes involved in drug absorption, distribution, metabolism, and excretion in organ development, tissue-specific gene expression, sexual dimorphism, and in the adaptive response to xenobiotic exposure, both therapeutic and toxic.

The articles also demonstrate that, in addition to genetic polymorphisms, epigenetics may also contribute to wide inter-individual variations in drug metabolism and transport. Identification of functionally relevant epigenetic biomarkers in human specimens has the potential to improve prediction of drug responses based on patient’s epigenetic profiles.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=157804

This study is published online in Drug Metabolism and Disposition

Part 8.  Pictorial Maps

 Prediction of intracellular metabolic states from extracellular metabolomic data

MK Aurich, G Paglia, Ottar Rolfsson, S Hrafnsdottir, M Magnusdottir, MM Stefaniak, BØ Palsson, RMT Fleming &

Ines Thiele

Metabolomics Aug 14, 2014;

http://dx.doi.org:/10.1007/s11306-014-0721-3

http://link.springer.com/article/10.1007/s11306-014-0721-3/fulltext.html#Sec1

http://link.springer.com/static-content/images/404/art%253A10.1007%252Fs11306-014-0721-3/MediaObjects/11306_2014_721_Fig1_HTML.gif

Metabolic models can provide a mechanistic framework

  • to analyze information-rich omics data sets, and are
  • increasingly being used to investigate metabolic alternations in human diseases.

An expression of the altered metabolic pathway utilization is the selection of metabolites consumed and released by cells. However, methods for the

  • inference of intracellular metabolic states from extracellular measurements in the context of metabolic models remain underdeveloped compared to methods for other omics data.

Herein, we describe a workflow for such an integrative analysis

  • emphasizing on extracellular metabolomics data.

We demonstrate,

  • using the lymphoblastic leukemia cell lines Molt-4 and CCRF-CEM,

how our methods can reveal differences in cell metabolism. Our models explain metabolite uptake and secretion by predicting

  • a more glycolytic phenotype for the CCRF-CEM model and
  • a more oxidative phenotype for the Molt-4 model,
  • which was supported by our experimental data.

Gene expression analysis revealed altered expression of gene products at

  • key regulatory steps in those central metabolic pathways, and

literature query emphasized the role of these genes in cancer metabolism.

Moreover, in silico gene knock-outs identified unique

  •  control points for each cell line model, e.g., phosphoglycerate dehydrogenase for the Molt-4 model.

Thus, our workflow is well suited to the characterization of cellular metabolic traits based on

  • -extracellular metabolomic data, and it allows the integration of multiple omics data sets
  • into a cohesive picture based on a defined model context.

Keywords Constraint-based modeling _ Metabolomics _ Multi-omics _ Metabolic network _ Transcriptomics

1 Introduction

Modern high-throughput techniques have increased the pace of biological data generation. Also referred to as the ‘‘omics avalanche’’, this wealth of data provides great opportunities for metabolic discovery. Omics data sets

  • contain a snapshot of almost the entire repertoire of mRNA, protein, or metabolites at a given time point or

under a particular set of experimental conditions. Because of the high complexity of the data sets,

  • computational modeling is essential for their integrative analysis.

Currently, such data analysis is a bottleneck in the research process and methods are needed to facilitate the use of these data sets, e.g., through meta-analysis of data available in public databases [e.g., the human protein atlas (Uhlen et al. 2010) or the gene expression omnibus (Barrett et al.  2011)], and to increase the accessibility of valuable information for the biomedical research community.

Constraint-based modeling and analysis (COBRA) is

  • a computational approach that has been successfully used to
  • investigate and engineer microbial metabolism through the prediction of steady-states (Durot et al.2009).

The basis of COBRA is network reconstruction: networks are assembled in a bottom-up fashion based on

  • genomic data and extensive
  • organism-specific information from the literature.

Metabolic reconstructions capture information on the

  • known biochemical transformations taking place in a target organism
  • to generate a biochemical, genetic and genomic knowledge base (Reed et al. 2006).

Once assembled, a

  • metabolic reconstruction can be converted into a mathematical model (Thiele and Palsson 2010), and
  • model properties can be interrogated using a great variety of methods (Schellenberger et al. 2011).

The ability of COBRA models

  • to represent genotype–phenotype and environment–phenotype relationships arises
  • through the imposition of constraints, which
  • limit the system to a subset of possible network states (Lewis et al. 2012).

Currently, COBRA models exist for more than 100 organisms, including humans (Duarte et al. 2007; Thiele et al. 2013).

Since the first human metabolic reconstruction was described [Recon 1 (Duarte et al. 2007)],

  • biomedical applications of COBRA have increased (Bordbar and Palsson 2012).

One way to contextualize networks is to

  • define their system boundaries according to the metabolic states of the system, e.g., disease or dietary regimes.

The consequences of the applied constraints can

  • then be assessed for the entire network (Sahoo and Thiele 2013).

Additionally, omics data sets have frequently been used

  • to generate cell-type or condition-specific metabolic models.

Models exist for specific cell types, such as

  1. enterocytes (Sahoo and Thiele2013),
  2. macrophages (Bordbar et al. 2010),
  3. adipocytes (Mardinoglu et al. 2013),
  4. even multi-cell assemblies that represent the interactions of brain cells (Lewis et al. 2010).

All of these cell type specific models, except the enterocyte reconstruction

  • were generated based on omics data sets.

Cell-type-specific models have been used to study

  • diverse human disease conditions.

For example, an adipocyte model was generated using

  • transcriptomic, proteomic, and metabolomics data.

This model was subsequently used to investigate metabolic alternations in adipocytes

  • that would allow for the stratification of obese patients (Mardinoglu et al. 2013).

The biomedical applications of COBRA have been

  1. cancer metabolism (Jerby and Ruppin, 2012).
  2. predicting drug targets (Folger et al. 2011; Jerby et al. 2012).

A cancer model was generated using

  • multiple gene expression data sets and subsequently used
  • to predict synthetic lethal gene pairs as potential drug targets
  • selective for the cancer model, but non-toxic to the global model (Recon 1),

a consequence of the reduced redundancy in the cancer specific model (Folger et al. 2011).

In a follow up study, lethal synergy between FH and enzymes of the heme metabolic pathway

  • were experimentally validated and resolved the mechanism by which FH deficient cells,
    e.g., in renal-cell cancer cells survive a non-functional TCA cycle (Frezza et al. 2011).

Contextualized models, which contain only the subset of reactions active in a particular tissue (or cell-) type,

  • can be generated in different ways (Becker and Palsson, 2008; Jerby et al. 2010).

However, the existing algorithms mainly consider

  • gene expression and proteomic data
  • to define the reaction sets that comprise the contextualized metabolic models.

These subset of reactions are usually defined

  • based on the expression or absence of expression of the genes or proteins (present and absent calls),
  • or inferred from expression values or differential gene expression.

Comprehensive reviews of the methods are available (Blazier and Papin, 2012; Hyduke et al. 2013). Only the compilation of a large set of omics data sets

  • can result in a tissue (or cell-type) specific metabolic model, whereas

the representation of one particular experimental condition is achieved

  • through the integration of omics data set generated from one experiment only (condition-specific cell line model).

Recently, metabolomic data sets have become more comprehensive and

  • using these data sets allow direct determination of the metabolic network components (the metabolites).

Additionally, metabolomics has proven to be stable, relatively inexpensive, and highly reproducible (Antonucci et al. 2012). These factors make metabolomic data sets particularly valuable for

  • interrogation of metabolic phenotypes.

Thus, the integration of these data sets is now an active field of research (Li et al. 2013; Mo et al. 2009; Paglia et al. 2012b; Schmidt et al. 2013).

Generally, metabolomic data can be incorporated into metabolic networks as

  • qualitative, quantitative, and thermodynamic constraints (Fleming et al. 2009; Mo et al. 2009).

Mo et al. used metabolites detected in the

  • spent medium of yeast cells to determine intracellular flux states through a sampling analysis (Mo et al. 2009),
  • which allowed unbiased interrogation of the possible network states (Schellenberger and Palsson 2009) and
  • prediction of internal pathway use.
Modes of transcriptional regulation during the YMC

Modes of transcriptional regulation during the YMC

Such analyses have also been used to reveal the effects of

  1. enzymopathies on red blood cells (Price et al. 2004),
  2. to study effects of diet on diabetes (Thiele et al. 2005) and
  3. to define macrophage metabolic states (Bordbar et al. 2010).

This type of analysis is available as a function in the COBRA toolbox (Schellenberger et al. 2011).

In this study, we established a workflow

  • for the generation and analysis of condition-specific metabolic cell line models
  • that can facilitate the interpretation of metabolomic data.

Our modeling yields meaningful predictions regarding

  • metabolic differences between two lymphoblastic leukemia cell lines (Fig. 1A).

Fig. 1

metabol leukem cell lines11306_2014_721_Fig1_HTML

metabol leukem cell lines11306_2014_721_Fig1_HTML

A Combined experimental and computational pipeline to study human metabolism.

  1. Experimental work and omics data analysis steps precede computational modeling.
  2. Model predictions are validated based on targeted experimental data.
  3. Metabolomic and transcriptomic data are used for model refinement and submodel extraction.
  4. Functional analysis methods are used to characterize the metabolism of the cell-line models and compare it to additional experimental data.
  5. The validated models are subsequently used for the prediction of drug targets.

B Uptake and secretion pattern of model metabolites. All metabolite uptakes and secretions that were mapped during model generation are shown.

  • Metabolite uptakes are depicted on the left, and
  • secreted metabolites are shown on the right.
  1. A number of metabolite exchanges mapped to the model were unique to one cell line.
  2. Differences between cell lines were used to set quantitative constraints for the sampling analysis.

C Statistics about the cell line-specific network generation.

D Quantitative constraints.

For the sampling analysis, an additional set of constraints was imposed on the cell line specific models,

  • emphasizing the differences in metabolite uptake and secretion between cell lines.

Higher uptake of a metabolite was allowed

  • in the model of the cell line that consumed more of the metabolite in vitro, whereas
  • the supply was restricted for the model with lower in vitro uptake.

This was done by establishing the same ratio between the models bounds as detected in vitro.

X denotes the factor (slope ratio) that distinguishes the bounds, and

  • which was individual for each metabolite.

(a) The uptake of a metabolite could be x times higher in CCRF-CEM cells,

(b) the metabolite uptake could be x times higher in Molt-4,

(c) metabolite secretion could be x times higher in CCRF-CEM, or

(d) metabolite secretion could be x times higher in Molt-4 cells.LOD limit of detection.

The consequence of the adjustment was, in case of uptake, that one model was constrained to a lower metabolite uptake (A, B), and the difference depended on the ratio detected in vitro. In case of secretion, one model

  • had to secrete more of the metabolite, and again
  • the difference depended on the experimental difference detected between the cell lines

2 Results

We set up a pipeline that could be used to infer intracellular metabolic states

  • from semi-quantitative data regarding metabolites exchanged between cells and their environment.

Our pipeline combined the following four steps:

  1. data acquisition,
  2. data analysis,
  3. metabolic modeling and
  4. experimental validation of the model predictions (Fig. 1A).

We demonstrated the pipeline and the predictive potential to predict metabolic alternations in diseases such as cancer based on

^two lymphoblastic leukemia cell lines.

The resulting Molt-4 and CCRF-CEM condition-specific cell line models could explain

^  metabolite uptake and secretion
^  by predicting the distinct utilization of central metabolic pathways by the two cell lines.
^  the CCRF-CEM model resembled more a glycolytic, commonly referred to as ‘Warburg’ phenotype,
^  our model predicted a more respiratory phenotype for the Molt-4 model.

We found these predictions to be in agreement with measured gene expression differences

  • at key regulatory steps in the central metabolic pathways, and they were also
  • consistent with additional experimental data regarding the energy and redox states of the cells.

After a brief discussion of the data generation and analysis steps, the results derived from model generation and analysis will be described in detail.

2.1 Pipeline for generation of condition-specific metabolic cell line models

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

2.1.1 Generation of experimental data

We monitored the growth and viability of lymphoblastic leukemia cell lines in serum-free medium (File S2, Fig. S1). Multiple omics data sets were derived from these cells.Extracellular metabolomics (exo-metabolomic) data,

integration of exometabolomic (EM) data

integration of exometabolomic (EM) data

^  comprising measurements of the metabolites in the spent medium of the cell cultures (Paglia et al. 2012a),
^ were collected along with transcriptomic data, and these data sets were used to construct the models.

2.1.4 Condition-specific models for CCRF-CEM and Molt-4 cells

To determine whether we had obtained two distinct models, we evaluated the reactions, metabolites, and genes of the two models. Both the Molt-4 and CCRF-CEM models contained approximately half of the reactions and metabolites present in the global model (Fig. 1C). They were very similar to each other in terms of their reactions, metabolites, and genes (File S1, Table S5A–C).

(1) The Molt-4 model contained seven reactions that were not present in the CCRF-CEM model (Co-A biosynthesis pathway and exchange reactions).
(2) The CCRF-CEM contained 31 unique reactions (arginine and proline metabolism, vitamin B6 metabolism, fatty acid activation, transport, and exchange reactions).
(3) There were 2 and 15 unique metabolites in the Molt-4 and CCRF-CEM models, respectively (File S1, Table S5B).
(4) Approximately three quarters of the global model genes remained in the condition-specific cell line models (Fig. 1C).
(5) The Molt-4 model contained 15 unique genes, and the CCRF-CEM model had 4 unique genes (File S1, Table S5C).
(6) Both models lacked NADH dehydrogenase (complex I of the electron transport chain—ETC), which was determined by the absence of expression of a mandatory subunit (NDUFB3, Entrez gene ID 4709).

Rather, the ETC was fueled by FADH2 originating from succinate dehydrogenase and from fatty acid oxidation, which through flavoprotein electron transfer

FADH2

FADH2

  • could contribute to the same ubiquinone pool as complex I and complex II (succinate dehydrogenase).

Despite their different in vitro growth rates (which differed by 11 %, see File S2, Fig. S1) and
^^^ differences in exo-metabolomic data (Fig. 1B) and transcriptomic data,
^^^ the internal networks were largely conserved in the two condition-specific cell line models.

2.1.5 Condition-specific cell line models predict distinct metabolic strategies

Despite the overall similarity of the metabolic models, differences in their cellular uptake and secretion patterns suggested distinct metabolic states in the two cell lines (Fig. 1B and see “Materials and methods” section for more detail). To interrogate the metabolic differences, we sampled the solution space of each model using an Artificial Centering Hit-and-Run (ACHR) sampler (Thiele et al. 2005). For this analysis, additional constraints were applied, emphasizing the quantitative differences in commonly uptaken and secreted metabolites. The maximum possible uptake and maximum possible secretion flux rates were reduced
^^^ according to the measured relative differences between the cell lines (Fig. 1D, see “Materials and methods” section).

We plotted the number of sample points containing a particular flux rate for each reaction. The resulting binned histograms can be understood as representing the probability that a particular reaction can have a certain flux value.

A comparison of the sample points obtained for the Molt-4 and CCRF-CEM models revealed

  • a considerable shift in the distributions, suggesting a higher utilization of glycolysis by the CCRF-CEM model
    (File S2, Fig. S2).

This result was further supported by differences in medians calculated from sampling points (File S1, Table S6).
The shift persisted throughout all reactions of the pathway and was induced by the higher glucose uptake (34 %) from the extracellular medium in CCRF-CEM cells.

The sampling median for glucose uptake was 34 % higher in the CCRF-CEM model than in Molt-4 model (File S2, Fig. S2).

The usage of the TCA cycle was also distinct in the two condition-specific cell-line models (Fig. 2). Interestingly,
the models used succinate dehydrogenase differently (Figs. 2, 3).

TCA_reactions

TCA_reactions

The Molt-4 model utilized an associated reaction to generate FADH2, whereas

  • in the CCRF-CEM model, the histogram was shifted in the opposite direction,
  • toward the generation of succinate.

Additionally, there was a higher efflux of citrate toward amino acid and lipid metabolism in the CCRF-CEM model (Fig. 2). There was higher flux through anaplerotic and cataplerotic reactions in the CCRF-CEM model than in the Molt-4 model (Fig. 2); these reactions include

(1) the efflux of citrate through ATP-citrate lyase,
(2) uptake of glutamine,
(3) generation of glutamate from glutamine,
(4) transamination of pyruvate and glutamate to alanine and to 2-oxoglutarate,
(5) secretion of nitrogen, and
(6) secretion of alanine.

energetics-of-cellular-respiration

energetics-of-cellular-respiration

The Molt-4 model showed higher utilization of oxidative phosphorylation (Fig. 3), again supported by
elevated median flux through ATP synthase (36 %) and other enzymes, which contributed to higher oxidative metabolism. The sampling analysis therefore revealed different usage of central metabolic pathways by the condition-specific models.

Fig. 2

Differences in the use of  the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

Differences in the use of the TCA cycle by the CCRF-CEM model (red) and the Molt-4 model (blue).

The table provides the median values of the sampling results. Negative values in histograms and in the table describe reversible reactions with flux in the reverse direction. There are multiple reversible reactions for the transformation of isocitrate and α-ketoglutarate, malate and fumarate, and succinyl-CoA and succinate. These reactions are unbounded, and therefore histograms are not shown. The details of participating cofactors have been removed.

Figure 3.

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Molt-4 has higher median flux through ETC reactions II–IV 11306_2014_721_Fig3_HTML

Atp ATP, cit citrate, adp ADP, pi phosphate, oaa oxaloacetate, accoa acetyl-CoA, coa coenzyme-A, icit isocitrate, αkg α-ketoglutarate, succ-coa succinyl-CoA, succ succinate, fumfumarate, mal malate, oxa oxaloacetate,
pyr pyruvate, lac lactate, ala alanine, gln glutamine, ETC electron transport chain

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

Ingenuity network analysis showing up (red) and downregulation (green) of miRNAs involved in PC and their target genes

metabolic pathways 1476-4598-10-70-1

metabolic pathways 1476-4598-10-70-1

Metabolic Systems Research Team fig2

Metabolic Systems Research Team fig2