Archive for the ‘Protein-energy malnutrition’ Category

Benefits of Fiber in Diet

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


UPDATED on 1/15/2019

This is How Much Daily Fiber to Eat for Better Health – More appears better in meta-analysis — as in more than 30 g/day

by Ashley Lyles, Staff Writer, MedPage Today

In the systematic review, observational data showed a 15% to 30% decline in cardiovascular-related death, all-cause mortality, and incidence of stroke, coronary heart disease, type 2 diabetes, and colorectal cancer among people who consumed the most dietary fiber compared to those consuming the lowest amounts.

Whole grain intake yielded similar findings.

Risk reduction associated with a range of critical outcomes was greatest when daily intake of dietary fibre was between 25 g and 29 g. Dose-response curves suggested that higher intakes of dietary fibre could confer even greater benefit to protect against cardiovascular diseases, type 2 diabetes, and colorectal and breast cancer.


Eating more dietary fiber was linked with lower risk of disease and death, a meta-analysis showed.

According to observational studies, risk was reduced most for a range of critical outcomes from all-cause mortality to stroke when daily fiber consumption was between 25 grams and 29 grams, reported Jim Mann, PhD, of University of Otago in Dunedin, New Zealand, and colleagues in The Lancet.

By upping daily intake to 30 grams or more, people had even greater prevention of certain conditions: colorectal and breast cancer, type 2 diabetes, and cardiovascular diseases, according to dose-response curves the authors created.

Quantitative guidelines relating to dietary fiber have not been available, the researchers said. With the GRADE method, they determined that there was moderate and low-to-moderate certainty of evidence for the benefits of dietary fiber consumption and whole grain consumption, respectively.

Included in the systematic review were 58 clinical trials and 185 prospective studies for a total of 4,635 adult participants with 135 million person-years of information (one trial in children was included, but analyzed separately from adults). Trials and prospective studies assessing weight loss, supplement use, and participants with a chronic disease were excluded.


Food is digested by bathing in enzymes that break down its molecules. Those molecular fragments then pass through the gut wall and are absorbed in our intestines. But our bodies make a limited range of enzymes, so that we cannot break down many of the tough compounds in plants. The term “dietary fiber” refers to those indigestible molecules. These dietary fibers are indigestible only to us. The gut is coated with a layer of mucus, on which sits a carpet of hundreds of species of bacteria, part of the human microbiome. Some of these microbes carry the enzymes needed to break down various kinds of dietary fibers.


Scientists at the University of Gothenburg in Sweden are running experiments that are yielding some important new clues about fiber’s role in human health. Their research indicates that fiber doesn’t deliver many of its benefits directly to our bodies. Instead, the fiber we eat feeds billions of bacteria in our guts. Keeping them happy means our intestines and immune systems remain in good working order. The scientists have recently reported that the microbes are involved in the benefits obtained from the fruits-and-vegetables diet. Research proved that low fiber diet decreases the gut bacteria population by tenfold.


Along with changes to the microbiome there were also rapid changes observed in the experimental mice. Their intestines got smaller, and its mucus layer thinner. As a result, bacteria wound up much closer to the intestinal wall, and that encroachment triggered an immune reaction. After a few days on the low-fiber diet, mouse intestines developed chronic inflammation. After a few weeks, they started putting on fat and developing higher blood sugar levels. Inflammation can help fight infections, but if it becomes chronic, it can harm our bodies. Among other things, chronic inflammation may interfere with how the body uses the calories in food, storing more of it as fat rather than burning it for energy.


In a way fiber benefits human health is by giving, indirectly, another source of food. When bacteria finished harvesting the energy in the dietary fiber, they cast off the fragments as waste. That waste — in the form of short-chain fatty acids — is absorbed by intestinal cells, which use it as fuel. But the gut’s microbes do more than just make energy. They also send messages. Intestinal cells rely on chemical signals from the bacteria to work properly. The cells respond to the signals by multiplying and making a healthy supply of mucus. They also release bacteria-killing molecules. By generating these responses, gut bacteria help to maintain a peaceful coexistence with the immune system. They rest on the gut’s mucus layer at a safe distance from the intestinal wall. Any bacteria that wind up too close get wiped out by antimicrobial poisons.


A diet of fiber-rich foods, such as fruits and vegetables, reduces the risk of developing diabetes, heart disease and arthritis. Eating more fiber seems to lower people’s mortality rate, whatever be the cause. Researchers hope that they will learn more about how fiber influences the microbiome to use it as a way to treat disorders. Lowering inflammation with fiber may also help in the treatment of immune disorders such as inflammatory bowel disease. Fiber may also help reverse obesity. They found that fiber supplements helped obese people to lose weight. It’s possible that each type of fiber feeds a particular set of bacteria, which send their own important signals to our bodies.

























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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published 

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included.  However, there is a substantial amount of material in the following categories:

  1. The systemic inflammatory response
  2. sepsis
  3. vasculitis
  4. neurodegenerative disease
  5. cancer immunology
  6. autoimmune diseases: rheumatoid arthritis, colitis, ileitis, …
  7. T cells in immunity

Proteomics, metabolomics and diabetes

























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A Reconstructed View of Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP


There has always been Personalized Medicine if you consider the time a physician spends with a patient, which has dwindled. But the current recognition of personalized medicine refers to breakthrough advances in technological innovation in diagnostics and treatment that differentiates subclasses within diagnoses that are amenable to relapse eluding therapies.  There are just a few highlights to consider:

  1. We live in a world with other living beings that are adapting to a changing environmental stresses.
  2. Nutritional resources that have been available and made plentiful over generations are not abundant in some climates.
  3. Despite the huge impact that genomics has had on biological progress over the last century, there is a huge contribution not to be overlooked in epigenetics, metabolomics, and pathways analysis.

A Reconstructed View of Personalized Medicine

There has been much interest in ‘junk DNA’, non-coding areas of our DNA are far from being without function. DNA has two basic categories of nitrogenous bases: the purines (adenine [A] and guanine [G]), and the pyrimidines (cytosine [C], thymine [T], and  no uracil [U]),  while RNA contains only A, G, C, and U (no T).  The Watson-Crick proposal set the path of molecular biology for decades into the 21st century, culminating in the Human Genome Project.

There is no uncertainty about the importance of “Junk DNA”.  It is both an evolutionary remnant, and it has a role in cell regulation.  Further, the role of histones in their relationship the oligonucleotide sequences is not understood.  We now have a large output of research on noncoding RNA, including siRNA, miRNA, and others with roles other than transcription. This requires major revision of our model of cell regulatory processes.  The classic model is solely transcriptional.

  • DNA-> RNA-> Amino Acid in a protein.

Redrawn we have

  • DNA-> RNA-> DNA and
  • DNA->RNA-> protein-> DNA.

Neverthess, there were unrelated discoveries that took on huge importance.  For example, since the 1920s, the work of Warburg and Meyerhoff, followed by that of Krebs, Kaplan, Chance, and others built a solid foundation in the knowledge of enzymes, coenzymes, adenine and pyridine nucleotides, and metabolic pathways, not to mention the importance of Fe3+, Cu2+, Zn2+, and other metal cofactors.  Of huge importance was the work of Jacob, Monod and Changeux, and the effects of cooperativity in allosteric systems and of repulsion in tertiary structure of proteins related to hydrophobic and hydrophilic interactions, which involves the effect of one ligand on the binding or catalysis of another,  demonstrated by the end-product inhibition of the enzyme, L-threonine deaminase (Changeux 1961), L-isoleucine, which differs sterically from the reactant, L-threonine whereby the former could inhibit the enzyme without competing with the latter. The current view based on a variety of measurements (e.g., NMR, FRET, and single molecule studies) is a ‘‘dynamic’’ proposal by Cooper and Dryden (1984) that the distribution around the average structure changes in allostery affects the subsequent (binding) affinity at a distant site.

What else do we have to consider?  The measurement of free radicals has increased awareness of radical-induced impairment of the oxidative/antioxidative balance, essential for an understanding of disease progression.  Metal-mediated formation of free radicals causes various modifications to DNA bases, enhanced lipid peroxidation, and altered calcium and sulfhydryl homeostasis. Lipid peroxides, formed by the attack of radicals on polyunsaturated fatty acid residues of phospholipids, can further react with redox metals finally producing mutagenic and carcinogenic malondialdehyde, 4-hydroxynonenal and other exocyclic DNA adducts (etheno and/or propano adducts). The unifying factor in determining toxicity and carcinogenicity for all these metals is the generation of reactive oxygen and nitrogen species. Various studies have confirmed that metals activate signaling pathways and the carcinogenic effect of metals has been related to activation of mainly redox sensitive transcription factors, involving NF-kappaB, AP-1 and p53.

I have provided mechanisms explanatory for regulation of the cell that go beyond the classic model of metabolic pathways associated with the cytoplasm, mitochondria, endoplasmic reticulum, and lysosome, such as, the cell death pathways, expressed in apoptosis and repair.  Nevertheless, there is still a missing part of this discussion that considers the time and space interactions of the cell, cellular cytoskeleton and extracellular and intracellular substrate interactions in the immediate environment.

There is heterogeneity among cancer cells of expected identical type, which would be consistent with differences in phenotypic expression, aligned with epigenetics.  There is also heterogeneity in the immediate interstices between cancer cells.  Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. In the case of breast cancer, there is interaction with estrogen , and we refer to an androgen-unresponsive prostate cancer.

Finally,  the interaction between enzyme and substrates may be conditionally unidirectional in defining the activity within the cell.  The activity of the cell is dynamically interacting and at high rates of activity.  In a study of the pyruvate kinase (PK) reaction the catalytic activity of the PK reaction was reversed to the thermodynamically unfavorable direction in a muscle preparation by a specific inhibitor. Experiments found that in there were differences in the active form of pyruvate kinase that were clearly related to the environmental condition of the assay – glycolitic or glyconeogenic. The conformational changes indicated by differential regulatory response were used to present a dynamic conformational model functioning at the active site of the enzyme. In the model, the interaction of the enzyme active site with its substrates is described concluding that induced increase in the vibrational energy levels of the active site decreases the energetic barrier for substrate induced changes at the site. Another example is the inhibition of H4 lactate dehydrogenase, but not the M4, by high concentrations of pyruvate. An investigation of the inhibition revealed that a covalent bond was formed between the nicotinamide ring of the NAD+ and the enol form of pyruvate.  The isoenzymes of isocitrate dehydrogenase, IDH1 and IDH2 mutations occur in gliomas and in acute myeloid leukemias with normal karyotype. IDH1 and IDH2 mutations are remarkably specific to codons that encode conserved functionally important arginines in the active site of each enzyme. In this case, there is steric hindrance by Asp279 where the isocitrate substrate normally forms hydrogen bonds with Ser94.

Personalized medicine has been largely viewed from a lens of genomics.  But genomics is only the reading frame.  The living activities of cell processes are dynamic and occur at rapid rates.  We have to keep in mind that personalized in reference to genotype is not complete without reconciliation of phenotype, which is the reference to expressed differences in outcomes.


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Excess Eating, Overweight, and Diabetic

Larry H Bernstein, MD, FCAP, Curator



You Did NOT Eat Your Way to Diabetes!



The myth that diabetes is caused by overeating also hurts the one out of five people who are not overweight when they contract Type 2 Diabetes. Because doctors only think “Diabetes” when they see a patient who fits the stereotype–the grossly obese inactive patient–they often neglect to check people of normal weight for blood sugar disorders even when they show up with classic symptoms of high blood sugar such as recurrent urinary tract infections or neuropathy.

Where Did This Toxic Myth Come From?

The way this myth originated is this: Because people with Type 2 Diabetes are often overweight and because many people who are overweight have a syndrome called “insulin resistance” in which their cells do not respond properly to insulin so that they require larger than normal amounts of insulin to lower their blood sugar, the conclusion was drawn years ago that insulin resistance was the cause of Type 2 Diabetes.

It made sense. Something was burning out the beta cells in these people, and it seemed logical that the something must be the stress of pumping out huge amounts of insulin, day after day. This idea was so compelling that it was widely believed by medical professionals, though few realized it had never been subjected to careful investigation by large-scale research.

That is why any time there is an article in the news about Type 2 Diabetes you are likely to read something that says, “While Type 1 diabetes (sometimes called Juvenile Diabetes) is a condition where the body does not produce insulin, Type 2 Diabetes is the opposite: a condition where the body produces far too much insulin because of insulin resistance caused by obesity.”

When your doctor tells you the same thing, the conclusion is inescapable: your overeating caused you to put on excess fat and that your excess fat is what made you diabetic.

Blaming the Victim

This line of reasoning leads to subtle, often unexpressed, judgmental decisions on the part of your doctor, who is likely to believe that had you not been such a pig, you would not have given yourself this unnecessary disease.

And because of this unspoken bias, unless you are able to “please” your doctor by losing a great deal of weight after your diagnosis you may find yourself treated with a subtle but callous disregard because of the doctor’s feeling that you brought this condition down on yourself. This bias is similar to that held by doctors who face patients who smoke a pack a day and get lung cancer and still refuse to stop smoking.

You also see this bias frequently expressed in the media. Articles on the “obesity epidemic” blame overeating for a huge increase in the number of people with diabetes, including children and teenagers who are pictured greedily gorging on supersized fast foods while doing no exercise more strenuous than channel surfing. In a society where the concepts “thin” and “healthy” have taken on the overtones of moral virtue and where the only one of the seven deadly sins that still inspires horror and condemnation is gluttony, being fat is considered by many as sure proof of moral weakness. So it is not surprising that the subtext of media coverage of obesity and diabetes is that diabetes is nothing less than the just punishment you deserve for being such a glutton.

Except that it’s not true.

Obesity Has Risen Dramatically While Diabetes Rates Have Not

The rate of obesity has grown alarmingly over the past decades, especially in certain regions of the U.S. The NIH reports that “From 1960-2 to 2005-6, the prevalence of obesity increased from 13.4 to 35.1 percent in U.S. adults age 20 to 74.7.”

If obesity was causing diabetes, you’d exect to see a similar rise in the diabetes rate. But this has not happened. The CDC reports that “From 1980 through 2010, the crude prevalence of diagnosed diabetes increased …from 2.5% to 6.9%.” However, if you look at the graph that accompanies this statement, you see that the rate of diabetes diagnoses rose only gradually through this period–to about 3.5% until it suddenly sped upward in the late 1990s. This sudden increase largely due to the fact that in 1998 the American Diabetes Association changed the criteria by which diabetes was to be diagnosed, lowering the fasting blood sugar level used to diagnose diabetes from 141 mg/dl to 126 mg/dl. (Details HERE)

Analyzing these statistics, it becomes clear that though roughtly 65 million more Americans became fat over this period, only 13 million more Americans became diabetic.

And to further confuse the matter, several factors other than the rise in obesity and the ADA’s lowering of the diagnostic cutoff also came into play during this period which also raised the rate of diabetes diagnoses:

Diabetes becomes more common as people age as the pancreas like other organs, becames less efficient. In 1950 only 12% of the U.S. population was over 65. By 2010 40% was, and of those 40%, 19% were over 75.(Details HERE.)

At the same time, the period during which the rate of diabetes rose was also the period in which doctors began to heavily prescribe statins, a class of drugs we now know raises the risk of developing diabetes. (Details HERE.)

Why Obesity Doesn’t Cause Diabetes: The Genetic Basis of Diabetes

While people who have diabetes are often heavy, one out of five people diagnosed with diabetes are thin or normal weight. And though heavy people with diabetes are, indeed, likely to be insulin resistant, the majority of people who are overweight will never develop diabetes. In fact, they will not develop diabetes though they are likely to be just as insulin resistant as those who do–or even more so.

The message that diabetes researchers in academic laboratories are coming up with about what really causes diabetes is quite different from what you read in the media. What they are finding is that to get Type 2 Diabetes you need to have some combination of a variety of already-identified genetic flaws which produce the syndrome that we call Type 2 Diabetes. This means that unless you have inherited abnormal genes or had your genes damaged by exposure to pesticides, plastics and other environmental toxins known to cause genetic damage, you can eat until you drop and never develop diabetes.

Now let’s look in more depth at what peer reviewed research has found about the true causes of diabetes

Twin Studies Back up a Genetic Cause for Diabetes

Studies of identical twins showed that twins have an 80% concordance for Type 2 Diabetes. In other words, if one twin has Type 2 Diabetes, the chance that the other will have it two are 4 out of 5. While you might assume that this might simply point to the fact that twins are raised in the same home by mothers who feed them the same unhealthy diets, studies of non-identical twins found NO such correlation. The chances that one non-identical twin might have Type 2 Diabetes if the other had it were much lower, though these non-identical twins, born at the same time and raised by the same caregivers were presumably also exposed to the same unhealthy diets.

This kind of finding begins to hint that there is more than just bad habits to blame for diabetes. A high concordance between identical twins which is not shared by non-identical twins is usually advanced as an argument for a genetic cause, though because one in five identical twins did not become diabetic, it is assumed that some additional factors beyond the inherited genome must come into play to cause the disease to appear. Often this factor is an exposure to an environmental toxin which knocks out some other, protective genetic factor.

The Genetic Basis of Type 2 Diabetes Mellitus: Impaired Insulin Secretion versus Impaired Insulin Sensitivity. John E. Gerich. Endocrine Reviews 19(4) 491-503, 1998.

The List of Genes Associated with Type 2 Keeps Growing

Here is a brief list of some of the abnormal genes that have been found to be associated with Type 2 Diabetes in people of European extraction: TCF7L2, HNF4-a, PTPN, SHIP2, ENPP1, PPARG, FTO, KCNJ11, NOTCh3, WFS1, CDKAL1, IGF2BP2, SLC30A8, JAZF1, and HHEX.

People from non-European ethnic groups have been found to have entirely different sets of diabetic genes than do Western Europeans, like the UCP2 polymorphism found in Pima Indians and the three Calpain-10 gene polymorphisms that have been found to be associated with diabetes in Mexicans. The presence of a variation in yet another gene, SLC16A11, was recently found to be associated with a 25% higher risk of a Mexican developing Type 2 diabetes.

The More Diabetes Genes You Have The Worse Your Beta Cells Perform

A study published in the Journal Diabetologia in November 2008 studied how well the beta cells secreted insulin in 1,211 non-diabetic individuals. They then screened these people for abnormalities in seven genes that have been found associated with Type 2 Diabetes.

They found that with each abnormal gene found in a person’s genome, there was an additive effect on that person’s beta cell dysfunction with each additional gene causing poorer beta cell function.

The impact of these genetic flaws becomes clear when we learn that in these people who were believed to be normal, beta cell glucose sensitivity and insulin production at meal times was decreased by 39% in people who had abnormalities in five genes. That’s almost half. And if your beta cells are only putting out half as much insulin as a normal person’s it takes a lot less stress on those cells to push you into becoming diabetic.

Beta cell glucose sensitivity is decreased by 39% in non-diabetic individuals carrying multiple diabetes-risk alleles compared with those with no risk alleles L. Pascoe et al. Diabetologia, Volume 51, Number 11 / November, 2008.

Gene Tests Predict Diabetes Independent of Conventional “Risk Factors”

A study of 16,061 Swedish and 2770 Finnish subjects found that

Variants in 11 genes (TCF7L2, PPARG, FTO, KCNJ11, NOTCh3, WFS1, CDKAL1, IGF2BP2, SLC30A8, JAZF1, and HHEX) were significantly associated with the risk of Type 2 Diabetes independently of clinical risk factors [i.e. family history, obesity etc.]; variants in 8 of these genes were associated with impaired beta-cell function.

Note that though the subjects here were being screened for Type 2 Diabetes, the defect found here was NOT insulin resistance, but rather deficient insulin secretion. This study also found that:

The discriminative power of genetic risk factors improved with an increasing duration of follow-up, whereas that of clinical risk factors decreased.

In short, the longer these people were studied, the more likely the people with these gene defects were to develop diabetes.

Clinical Risk Factors, DNA Variants, and the Development of Type 2 Diabetes Valeriya Lyssenko, M.D. et. al. New England Journal of Medicine, Volume 359:2220-2232, November 20, 2008,Number 21.

What A Common Diabetes Gene Does

A study published in July of 2009 sheds light on what exactly it is that an allele (gene variant) often found associated with diabetes does. The allele in question is one of TCF7L2 transcription factor gene. The study involved 81 normal healthy young Danish men whose genes were tested. They were then given a battery of tests to examine their glucose metabolisms. The researchers found that:

Carriers of the T allele were characterised by reduced 24 h insulin concentrations … and reduced insulin secretion relative to glucose during a mixed meal test … but not during an IVGTT [intravenous glucose tolerance test].

This is an interesting finding, because what damages our bodies is the blood sugar we experience after eating “a mixed meal” but so much research uses the artificial glucose tolerance (GTT) test to assess blood sugar health. This result suggests that the GTT may be missing important signs of early blood sugar dysfunction and that the mixed meal test may be a better diagnostic test than the GTT. I have long believed this to be true, since so many people experience reactive lows when they take the GTT which produces a seemingly “normal reading” though they routinely experience highs after eating meals. These highs are what damage our organs.

Young men with the TCF7L2 allele also responded with weak insulin secretion in response to the incretin hormone GLP-1 and “Despite elevated hepatic [liver] glucose production, carriers of the T allele had significantly reduced 24 h glucagon concentrations … suggesting altered alpha cell function.”

Here again we see evidence that long before obesity develops, people with this common diabetes gene variant show highly abnormal blood sugar behavior. Abnormal production of glucose by the liver may also contribute to obesity as metformin, a drug that that blocks the liver’s production of glucose blocks weight gain and often causes weight loss.

The T allele of rs7903146 TCF7L2 is associated with impaired insulinotropic action of incretin hormones, reduced 24 h profiles of plasma insulin and glucagon, and increased hepatic glucose production in young healthy men. K. Pilgaard et al. Diabetologia, Issue Volume 52, Number 7 / July, 2009. DOI 10.1007/s00125-009-1307-x

Genes Linked to African Heritage Linked to Poor Carbohydrate Metabolism

It has long been known that African-Americans have a much higher rate of diabetes and metabolic syndrome than the American population as a whole. This has been blamed on lifestyle, but a 2009 genetic study finds strong evidence that the problem is genetic.

The study reports,

Using genetic samples obtained from a cohort of subjects undergoing cardiac-related evaluation, a strict algorithm that filtered for genomic features at multiple levels identified 151 differentially-expressed genes between Americans of African ancestry and those of European ancestry. Many of the genes identified were associated with glucose and simple sugar metabolism, suggestive of a model whereby selective adaptation to the nutritional environment differs between populations of humans separated geographically over time.

In the full text discussion the authors state,

These results suggest that differences in glucose metabolism between Americans of African and European may reside at the transcriptional level. The down-regulation of these genes in the AA cohorts argues against these changes being a compensatory response to hyperglycemia and suggests instead a genetic adaptation to changes in the availability of dietary sugars that may no longer be appropriate to a Western Diet.

In conclusion the authors note that the vegetarian diet of the Seventh Day Adventists, often touted as proof of the usefulness of the “Diet Pyramid” doesn’t provide the touted health benefits to people of African American Heritage. Obviously, when hundreds of carbohydrate metabolizing genes aren’t working properly the diet needed is a low carbohydrate diet.

The study is available in full text here:

Stable Patterns of Gene Expression Regulating Carbohydrate Metabolism Determined by Geographic AncestryJonathan C. Schisler et. al. PLoS One 4(12): e8183. doi:10.1371/journal.pone.0008183

Gene that Disrupts Circadian Clock Associated with Type 2 Diabetes

It has been known for a while that people who suffer from sleep disturbances often suffer raised insulin resistance. In December of 2008, researchers identified a gene, “rs1387153, near MTNR1B (which encodes the melatonin receptor 2 (MT2)), as a modulator of fasting plasma glucose.” They conclude,

Our data suggest a possible link between circadian rhythm regulation and glucose homeostasis through the melatonin signaling pathway.

Melatonin levels appear to control the body clock which, in turn, regulates the secretion of substances that modify blood pressure, hormone levels, insulin secretion and many other processes throughout the body.

A variant near MTNR1B is associated with increased fasting plasma glucose levels and type 2 diabetes risk. Nabila Bouatia-Naji et al. Nature Genetics Published online: 7 December 2008, doi:10.1038/ng.277

There’s an excellent translation of what this study means, translated into layman’s terms at Science Daily:

Body Clock Linked to Diabetes And High Blood Sugar In New Genome-wide Study


The Environmental Factors That Push Borderline Genes into Full-fledged Diabetes

We’ve seen so far that to get Type 2 Diabetes you seem to need to have some diabetes gene or genes, but that not everyone with these genes develops diabetes. There are what scientists call environmental factors that can push a borderline genetic case into full fledged diabetes. Let’s look now at what the research has found about what some of these environmental factors might be.


Your Mother’s Diet During Pregnancy May Have Caused Your Diabetes

Many “environmental factors” that scientists explore occur in the environment of the womb. Diabetes is no different, and the conditions you experienced when you were a fetus can have life-long impact on your blood sugar control.

Researchers following the children of mothers who had experienced a Dutch famine during World War II found that children of mothers who had experienced famine were far more likely to develop diabetes in later life than a control group from the same population whose mothers had been adequately fed.

Glucose tolerance in adults after prenatal exposure to famine. Ravelli AC et al.Lancet. 1998 Jan 17;351(9097):173-7.,

A study of a Chinese population found a link between low birth weight and the development of both diabetes and impaired glucose regulation (i.e. prediabetes) that was independent of “sex, age, central obesity, smoking status, alcohol consumption, dyslipidemia, family history of diabetes, and occupational status.” Low birth weight in this population may well be due to less than optimal maternal nutrition during pregnancy.

Evidence of a Relationship Between Infant Birth Weight and Later Diabetes and Impaired Glucose Regulation in a Chinese Population Xinhua Xiao et. al. Diabetes Care31:483-487, 2008.

This may not seem all that relevant to Americans whose mothers have not been exposed to famine conditions. But to conclude this is to forget how many American teens and young women suffer from eating disorders and how prevalent crash dieting is in the group of women most likely to get pregnant.

It is also true that until the 1980s obstetricians routinely warned pregnant women against gaining what is now understood to be a healthy amount of weight. When pregnant women started to gain weight, doctors often put them on highly restrictive diets which resulted in many case in the birth of underweight babies.

Your Mother’s Gestational Diabetes May Have Caused Your Diabetes

Maternal starvation is not the only pre-birth factor associated with an increased risk of diabetes. Having a well-fed mother who suffered gestational diabetes also increases a child’s risk both of obesity and of developing diabetes.

High Prevalence of Type 2 Diabetes and Pre-Diabetes in Adult Offspring of Women With Gestational Diabetes Mellitus or Type 1 Diabetes The role of intrauterine hyperglycemia Tine D. Clausen, MD et al. Diabetes Care 31:340-346, 2008

Pesticides and PCBs in Blood Stream Correlate with Incidence of Diabetes

A study conducted among members of New York State’s Mohawk tribe found that the odds of being diagnosed with diabetes in this population was almost 4 times higher in members who had high concentrations of PCBs in their blood serum. It was even higher for those with high concentrations of pesticides in their blood.

Diabetes in Relation to Serum Levels of Polychlorinated Biphenyls and Chlorinated Pesticides in Adult Native Americans Neculai Codru, Maria J. Schymura,Serban Negoita,Robert Rej,and David O. Carpenter.Environ Health Perspect. 2007 October; 115(10): 1442-1447.Published online 2007 July 17. doi: 10.1289/ehp.10315.

It is very important to note that there is no reason to believe this phenomenon is limited to people of Native American heritage. Upstate NY has a well-known and very serious PCB problem–remember Love Canal? And the entire population of the U.S. has been overexposed to powerful pesticides for a generation.

More evidence that obesity may be caused by exposure to toxic pollutants which damage genes comes in a study published January of 2009. This study tracked the exposure of a group of pregnant Belgian woman to several common pollutants: hexachlorobenzene, dichlorodiphenyldichloroethylene (DDE) , dioxin-like compounds, and polychlorinated biphenyls (PCBs). It found a correlation between exposure to PCBs and DDE and obesity by age 3, especially in children of mothers who smoked.

Intrauterine Exposure to Environmental Pollutants and Body Mass Index during the First 3 Years of Life Stijn L. Verhulst et al., Environmental Health Perspectives. Volume 117, Number 1, January 2009

These studies, which garnered no press attention at all, probably have more to tell us about the reason for the so-called “diabetes epidemic” than any other published over the last decade.

BPA and Plasticizers from Packaging Are Strongly Linked to Obesity and Insulin Resistance

BPA, the plastic used to line most metal cans has long been suspected of causing obesity. Now we know why. A study published in 2008 reported that BPA suppresses a key hormone, adiponectin, which is responsible for regulating insulin sensitivity in the body and puts people at a substantially higher risk for metabolic syndrome.

Science Daily: Toxic Plastics: Bisphenol A Linked To Metabolic Syndrome In Human Tissue

The impact of BPA on children is dramatic. Analysis of 7 years of NHANES epidemiological data found that having a high urine level of BPA doubles a child’s risk of being obese.

Bisphenol A and Chronic Disease Risk Factors in US Children. Eng, Donna et al.Pediatrics Published online August 19, 2013. doi: 10.1542/peds.2013-0106

You, and your children are getting far more BPA from canned foods than what health authorities assumed they were getting. A research report published in 2011 reported that the level of BPA actually measured in people’s bodies after they consumed canned soup turned out to be extremely high. People who ate a serving of canned soup every day for five days had BPA levels of 20.8 micrograms per liter of urine, whereas people who instead ate fresh soup had levels of 1.1 micrograms per liter.

Canned Soup Consumption and Urinary Bisphenol A: A Randomized Crossover Trial Carwile, JL et al. JAMA. November 23/30, 2011, Vol 306, No. 20

Nevertheless, the FDA caved in to industry pressure in 2012 and refused to regulate BPA claiming that, as usual, more study was needed. (FDA: BPA)

BPA is not the only toxic chemical associated with plastics that may be promoting insulin resistance. . Phthalates are compounds added to plastic to make it flexible. They rub off on our food and are found in our blood and urine. A study of 387 Hispanic and Black, New York City children who were between six and eight years old measured the phthalates in their urine and found that the more phthalates in their urine, the fatter the child was a year later.

Associations between phthalate metabolite urinary concentrations and body size measures in New York City children.
Susan L. Teitelbaum et al.Environ Res. 2012 Jan;112:186-93.

This finding was echosed by another study:

Urinary phthalates and increased insulin resistance in adolescents Trasande L, et al. Pediatrics 2013; DOI: 10.1542/peds.2012-4022.

And phthalates are everywhere. A study of 1,016 Swedes aged 70 years and older found that four phthalate metabolites were detected in the blood serum of almost all the participants. High levels of three of these were associated with the prevalence of diabetes. The researchers explain that one metabolite was mainly related to poor insulin secretion, whereas two others were related to insulin resistance. The researchers didn’t check to see whether this relationship held for prediabetes.

Circulating Levels of Phthalate Metabolites Are Associated With Prevalent Diabetes in the Elderly.Lind, MP et al. Diabetes. Published online before print April 12, 2012, doi: 10.2337/dc11-2396

Chances are very good that these same omnipresent phthalates are also causing insulin resistance and damaging insulin secretion in people whose ages fall between those of the two groups studied here.

Use of Herbicide Atrazine Maps to Obesity, Causes Insulin Resistance

A study published in April of 2009 mentions that “There is an apparent overlap between areas in the USA where the herbicide, atrazine (ATZ), is heavily used and obesity-prevalence maps of people with a BMI over 30.”

It found that when rats were given low doses of this pesticide in thier water, “Chronic administration of ATZ decreased basal metabolic rate, and increased body weight, intra-abdominal fat and insulin resistance without changing food intake or physical activity level.” In short the animals got fat even without changing their food intake. When the animals were fed a high fat,high carb diet, the weight gain was even greater.

Insulin resistance was increased too, which if it happens in people, means that people who have genetically-caused borderline capacity to secrete insulin are more likely to become diabetic when they are exposed to this chemical via food or their drinking water.

Chronic Exposure to the Herbicide, Atrazine, Causes Mitochondrial Dysfunction and Insulin Resistance PLoS ONE Published 13 Apr 2009

2,4-D A Common Herbicide Blocks Secretion of GLP-1–A Blood Sugar Lowering Gastric Peptide

In 2007 scientists at New York’s Mount Sinai Hospital discovered that the intestine has receptors for sugar identical to those found on the tongue and that these receptors regulate secretion of glucagon-like peptide-1 (GLP-1). GLP-1 is the peptide that is mimicked by the diabetes drug Byetta and which is kept elevated by Januvia and Onglyza. You can read about that finding in this Science Daily report:

Science Daily: Your Gut Has Taste Receptors

In November 2009, these same scientists reported that a very common herbicide 2,4 D blocked this taste receptor, effectively turning off its ability to stimulate the production GLP-1. The fibrate drugs used to lower cholesterol were also found to block the receptor.

Science Daily: Common Herbicides and Fibrates Block Nutrient-Sensing Receptor Found in Gut and Pancreas

What was even more of concern was the discovery that the ability of these compounds to block this gut receptor “did not generalize across species to the rodent form of the receptor.” The lead researcher was quoted as saying,

…most safety tests were done using animals, which have T1R3 receptors that are insensitive to these compounds,

This takes on additional meaning when you realize that most compounds released into the environment are tested only on animals, not humans. It may help explain why so many supposedly “safe” chemicals are damaging human glucose metabolisms.

Trace Amounts of Arsenic in Urine Correlate with Dramatic Rise in Diabetes

A study published in JAMA in August of 2008 found of 788 adults who had participated in the 2003-2004 National Health and Nutrition Examination Survey (NHANES) found those who had the most arsenic in their urine, were nearly four times more likely to have diabetes than those who had the least amount.

The study is reported here:

Arsenic Exposure and Prevalence of Type 2 Diabetes in US Adults. Ana Navas-Acien et al. JAMA. 2008;300(7):814-822.

The New York Times report about this study (no longer online) added this illuminating bit of information to the story:

Arsenic can get into drinking water naturally when minerals dissolve. It is also an industrial pollutant from coal burning and copper smelting. Utilities use filtration systems to get it out of drinking water.

Seafood also contains nontoxic organic arsenic. The researchers adjusted their analysis for signs of seafood intake and found that people with Type 2 Diabetes had 26 percent higher inorganic arsenic levels than people without Type 2 Diabetes.

How arsenic could contribute to diabetes is unknown, but prior studies have found impaired insulin secretion in pancreas cells treated with an arsenic compound.

Prescription Drugs, Especially SSRI Antidepressants Cause Obesity and Possibly Diabetes

Another important environmental factor is this: Type 2 Diabetes can be caused by some commonly prescribed drugs. Beta blockers and atypical antipsychotics like Zyprexa have been shown to cause diabetes in people who would not otherwise get it. This is discussed here.

There is some research that suggests that SSRI antidepressants may also promote diabetes. It is well known that antidepressants cause weight gain.

Spin doctors in the employ of the drug companies who sell these high-profit antidepressants have long tried to attribute the relationship between depression and obesity to depression, rather than the drugs used to treat the condition.

However, a new study published in June 2009 used data from the Canadian National Population Health Survey (NPHS), a longitudinal study of a representative cohort of household residents in Canada and tracked the incidence of obesity over ten years.

The study found that, “MDE [Major Depressive Episode] does not appear to increase the risk of obesity. …Pharmacologic treatment with antidepressants may be associated with an increased risk of obesity. [emphasis mine]. The study concluded,

Unexpectedly, significant effects were seen for serotonin-reuptake-inhibiting antidepressants [Prozac,Celexa, Lovox, Paxil, Zoloft] and venlafaxine [Effexor], but neither for tricyclic antidepressants nor antipsychotic medications.

Scott B. Patten et al. Psychother Psychosom 2009;78:182-186 (DOI: 10.1159/000209349)

Here is an article posted by the Mayo Clinic that includes the statement “weight gain is a reported side effect of nearly all antidepressant medications currently available.

Antidepressants and weight gain – Mayoclinic.com

Here is a report about a paper presented at the 2006 ADA Conference that analyzed the Antidepressant-Diabetes connection in a major Diabetes prevention study:

Medscape: Antidepressant use associated with increased type 2 diabetes risk.

Treatment for Cancer, Especially Radiation, Greatly Increases Diabetes Risk Independent of Obesity or Exercise Level

A study published in August 2009 analyzed data for 8599 survivors in the Childhood Cancer Survivor Study. It found that after adjusting for body mass and exercise levels, survivors of childhood cancer were 1.8 times more likely than the siblings to report that they had diabetes.

Even more significantly, those who had had full body radiation were 7.2 times more likely to have diabetes.

This raises the question of whether exposure to radiation in other contexts also causes Type 2 diabetes.

Diabetes Mellitus in Long-term Survivors of Childhood Cancer: Increased Risk Associated With Radiation Therapy: A Report for the Childhood Cancer Survivor Study.Lillian R. Meacham et al. Arch. Int. Med.Vol. 169 No. 15, Aug 10/24, 2009.

More Insight into the Effect of Genetic Flaws

Now that we have a better idea of some of the underlying physiological causes of diabetes, lets look more closely at the physiological processes that takes place as these genetic flaws push the body towards diabetes.

Insulin Resistance Develops in Thin Children of People with Type 2 Diabetes

Lab research has come up with some other intriguing findings that challenge the idea that obesity causes insulin resistance which causes diabetes. Instead, it looks like the opposite happens: Insulin resistance precedes the development of obesity.

One of these studies took two groups of thin subjects with normal blood sugar who were evenly matched for height and weight. The two groups differed only in that one group had close relatives who had developed Type 2 Diabetes, and hence, if there were a genetic component to the disorder, they were more likely to have it. The other group had no relatives with Type 2 Diabetes. The researchers then and examined the subjects’ glucose and insulin levels during a glucose tolerance test and calculated their insulin resistance. They found that the thin relatives of the people with Type 2 Diabetes already had much more insulin resistance than did the thin people with no relatives with diabetes.

Insulin resistance in the first-degree relatives of persons with Type 2 Diabetes. Straczkowski M et al. Med Sci Monit. 2003 May;9(5):CR186-90.

This result was echoed by a second study published in November of 2009.

That study compared detailed measurements of insulin secretion and resistance in 187 offspring of people diagnosed with Type 2 diabetes against 509 controls. Subjects were matched with controls for age, gender and BMI. It concluded:

The first-degree offspring of type 2 diabetic patients show insulin resistance and beta cell dysfunction in response to oral glucose challenge. Beta cell impairment exists in insulin-sensitive offspring of patients with type 2 diabetes, suggesting beta cell dysfunction to be a major defect determining diabetes development in diabetic offspring.

Beta cell (dys)function in non-diabetic offspring of diabetic patients M. Stadler et al. Diabetologia Volume 52, Number 11 / November, 2009, pp 2435-2444. doi 10.1007/s00125-009-1520-7

Mitochondrial Dysfunction is Found in Lean Relatives of People with Type 2 Diabetes

One reason insulin resistance might precede obesity was explained by a landmark 2004 study which looked at the cells of the “healthy, young, lean” but insulin-resistant relatives of people with Type 2 Diabetes and found that their mitochondria, the “power plant of the cells” that is the part of the cell that burns glucose, appeared to have a defect. While the mitochondria of people with no relatives with diabetes burned glucose well, the mitochondria of the people with an inherited genetic predisposition to diabetes were not able to burn off glucose as efficiently, but instead caused the glucose they could not burn and to be stored in the cells as fat.

Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. Petersen KF et al. New England J Med 2004 Feb 12; 350(7);639-41

More Evidence that Abnormal Insulin Resistance Precedes Weight Gain and Probably Causes It

A study done by the same researchers at Yale University School of Medicine who discovered the mitochondrial problem we just discussed was published in Proceedings of the National Academy of Science (PNAS) in July 2007. It reports on a study that compared energy usage by lean people who were insulin resistant and lean people who were insulin sensitive.

The role of skeletal muscle insulin resistance in the pathogenesis of the metabolic syndrome Petersen,KF et al. PNAS July 31, 2007 vol. 104 no. 31 12587-12594.

Using new imaging technologies, the researchers found that lean but insulin resistant subjects converted glucose from high carbohydrate meals into triglycerides–i.e. fat. Lean insulin-sensitive subjects, in contrast, stored the same glucose in the form of muscle and liver glycogen.

The researchers conclude that:

the insulin resistance, in these young, lean, insulin resistant individuals, was independent of abdominal obesity and circulating plasma adipocytokines, suggesting that these abnormalities develop later in the development of the metabolic syndrome.”

In short, obesity looked to be a result, not a cause of the metabolic flaw that led these people to store carbohydrate they ate in the form of fat rather than burn it for energy.

The researchers suggested controlling insulin resistance with exercise. It would also be a good idea for people who are insulin resistant, or have a family history of Type 2 Diabetes to cut back on their carb intake, knowing that the glucose from the carbs they eat is more likely to turn into fat.

Beta Cells Fail to Reproduce in People with Diabetes

A study of pancreas autopsies that compared the pancreases of thin and fat people with diabetes with those of thin and fat normal people found that fat, insulin-resistant people who did not develop diabetes apparently were able to grow new beta-cells to produce the extra insulin they needed. In contrast, the beta cells of people who developed diabetes were unable to reproduce. This failure was independent of their weight.

Beta-Cell Deficit and Increased Beta-Cell Apoptosis in Humans With Type 2 Diabetes. Alexandra E. Butler, et al. Diabetes 52:102-110, 2003

Once Blood Sugars Rise They Impair a Muscle Gene that Regulates Insulin Sensitivity

Another piece of the puzzle falls into place thanks to a research study published on Feb 8, 2008.

Downregulation of Diacylglycerol Kinase Delta Contributes to Hyperglycemia-Induced Insulin Resistance. Alexander V. Chibalin et. al. Cell, Volume 132, Issue 3, 375-386, 8 February 2008.

As reported in Diabetes in Control (which had access to the full text of the study)

The research team identified a “fat-burning” gene, the products of which are required to maintain the cells insulin sensitivity. They also discovered that this gene is reduced in muscle tissue from people with high blood sugar and type 2-diabetes. In the absence of the enzyme that is made by this gene, muscles have reduced insulin sensitivity, impaired fat burning ability, which leads to an increased risk of developing obesity.

“The expression of this gene is reduced when blood sugar rises, but activity can be restored if blood sugar is controlled by pharmacological treatment or exercise”, says Professor Juleen Zierath. “Our results underscore the importance of tight regulation of blood sugar for people with diabetes.”

In short, once your blood sugar rises past a certain point, you become much more insulin resistant. This, in turn, pushes up your blood sugar more.

A New Model For How Diabetes Develops

These research findings open up a new way of understanding the relationship between obesity and diabetes.

Perhaps people with the genetic condition underlying Type 2 Diabetes inherit a defect in the beta cells that make those cells unable to reproduce normally to replace cells damaged by the normal wear and tear of life.Or perhaps exposure to an environmental toxin damages the related genes.

Perhaps, too, a defect in the way that their cells burn glucose inclines them to turn excess blood sugar into fat rather than burning it off as a person with normal mitochondria might do.

Put these facts together and you suddenly get a fatal combination that is almost guaranteed to make a person fat.

Studies have shown that blood sugars only slightly over 100 mg/dl are high enough to render beta cells dysfunctional.

Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Gastaldelli A, et al. Diabetologia. 2004 Jan;47(1):31-9. Epub 2003 Dec 10.

In a normal person who had the ability to grow new beta cells, any damaged beta cells would be replaced by new ones, which would keep the blood sugar at levels low enough to avoid further damage. But the beta cells of a person with a genetic heritage of diabetes are unable to reproduce So once blood sugars started to rise, more beta cells would succumb to the resulting glucose toxicity, and that would, in turn raise blood sugar higher.

As the concentration of glucose in their blood rose, these people would not be able to do what a normal person does with excess blood sugar–which is to burn it for energy. Instead their defective mitochondria will cause the excess glucose to be stored as fat. As this fat gets stored in the muscles it causes the insulin resistance so often observed in people with diabetes–long before the individual begins to gain visible weight. This insulin resistance puts a further strain on the remaining beta cells by making the person’s cells less sensitive to insulin. Since the person with an inherited tendency to diabetes’ pancreas can’t grow the extra beta cells that a normal person could grow when their cells become insulin resistant this leads to ever escalating blood sugars which further damage the insulin-producing cells, and end up in the inevitable decline into diabetes.

Low Fat Diets Promote the Deterioration that Leads to Diabetes in People with the Genetic Predisposition

In the past two decades, when people who were headed towards diabetes begin to gain weight, they were advised to eat a low fat diet. Unfortunately, this low fat diet is also a high carbohydrate diet–one that exacerbates blood sugar problems by raising blood sugars dangerously high, destroying more insulin-producing beta-cells, and catalyzing the storage of more fat in the muscles of people with dysfunctional mitochondria. Though they may have stuck to diets to low fat for weeks or even months these people were tormented by relentless hunger and when they finally went off their ineffective diets, they got fatter. Unfortunately, when they reported these experiences to their doctors, they were almost universally accused of lying about their eating habits.

It has only been documented in medical research during the past two years that that many patients who have found it impossible to lose weight on the low fat high carbohydrate can lose weight–often dramatically–on a low carbohydrate diet while improving rather than harming their blood lipids.

Very low-carbohydrate and low-fat diets affect fasting lipids and postprandial lipemia differently in overweight men. Sharman MJ, et al. J Nutr. 2004 Apr;134(4):880-5.

An isoenergetic very low carbohydrate diet improves serum HDL cholesterol and triacylglycerol concentrations, the total cholesterol to HDL cholesterol ratio and postprandial lipemic responses compared with a low fat diet in normal weight, normolipidemic women. Volek JS, et al. J Nutr. 2003 Sep;133(9):2756-61.

The low carb diet does two things. By limiting carbohydrate, it limits the concentration of blood glucose which often is enough to bring moderately elevated blood sugars down to normal or near normal levels. This means that there will be little excess glucose left to be converted to fat and stored.

It also gets around the mitochondrial defect in processing glucose by keeping blood sugars low so that the body switches into a mode where it burns ketones rather than glucose for muscle fuel.

Relentless Hunger Results from Roller Coaster Blood Sugars

There is one last reason why you may believe that obesity caused your diabetes, when, in fact, it was undiagnosed diabetes that caused your obesity.

Long before a person develops diabetes, they go through a phase where they have what doctors called “impaired glucose tolerance.” This means that after they eat a meal containing carbohydrates, their blood sugar rockets up and may stay high for an hour or two before dropping back to a normal level.

What most people don’t know is that when blood sugar moves swiftly up or down most people will experience intense hunger. The reasons for this are not completely clear. But what is certain is that this intense hunger caused by blood sugar swings can develop years before a person’s blood sugar reaches the level where they’ll be diagnosed as diabetic.

This relentless hunger, in fact, is often the very first diabetic symptom a person will experience, though most doctors do not recognize this hunger as a symptom. Instead, if you complain of experiencing intense hunger doctors may suggest you need an antidepressant or blame your weight gain, if you are female, on menopausal changes.

This relentless hunger caused by impaired glucose tolerance almost always leads to significant weight gain and an increase in insulin resistance. However, because it can take ten years between the time your blood sugar begins to rise steeply after meals and the time when your fasting blood sugar is abnormal enough for you to be diagnosed with diabetes, most people are, indeed, very fat at the time of diagnosis.

With better diagnosis of diabetes (discussed here) we would be able to catch early diabetes before people gained the enormous amounts of weight now believed to cause the syndrome. But at least now people with diabetic relatives who are at risk for developing diabetes can go a long way towards preventing the development of obesity by controlling their carbohydrate intake long before they begin to put on weight.

You CAN Undo the Damage

No matter what your genetic heritage or the environmental insults your genes have survived, you can take steps right now to lower your blood sugar, eliminate the secondary insulin resistance caused by high blood sugars, and start the process that leads back to health. The pages linked here will show you how.

How To Get Your Blood Sugar Under Control

What Can You Eat When You Are Cutting The Carbs?

What is a Normal Blood Sugar

Research Connecting Blood Sugar Level with Organ Damage

The 5% Club: They Normalized Their Blood Sugar and So Can You

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microglia and brain maintenance

Larry H. Bernstein, MD, FCAP, Curator



Mapping mosaicism: Tracing subtle mutations in our brains

Posted on January 14, 2015 by Nancy Fliesler

Posted in All PostsInformation technology

More On: brain developmentDNA sequencinggeneticsmosaicismneurosciencesomatic mutations

DNA sequences were once thought to be the same in every cell, but the story is now known to be more complicated than that. The brain is a case in point: Mutations can arise at different times in brain development and affect only a percentage of neurons, forming a mosaic pattern.

Now, thanks to new technology described last week in Neuron, these subtle “somatic” brain mutations can be mapped spatially across the brain and even have their ancestry traced.

Like my family, who lived in Eastern Europe, migrated to lower Manhattan and branched off to Boston, California and elsewhere, brain mutations can be followed from the original mutant cells as they divide and migrate to their various brain destinations, carrying their altered DNA with them.

“Some mutations may occur on one side of the brain and not the other,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s Hospital and co-senior author on the paper. “Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

This tracking capability represents a significant advance for genetics research. And for neuroscientists, it provides a new way to study both the normal brain and brain disorders like epilepsy, autism and intellectual disability.

Walsh and colleagues studied normal brain tissue from a teenage boy who had passed away from other causes. Sampling in more than 30 brain locations, they used deep, highly sensitive, whole-genome sequencing of one neuron at a time—unlike usual methods, which sequence thousands or millions of cells mixed together and simply read out an average.


The blue and green boxes indicate different degrees of mosaicism (based on proportion of cells affected) in the left half of this teen’s normal brain. The blue shaded area indicates that retrotransposon mutation #1 (blue boxes) is limited to a focal area in the middle frontal gyrus. The empty boxes indicate areas where mutation #1 was not detected. (Courtesy Gilad Evrony, PhD, Boston Children’s Hospital)

Next, using technology developed by Alice (Eunjung) Lee in the lab of Peter Park, PhD, at Harvard Medical School’s Center for Biomedical Informatics, they zeroed in on inserted bits of DNA caused by retrotransposons, one type of mutation that can arise as the brain develops. These essentially served as markers that allowed cell lineages to be traced.

“Our findings are intriguing because they suggest that every normal brain may in fact be a mosaic patchwork of focal somatic mutations, though in normal individuals most are likely silent or harmless,” says Gilad Evrony, PhD, in the Walsh Lab.


This model illustrates the origins of two somatic retrotransposon mutations during prenatal development and their subsequent dissemination in the brain. Insertion #2 (in green) occurred soon after conception; #1 (in blue) happened sometime later during brain development. The ‘pie slices’ show a closeup of the layers of the cerebral cortex. Later in development, additional somatic mutations occurred inside insertions #1 and #2, creating new, smaller sublineages of cells. (Courtesy Gilad Evrony, PhD)

A parallel study from Walsh’s lab in 2014 used single-neuron sequencing to find copy number variants— a different type of mutation affecting the number of copies of chromosomes or chromosome fragments. It, too, found the mutations to be present in normal brains as well as neurologically diseased brains.

Walsh and others speculate that some somatic brain mutations might play a role in autism, epilepsy, schizophrenia and other unsolved neuropsychiatric diseases whose causes are mostly still a mystery.

“It is possible that a whole new class of brain disorders may exist that has not been previously recognized,” says Evrony. “In such disorders, a somatic mutation may subtly affect only one small part of the brain involved in a specific ability, for example language, while sparing the rest of the brain.”

Read more:


Tracking subtle brain mutations, systematicallyTool can trace and spatially map “mosaic” mutations in the brain


BOSTON, Jan. 7, 2015 /PRNewswire-USNewswire/ — DNA sequences were once thought to be identical from cell to cell, but it’s increasingly understood that mutations can arise during brain development that affect only certain groups of brain cells. A technique developed at Boston Children’s Hospital allows these subtle mutation patterns to be traced and mapped spatially for the first time. This capability is a significant advance for genetics research and provides a new way to study both the normal brain and brain disorders such as epilepsy and autism.

Described in the January 7th issue of Neuron, the technique uses “deep,” highly sensitive whole-genome sequencing of single neurons and a new technology that identifies inserted bits of DNA caused by retrotransposons, one of several kinds of so-called somatic mutations that can arise as the brain develops.

The technique picks up somatic mutations that affect just a fraction of the brain’s cells, in a “mosaic” pattern. It also allows “lineage tracing,” showing when during brain development the mutations arise and how they spread through brain tissue as the mutated cells grow, replicate and migrate, carrying the mutation with them.

“There is a lot of genetic diversity from one neuron to the other, and this work gets at how somatic mutations are distributed in the brain,” says Christopher Walsh, MD, PhD, chief of Genetics and Genomics at Boston Children’s and co-senior author on the paper. “Some mutations may occur on one side of the brain and not the other. Some may be ‘clumped,’ affecting just one gyrus [fold] of the brain, disrupting just a little part of the cortex at a time.”

The study examined brain tissue from a deceased 17-year-old who had been neurologically normal, sampling in more than 30 brain locations. It builds on work published by the Walsh lab in 2012, which developed methods to sequence the genomes of single neurons, and represents the first time single neurons have been sequenced in their entirety. The single-cell technique is better at detecting subtle mosaicism than usual DNA sequencing methods, which sequence many thousands or millions of cells mixed together and read out an average for the sample.

Somatic brain mutations, affecting just pockets of cells, can be harmful, and have been suggested as a possible cause of neurodevelopmental disorders such as autism, epilepsy or intellectual disability (see this review article for further background). But they also can be completely benign or have just a subtle effect.

“Our findings are intriguing because they suggest that every normal brain may in fact be a mosaic patchwork of focal somatic mutations, though in normal individuals most are likely silent or harmless,” says Gilad Evrony, PhD, in the Walsh Lab, co-first author on the Neuron paper. “These same technologies can now be used to study the brains of people who died from unexplained neuropsychiatric diseases to determine whether somatic mutations may be the cause.”

Finally, says Evrony, the findings provide a proof-of-principle for a systematic way of studying how brain cells disperse and migrate during development, “something that has not been possible to do before in humans,” he says.

Co-first author Alice Eunjung Lee, PhD, from the lab of Peter Park, PhD, at the Center for Biomedical Informatics at Harvard Medical School, developed the study’s retrotransposon analysis tool, which detects somatic retrotransposon mutations in single-cell sequencing data.

Mirroring these findings, study published by Walsh’s lab in 2014 used single-neuron sequencing to detect copy number variants—another type of mutation affecting the number of copies of chromosomes or chromosome fragments. The study found that these mutations can occur in both normal and neurologically diseased brains.

Evrony and Lee are first authors on the Neuron paper; Walsh and Park are senior authors. The research was supported by the National Institutes of Health (MSTP grant T32GM007753), the National Institute of Neurological Disorders and Stroke (R01 NS079277 and R01 NS032457), the Louis Lange III Scholarship in Translational Research, the Eleanor and Miles Shore Fellowship, the Research Connection and the Manton Center for Orphan Disease Research at Boston Children’s Hospital, the Paul G. Allen Family Foundation and the Howard Hughes Medical Institute.

SOURCE Boston Children’s Hospital


Beth Stevens: A transformative thinker in neuroscience

Posted on September 29, 2015 by Nancy Fliesler

Posted in All PostsDrug discoveryProfiles


More On: Alzheimer’s diseaseautismFM Kirby Neurobiology Centerglial cellsneurosciencesynapse development



When 2015 MacArthur “genius” grant winner Beth Stevens, PhD, began studying the role of glia in the brain in the 1990s, these cells—“glue” from the Greek—weren’t given much thought. Traditionally, glia were thought to merely protect and support neurons, the brain’s real players.

But Stevens, from the Department of Neurology and the F.M. Kirby Neurobiology Center at Boston Children’s Hospital, has made the case that glia are key actors in the brain, not just caretakers. Her work—at the interface between the nervous and immune systems—is helping transform how neurologic disorders like autism, amyotrophic lateral sclerosis (ALS), Alzheimer’s disease and schizophrenia are viewed.

Soon after college graduation in 1993, without prior experience in neuroscience, she helped discoveran interplay between neurons and glial cells known as Schwann cells that controlled production of the nerve insulation known as myelin It was one of the early pieces of evidence that glia and neurons talk to each other.

In 2007, while still a postdoctoral fellow, Stevens showed how star-shaped glial cells called astrocytes influence the development of synapses, or brain connections. Studying neurons, her lab showed that a gene called C1q was markedly more active when astrocytes were present. C1q is an immune gene, one nobody had expected to see in a normal brain. In the context of disease, it initiates the complement cascade, an immunologic pathway for tagging unwanted cells and debris for clearance by other immune cells.

But in healthy developing brains, Stevens showed, C1q was concentrated at developing synapses, or brain connections, apparently marking certain synapses for pruning.

Then in 2012, the Stevens lab showed that microglia—another type of glia usually thought of as immune cells themselves—actively sculpt the brain’s wiring. They literally trim away unwanted, inappropriate synapses by eating them—in the same way they’d engulf and destroy invading bacteria.


That paper was cited by the journal Neuron as the year’s most influential paper.

The same year, she received a Presidential Early Career Award for Scientists and Engineers, honoring her innovative research and scientific leadership.

Stevens’s current investigations are looking at synapse loss—a hallmark of neurodegenerative conditions such as Alzheimer’s—and trying to understand why it occurs. Her lab’s recent work suggests that normal pruning mechanisms that are active during early brain development get re-activated later in life. Intervening with this activation could lead to a new treatment approach, she believes.

Stevens isn’t the only brain researcher at Boston Children’s to become a MacArthur fellow. Neurosurgeon Benjamin Warf, MD, received the honor in 2012.

For more:



Immune cells “sculpt” brain circuits — by eating excess connections

Posted on June 5, 2012 by Nancy Fliesler

Posted in All PostsDrug discoveryPediatrics

More On: ALSAlzheimer’s diseaseautismbrain developmentepilepsyglaucomaHuntington’s diseaseLou Gehrig’s disease,Parkinson’s diseasesynapse development


The above movie shows an immune cell caught in the act of tending the brain—it’s just eaten away unnecessary connections, or synapses, between neurons.

That’s not something these cells, known as microglia, were previously thought to do. As immune cells, it was thought that their job was to rid the body of unwanted pathogens and debris, by engulfing and digesting them.

The involvement of microglia in the brain’s development has started to be recognized only recently. The latest research finds that microglia tune into the brain’s cues, akin to the way they survey their environment for invading microbes, and get rid of excess synapses the same way they’d dispatch these invaders—by eating them.

It’s a whole other way of understanding how the healthy brain develops—at the hands of cells that were once thought to be merely nerve “glue” (the literal meaning of “glia” from the Greek), playing a protective role to neurons, say investigators Beth Stevens, PhD, and Dori Schafer, PhD, of the F.M. Kirby Neurobiology Center at Boston Children’s Hospital.

“In the field of neuroscience, glia have often been ignored,” says Stevens. “But glia aren’t the nerve glue, they’re actively communicating with neurons. People have gotten a new respect for glia and are hungry to know more about them.”

Such knowledge could eventually shed light on brain disorders ranging from autism to Alzheimer’s.

The “eat me” sign

We’re all born with more brain connections than we need. As we begin to encounter our world, they’re trimmed back to fine-tune our circuitry. It’s a bit of an oversimplification, but Stevens and Schafer demonstrated last week in the journal Neuron that when two neurons start talking to each other less – because their connection is no longer important to our lives– the microglia notice that and prune the synapse away.

To study microglia’s pruning activity, Stevens and Schafer used a time-honored model: the visual system. When you cover one eye soon after birth, you force the brain to rewire: Brain connections with the covered eye weaken and those synapses eventually get eliminated.

Using this model, Stevens and Schafer showed that microglia take their cues from a set of signals also used by the immune system, known as the complement cascade. Specifically, microglia carry receptors that recognize the complement protein C3—the same protein found on synapses that are destined for elimination.

“We think that weaker synapses are being tagged with C3, and that microglia are eliminating them just as macrophages would eliminate bacteria,” says Schafer.  “C3 is like an ‘eat me’ signal.”

As a postdoctoral fellow in 2007, Stevens showed that neurons are loaded with complement proteins soon after birth, just when pruning is at its peak. In the new study, she and Schafer deliberately disrupted complement signaling in mice—stripping the microglia of C3 receptors, or blocking those receptors with a drug. When they did so, pruning of irrelevant synapses didn’t occur.

Stevens thinks their findings might have relevance for brain disorders. Developmental brain disorders such as autism, epilepsy or schizophrenia are increasingly seen as disorders of synapse development, and some data suggest that microglia and/or the complement cascade are involved.

At the other end of the spectrum, scientists have noted that microglia—normally in a resting state in adults—are activated in neurodegenerative diseases like glaucoma, Alzheimer’s disease, Lou Gehrig’s disease, Huntington’s disease and Parkinson’s disease. Subtle changes have been found in synapses that might cause them to be targeted for elimination.

So could targeting microglia or the complement cascade prevent synapse loss or alter pruning in these diseases?  “All this is still very speculative,” Stevens cautions. “We first need to understand normal brain development.”


Beth Stevens


Assistant Professor of Neurology, F. M. Kirby Neurobiology Center, Boston Children’s Hospital

Department of Neurology, Harvard Medical School

Boston, Massachusetts

Age: 45

Published September 28, 2015


Beth Stevens is a neuroscientist whose research on microglial cells is prompting a significant shift in thinking about neuron communication in the healthy brain and the origins of adult neurological diseases. Until recently, it was believed that the primary function of microglia was immunological; they protected the brain by reducing inflammation and removing foreign bodies.

Stevens identified an additional, yet critical, role: the microglia are responsible for the “pruning” or removal of synaptic cells during brain development. Synapses form the connections, or means of communication, between nerve cells, and these pathways are the basis for all functions or jobs the brain performs. Using a novel model system that allows direct visualization of synapse pruning at various stages of brain development, Stevens demonstrated that the microglia’s pruning depends on the level of activity of neural pathways. She identified immune proteins called complement that “tag” (or bind) excess synapses with an “eat me” signal in the healthy developing brain. Through a process of phagocytosis, the microglia engulf or “eat” the synapses identified for elimination. This pruning optimizes the brain’s synaptic arrangements, ensuring that it has the most efficient “wiring.”

Stevens’s discoveries indicate that our adult neural circuitry is determined not only by the nerve cells but also by the brain’s immune cells. Her work suggests that adult diseases caused by deficient neural architecture (such as autism and schizophrenia) or states of neurodegeneration (such as Alzheimer’s or Huntington’s disease) may be the result of impaired microglial function and abnormal activation of this pruning pathway. Stevens is redefining our understanding of how the wiring in the brain occurs and changes in early life and shedding new light on how the nervous and immune systems interact in the brain, both in health and disease.

Beth Stevens received B.S. (1993) from Northeastern University and a Ph.D. (2003) from the University of Maryland. She was a postdoctoral fellow (2005–2008) at Stanford University and is currently an assistant professor in the Department of Neurology at Harvard Medical School and the F. M. Kirby Neurobiology Center at Boston Children’s Hospital. She is also an Institute Member of the Broad Institute of MIT and Harvard. Her scientific papers have appeared in such journals as NeuronScienceProceedings of the National Academy of Sciences, and Nature Neuroscience, among others.

– See more at: https://www.macfound.org/fellows/946/#sthash.GpHuiEC6.dpuf

Portraits of scientists who are making a mark on autism research.


Beth Stevens: Casting immune cells as brain sculptors


Shortly after Beth Stevens launched her lab at Boston Children’s Hospital in 2008, she invited students from the Newton Montessori School, in a nearby suburb, to come for a visit. The children peered at mouse and rat brains bobbing in fluid-filled jars. They also learned how to position delicate slices of brain tissue on glass slides and inspect them with a microscope.

This visit sparked a running relationship with the school, with a steady stream of students visiting the growing lab each year. Soon it became too complicated to bring so many children to the lab, so Stevens decided to take her neuroscience lessons on the road, visiting a number of local elementary schools each year. Last year, she dropped in on the classrooms of her 5- and 8-year-old daughters, Zoe and Riley.

“The kids got really excited,” Stevens says. “It’s become such a thing that the principal wants me to come back for the whole school.”

Stevens’ enthusiasm for science has left a lasting impression on researchers, too. Her pioneering work points to a surprise role in brain development formicroglia, a type of cell once considered to simply be the brain’s immune defense system, cleaning up cellular debris, damaged tissue and pathogens. But thanks to Stevens, researchers now appreciate that these non-neuronal cells also play a critical role in shaping brain circuits.

In a 2012 discovery that created a buzz among autism researchers, Stevens and her colleagues discovered that microglia prune neuronal connections, calledsynapses, in the developing mouse brain. The trimming of synapses is thought to go awry in autism. And indeed, emerging work from Stevens’ lab hints at a role for microglia in the disorder.

Stevens has already earned praise and several prizes for her work. In 2012, shereceived the Presidential Early Career Award for Scientists and Engineers, the most prestigious award that the U.S. government bestows on young scientists. And in October, she’ll deliver one of four presidential lectures at the world’s largest gathering of neuroscientists — the annual meeting of the Society for Neuroscience — an honor she shares with three neuroscience heavyweights, including two Nobel laureates.

“The field is probably expecting a lot from Beth,” says Jonathan Kipnis, professor of neuroscience at the University of Virginia. Stevens has put microglia at the forefront, Kipnis says. “What used to be a stepchild of neuroscience research is now getting a lot of attention, and I think in part it’s due to her research.”

Curious mind:

Stevens was born in 1970 in Brockton, Massachusetts, where her mother taught elementary school and her father was the school’s principal. As a child, she was deeply inquisitive, eager to understand how things work. She enjoyed collecting bugs and worms, and would analyze these precious specimens in makeshift labs in her backyard.

But a career in science wasn’t on her radar until high school, when she took a biology class with an inspiring teacher named Anthony Cabral. “He totally made me realize that this could be a career, that I could be a scientist,” Stevens says. “It was that one class that changed it, and I’m like, ‘Okay, I’m going to do this.’”

In 1988, she began studying biology at Northeastern University in Boston, which offered an unusual opportunity. It had a unique cooperative education program that allowed Stevens to spend several semesters working full time in medical labs after finishing her coursework.

After that experience, Stevens knew she wanted to find a job in a research lab. After graduating in 1993, she joined her then-boyfriend Rob Graham, now her husband, in Washington, D.C., where he had landed a job in the U.S. Senate. Stevens headed to the National Institutes of Health (NIH) in Rockville, Maryland, to apply for a job as a research assistant.

At around the same time, neuroscientist R. Douglas Fields was launching his lab at the NIH. He studied how neural impulses influence glia — a class of non-neuronal cells that includes microglia — and shape the structure of the developing brain. Fields readily hired Stevens despite her lack of expertise in neuroscience. “I was impressed with her work ethic, energy and drive,” he says.

Stimulating research:

In Fields’ lab, Stevens used a multi-compartment cell culture system to investigate whether stimulating neurons influences the activity of Schwann cells, glial cells that produce a fatty substance called myelin, which insulates nerves1. She discovered that patterns of neural impulses similar to those that occur during early development influence the maturation of Schwann cells and the production of myelin.

The findings added to mounting evidence that glia and neurons communicate with each other, a newly emerging concept at the time.

“What I loved about the glia research was that there were so few neuroscientists studying it; it was such a mysterious part of neuroscience,” Stevens says. “Those years in Doug’s lab were really exciting because it was a new field.”

Stevens spent five years in Fields’ lab. “She was doing extraordinary work,” Fields says. “She had the potential and the interest to do neuroscience research, and I recommended that she should consider going to graduate school.”

But Stevens didn’t want to give up her position in the lab, and at that time, the NIH did not allow its researchers to have graduate students. So she and Fields convinced the University of Maryland, College Park, just 10 miles away, to allow her to take graduate courses in neuroscience while completing the necessary research for her Ph.D. in Fields’ lab.

In 2000, less than two years after starting graduate school, Stevens published a paper in Science showing that nerves in the peripheral nervous system (located outside the brain and spinal cord) use chemical signals to communicate with Schwann cells2. Two years later, she reported in Neuron that a similar form of communication occurs in the brain, between neurons and oligodendrocytes, the myelin-producing cells in the brain3.

As she was closing in on her Ph.D., Stevens sought career advice from Story Landis, then-director of the National Institute of Neurological Disorders and Stroke. Landis turned Stevens on to the possibility of starting her own lab one day. “I convinced her that she really had the abilities and energy and intelligence to run an independent research program,” Landis says.

In 2004, Stevens sought a postdoctoral fellowship with neurobiologist Ben Barres at Stanford University. “She was already seen as a leading researcher in the glial field,” recalls Barres, who promptly hired her. “She had done all sorts of beautiful work on glia.”

In Barres’ lab, Stevens continued to explore the dialogue between neurons and glia, turning her attention to star-shaped glia called astrocytes. Barres and his team had discovered that astrocytes help neurons form synapses4. To get a better handle on this process, Stevens examined how astrocytes influence gene expression in neurons in the developing mouse brain.

To her surprise, she found that astrocytes trigger neurons to produce a ‘complement’ protein that is best known for its role in the immune system. There, the protein serves as an ‘eat me’ signal, flagging pathogens and debris for removal. She found that neurons deposit this tag around immature synapses, but not mature ones, in mouse brain tissue, and mice that lack this protein have too many immature synapses. The findings suggested that astrocytes might help eliminate synapses by triggering the complement cascade5.



Young recruit: Beth Stevens’ daughter Riley inspects brain tissue during a visit to her mother’s lab. | Courtesy of Beth Stevens

But it was still unclear exactly how the tagged synapses are cleared. The prime suspects were microglia, the only cells in the brain known to have the receptor for the ‘eat me’ signal.

Stevens set out to test this hypothesis in her own lab: After four years as a postdoc, she had decided to branch out on her own. In 2008, neuroscientist Michael Greenberg — chair of the neurobiology department at Harvard — recruited her to the Harvard-affiliated Boston Children’s Hospital. Even when her lab was in its infancy, she had little trouble convincing new staff to join her.

“A lot of people might be a little hesitant to join a new lab,” says Dorothy Schafer, a former postdoctoral fellow in Stevens’ lab who is now assistant professor of neurobiology at the University of Massachusetts-Worcester. “But I was so excited by the research, and she was so energetic and extremely positive, and just seemed like a very nice person.”

One decision Stevens made early on was to continue to studying microglia in mice rather than experiment with new model systems. “You’ll never see her working on songbirds, because she has this aversion to birds,” Schafer says. “I think they think her curly blond hair is a nest or something, and she’s had really bad experiences with many types of birds dive-bombing her head.”

Just four years into her foray as an independent researcher, Stevens found the proof she had been looking for. In 2012, her team published evidence that microglia eat synapses, especially those that are weak and unused6.

The findings pinned down a new role for microglia in wiring the brain. They also helped to explain how the brain, which starts out with a surplus of neurons, trims some of the excess away. Neuron named the paper its most influential publication of 2012.

Stevens continues to study the function of microglia in the healthy brain, most recently uncovering preliminary evidence that a certain protein serves as a ‘don’t eat me’ tag that protects synapses from being engulfed by microglia. She is also exploring the role of microglia in disorders such as autism.

Several studies suggest that microglia are more active and more numerous in the brains of people with autism than in controls. Stevens and her team are looking at whether the activity of microglia is altered during brain development in mouse models of autism.


Immunodulatory Thalidomides in ~ conjugants unleash proteasome degradation on ~ oncoproteins with distinct mechanisms- BRD4,MYC & PIM1 & little collteral damage to 7429 other proteins!

Imagine being able to specifically target a cancer protein for immediate destruction, slipping Robert Louis Stevenson’s notorious black spot into a crevice in the secondary structure and spelling imminent death. Well, this is what Winter et al. (2015) describe in a recent drug discovery report for Science.1 Using phthalimide conjugation, the researchers not only specifically marked BRD4, a transcriptional coactivator important in MYC oncogene upregulation, for proteasomal degradation, but also achieved reduced tumor burdens in vivo.

The research team combined two drugs, thalidomide and JQ1, exploiting the properties of each to create a bifunctional compound, dBET1, that drives the proteasomal degradation of BRD4. JQ1, which in itself is anti-oncogenic, selectively binds BET bromodomains on the transcription factor, thus competitively inhibiting BRD4 activity on chromatin. Thalidomide, a phthalimide-based drug with immunomodulatory properties, binds cereblon (CRBN) in the cullin-RING ubiquitin ligase (CRL) complex, which is important in proteasomal protein degradation.

After confirming that the new phthalimide conjugate, dBET1, retained affinity for BRD4 and that this binding was specific, the team used a human acute myelocytic leukemia (AML) cell line, MV4;11, to show that treatment with the conjugate over 18 hours reduced BRD4 abundance. The researchers also found this with dBET1 treatment of other human cancer cell lines (SUM159, MOLM13). Following this, Winter et al. investigated the mechanisms by which dBET1 inhibits BRD4. By focusing primarily on proteasome function, the researchers determined that the reduction in BRD4 abundance in MV4;11 cells is proteasomal and dependent on CRBN binding activity.

Having established targeted proteasomal degradation using the dBET1 conjugate, Winter et al. then investigated the proteomic consequences of treatment in MV4;11 cells. Scientists at the Thermo Fisher Scientific Center for Multiplexed Proteomics (Harvard Medical School) used quantitative proteomics analysis with an isobaric tagging approach to compare the immediate effects of dBET1 treatment following two hours of incubation with the responses to JQ1 and vehicle control. Spectral data analysis identified 7,429 proteins with few differences in response to either treatment. JQ1 treatment reduced levels of MYC and oncoprotein PIM1 similarly to the response following dBET1 incubation. However, treatment with the latter also reduced BRD2, BRD3 and BRD4 abundance, findings that the research team confirmed with specific immunoblotting. Measuring expression of mRNA showed that both treatments reduced levels of MYC and PIM1 abundance. However, Winter et al. found no difference in BRD3 and BRD4, suggesting that dBET1 reduces the protein levels by post-transcriptional regulation.

Investigating the antiproliferative potential of the phthalimide conjugate, dBET1, Winter and coauthors examined apoptotic response in both MV4;11 and DHL4 lymphoma cells, and in primary human AML blast cultures. Compared to JQ1 treatment, dBET1 stimulated a profound and prolonged apoptotic response in both cell lines, suggesting that targeted degradation could be a more effective treatment than target inhibition.

shapes of proteins as they shift from one stable shape to a different, folded one Protein-structural-changes

shapes of proteins as they shift from one stable shape to a different, folded one Protein-structural-changes

Orchestrating the unfolded protein response in health and disease

Randal J. Kaufman Department of Biological Chemistry,
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor, Michigan, USA J. Clin. Invest. 110:1389–1398 (2002).   http://dx.doi.org:/10.1172/JCI200216886

The endoplasmic reticulum (ER), the entrance site for proteins destined to reside in the secretory pathway or the extracellular environment, is also the site of biosynthesis for steroids and for cholesterol and many lipids. Given the considerable number of resident structural proteins and biosynthetic enzymes and the high expression of many secreted proteins, the total concentration of proteins in the this organelle can reach 100 mg/ml. The ER relies on an efficient system of protein chaperones that prevent the accumulation of unfolded or aggregated proteins and correct misfolded proteins that are caught in low-energy kinetic traps (see Horwich, this Perspective series, ref. 1).

These chaperone-mediated processes expend metabolic energy to ensure high-fidelity protein folding in the lumen of the ER. For example, the most abundant ER chaperone, BiP/GRP78, uses the energy from ATP hydrolysis to promote folding and prevent aggregation of proteins within the ER. In addition, the oxidizing environment of the ER creates a constant demand for cellular protein disulfide isomerases to catalyze and monitor disulfide bond formation in a regulated and ordered manner. Operating in parallel with chaperone dependent protein folding are several “quality control” mechanisms, which ensure that, of all proteins translocated into the ER lumen, only those that are properly folded transit to the Golgi compartment. Proteins that are misfolded in the ER are retained until they reach their native conformation or are retrotranslocated back into the cytosol for degradation by the 26S proteasome. The ER has evolved highly specific signaling pathways to ensure that its protein-folding capacity is not overwhelmed. These pathways, collectively termed the unfolded protein response (UPR), are required if the cell is to survive the ER stress (see Ron, this Perspective series, ref. 2) that can result from perturbation in calcium homeostasis or redox status, elevated secretory protein synthesis, expression of misfolded proteins, sugar/glucose deprivation, or altered glycosylation. Upon accumulation of unfolded proteins in the ER lumen, the UPR is activated, reducing the amount of new protein translocated into the ER lumen, increasing retrotranslocation and degradation of ER-localized proteins, and bolstering the protein-folding capacity of the ER. The UPR is orchestrated by the coordinate transcriptional activation of multiple genes, a general decrease in translation initiation, and a concomitant shift in the mRNAs that are translated.

The recent discovery of the mechanisms of ER stress signaling, coupled with the ability to genetically engineer model organisms, has led to major new insights into the diverse cellular and physiological processes that are regulated by the UPR. Here, I summarize current discoveries that have offered insights into the complex regulation of the UPR and its relevance to human physiology and disease.

Glucose and protein folding Early studies demonstrated that both viral transformation and glucose depletion induce transcription of a set of related genes that were termed glucose-regulated proteins (GRPs) (3). Since viral transformation increases both the cellular metabolic rate and ATP utilization, it became evident that, in both cases, this signal emanates from the ER as a consequence of energy deprivation. Because proteins have different ATP requirements for protein folding prior to export, it has been proposed that the threshold for UPR activation might differ among various cell types, depending on their energy stores and the amount and nature of the secretory proteins they produce (4). Glucose not only provides the metabolic energy needed by cells but also participates directly in glycoprotein folding as a component of oligosaccharide structures.

The recognition and modification of oligosaccharide structures in the lumen of the ER is intimately coupled to polypeptide folding (5). As the growing nascent chain is translocated into the lumen of the ER, a 14-oligosaccharide core (GlcNAc2Man9Glc3) is added to consensus asparagine residues. Immediately after the addition of this core, the three terminal glucose residues are cleaved by the sequential action of glucosidases I and II to yield a GlcNAc2Man9 structure. If the polypeptide is not folded properly, a UDP-glucose:glycoprotein glucosyltransferase (UGGT) recognizes the unfolded nature of the glycoprotein and reglucosylates the core structure to re-establish the glucose-α(1, 3)–mannose glycosidic linkage. Monoglucosylated oligosaccharides containing this bond bind to the ER-resident protein chaperones calnexin and calreticulin.

This quality control process ensures that unfolded glycoproteins do not exit the ER. Treatment of cells with castanospermine, a transition-state analogue inhibitor of glucosidases I and II, inhibits this monoglucosylation cycle, prevents interaction of unfolded glycoproteins with calnexin and calreticulin, and activates the UPR. Genetic alterations that reduce the nucleotide sugar precursor pool or glycosyltransferase reactions likewise activate the UPR (6). Therefore, the recognition of altered carbohydrate structures is in some manner linked to UPR activation.

The UPR in yeast and higher eukaryotes On a cellular level, the accumulation of unfolded proteins in the ER lumen induces the transcription of a large set of genes whose products increase the ER’s volume or its capacity for protein folding or promote the degradation of misfolded proteins through the process of ER-associated protein degradation (ERAD) (7). For example, transcription of the ER protein chaperone BiP is a classical marker for UPR activation in yeast and mammalian cells (8). BiP binds hydrophobic exposed patches on the surfaces of unfolded proteins and interactive sites on unassembled protein subunits, and it releases its polypeptide substrates upon ATP binding.

In parallel, as Ron (this Perspective series, ref. 2) details in his accompanying article, translation is attenuated to decrease the protein-folding load. The complex network of physiological responses to ER stress is regulated by only a few ER transmembrane proteins: IRE1, PERK, and ATF6 (9). IRE1, PERK, and ATF6 are proximal sensors that regulate the production and/or quality of basic leucine zipper–containing (bZIP-containing) transcription factors that may form homo- and heterodimers. Combinatorial interactions of these factors generate diversity in responses for different subsets of UPRresponsive genes. In multicellular organisms, if these adaptive responses are not sufficient to relieve ER stress, the cell dies through apoptosis or necrosis.

IRE1-dependent splicing The UPR-signaling pathway was first described less than ten years ago in the budding yeast Saccharomyces cerevisiae. Elegant studies identified IRE1 as the sensor of unfolded proteins in the ER lumen. IRE1 is a type 1 transmembrane Ser/Thr protein kinase that also has a site-specific endoribonuclease (RNase) activity. The presence of unfolded proteins in the ER lumen promotes dimerization and trans-autophosphorylation, rendering IRE1 active as an RNase, and allowing it to cleave a 252-base intron from the mRNA encoding the transcription factor HAC1 (10). The 5′ and 3′ ends of HAC1 mRNA are spliced together by tRNA ligase in a process that is independent of the spliceosome and the usual intranuclear machinery for mRNA splicing. Splicing of HAC1 mRNA increases its translational efficiency and alters sequence of the encoded HAC1 protein, yielding a potent transcriptional activator (11) that can bind and activate the UPR elements (UPREs) upstream of many UPR-inducible genes. In S. cerevisiae, the UPR activates transcription of approximately 381 genes (7).

All eukaryotic cells appear to have maintained the essential and unique properties of the UPR present in S. cerevisiae, but higher eukaryotes possess additional sensors that generate diverse, coordinately regulated responses that promote stress adaptation or cell death. The mammalian genome contains two homologues of yeast IRE1 — IRE1α and IRE1β. Whereas IRE1α is expressed in most cells and tissues, with high-level expression in the pancreas and placenta (12), IRE1β expression is prominent only in intestinal epithelial cells (13). Both IRE1 molecules respond to the accumulation of unfolded proteins in the ER, which activate their kinase and, thereby, their RNase activities. The cleavage specificities of IRE1α and IRE1β are similar, if not identical, suggesting that they do not recognize different sets of substrates but rather generate temporally specific and tissue-specific expression (14, 15).

Searching for transcription factors that mediate the UPR, Yoshida et al. defined a mammalian ER stress response element [ERSEI; CCAAT(N9)CCACG] that is necessary and sufficient for UPR gene activation. Using a yeast one-hybrid screen, these authors isolated XBP1, a bZIP transcription factor X-box DNA binding protein (16). Subsequently, several groups demonstrated that XBP1 mRNA is a substrate for mammalian IRE1, much as the HAC1 mRNA in S. cerevisiae is processed by the yeast IRE1; this pathway is also conserved in Caenorhabditis elegans (17–20). On activation of the UPR, XBP1 mRNA is cleaved by IRE1 to remove a 26-nucleotide intron and generate a translational frameshift. As expected given the precedent of HAC1 regulation in yeast, the resulting processed mRNA encodes a protein with a novel carboxy-terminus that acts as a potent transcriptional activator.

Overexpression of either IRE1α or IRE1β is sufficient to activate transcription from a BiP promoter reporter construct (15). Analysis of a minimal UPRE motif (TGACGTGC/A) (21) uncovered a transcriptional defect in IRE1α-null mouse embryo fibroblasts that could be complemented by expression of spliced XBP1 mRNA (20), and Yoshida et al. (unpublished data) recently identified a UPR-inducible gene that uniquely requires IRE1α-mediated splicing of XBP1 mRNA. However, neither IRE1α nor IRE1β is necessary for transcriptional activation of the BiP gene, as judged by the phenotype of IRE1α/β–deleted murine cells (20, 22, 23). These results indicate that a subset of UPR targets require IRE1 but that at least one IRE1-independent pathway exists for UPR-mediated transcriptional induction. Deletion of IRE1α causes embryonic lethality at embryonic day 10.5 (E10.5) (20, 22, 23). Therefore, although IRE1α is not required for the UPR, it is clearly required for mammalian embryogenesis. XBP1 deletion also causes embryonic lethality, but the mutant embryos can survive up to day E14.5, consistent with the notion that XBP1 acts downstream of IRE1α. XBP1 deletion causes cardiomyopathy and liver hypoplasia (24, 25). In contrast, IRE1β-null mice develop normally but exhibit increased susceptibility to experimentally induced colitis, a phenotype that is consistent with the specific expression of this kinase in the intestinal epithelium (26).

Activation of ATF6 and PERK by ER stress The activating transcription factor ATF6 (16) has been identified as another regulatory protein that, like XBP1, can bind ERSEI elements in the promoters of UPRresponsive genes. There are two forms of ATF6, both synthesized as ER transmembrane proteins. ATF6α (90 kDa) and ATF6β (110 kDa, also known as CREB-RP) both require the presence of the transcription factor CBF (also called NF-Y) to bind ERSEI (27–30).

On activation of the UPR, both forms of ATF6 are processed to generate 50- to 60-kDa cytosolic, bZIP containing transcription factors that migrate to the nucleus (27). Processing of ATF6 by site-1 protease (S1P) and site-2 protease (S2P) occurs within the transmembrane segment and at an adjacent site exposed to the ER lumen. S1P and S2P are the processing enzymes that cleave the ER-associated transmembrane sterolresponse element–binding protein (SREBP) upon cholesterol deprivation (31). The cytosolic fragment of cleaved SREBP migrates to the nucleus to activate transcription of genes required for sterol biosynthesis. Interestingly, although the mechanism regulating ATF6 processing is similar to that regulating SREBP processing (32), the UPR only elicits ATF6 processing, whereas sterol deprivation alone induces SREBP processing. The SREBP cleavage–activating protein (SCAP) confers specificity for SREBP transport to the Golgi compartment, and consequently cleavage in response to sterol deprivation (33). It is unknown whether another cleavage-activating protein, analogous to SCAP but active only following induction of the UPR, promotes the specific cleavage and activation of ATF6 by S1P and S2P.

Transcription of UPR-responsive genes is induced when the cleaved form of ATF6 activates the XBP1 promoter. Therefore, signaling through ATF6 and IRE1 merges to induce XBP1 transcription and mRNA splicing, respectively (Figure 1, a and b). ATF6 increases XBP1 transcription to produce more substrate for IRE1- mediated splicing that generates more active XBP1, providing a positive feedback for UPR activation. However, cells that lack either IRE1α or ATF6 cleavage can induce XBP1 mRNA (20). These two pathways may thus provide parallel signaling pathways for XBP1 transcriptional induction. Alternatively, another pathway — possibly mediated by the ER-localized protein kinase PERK (see Ron, this Perspective series, ref. 2) — may also contribute to induction of XBP1 mRNA. The binding specificities of XBP1 and ATF6 are similar, although ATF6 binding requires CBF binding to an adjacent site, whereas XBP1 binds independently (17, 20, 21, 34). These binding specificities provide another avenue for complementary interaction between the IRE1-XBP1 and ATF6 pathways at the level of transcriptional activation. In addition, these transcription factors might regulate transcription from a second ERSE (ERSEII), which also contains a CCACG motif (35).

In parallel with the activation of ATF6 processing and the consequent changes in gene transcription, the accumulation of unfolded proteins in the ER also alters cellular patterns of translation. The protein kinase PERK has been implicated in this aspect of the ER stress response (see Ron, this Perspective series, ref. 2). Activated PERK phosphorylates the α subunit of eukaryotic translation initiation factor 2 (eIF2α) and attenuates general protein synthesis. Inactivation of the PERK-eIF2α phosphorylation pathway decreases cells’ ability to survive ER stress (36, 37). The PERK pathway promotes cell survival not only by limiting the protein-folding load on the ER, but also by inducing transcription of UPR- activated genes, one-third of which require phosphorylation of eIF2α for their induction (36). Preferential translation of the transcription factor ATF4 allows for continued activation of these genes under conditions of stress, when general protein synthesis is inhibited (36, 37).

A coordinated mechanism for activation One puzzling question about the UPR is how three independent sensors are activated by a common stimulus, the accumulation of unfolded proteins in the ER lumen. BiP, which negatively regulates the UPR, interacts with all three sensors, IRE1, PERK, and ATF6, under nonstressed conditions and may indeed be the master regulator of UPR activation.

Upon accumulation of unfolded proteins in the ER, BiP is released from IRE1, PERK, and ATF6. It is believed that the unfolded proteins bind BiP and sequester it from interacting with IRE1, PERK, and ATF6 to elicit their activation. In this manner, BiP senses both the level of unfolded proteins and the energy (ATP) level in the cell in regulating the UPR. Following release from BiP, IRE1 and PERK are each free to undergo spontaneous homodimerization mediated by their lumenal domains and to become phosphorylated by their endogenous kinase activities (38, 39). BiP interaction with ATF6 prevents trafficking of ATF6 to the Golgi compartment. For this reason, BiP release permits ATF6 transport to the Golgi compartment, where it gains access to S1P and S2P proteases (32). The regulation of signaling through the free level of BiP is an attractive hypothesis providing a direct mechanism by which all three ER stress sensors could be activated by the same stimulus. In addition, the increase in BiP during the UPR would provide a negative feedback to turn off UPR signaling. However, in certain cells, different stress conditions can selectively activate only one or two of the ER stress sensors. For example, in pancreatic β cells, glucose limitation appears to activate PERK prior to activation of IRE1 (D. Scheuner and R.J. Kaufman, unpublished results). It will be important to elucidate how general BiP repression permits the selective activation of individual components of the UPR that mediate various downstream effects.

Signaling the UPR in eukaryotes

Signaling the UPR in eukaryotes

Figure 1 Signaling the UPR in eukaryotes.


Three proximal sensors, IRE1, PERK, and ATF6, coordinately regulate the UPR through their various signaling pathways. Whereas IRE1 and PERK are dispensable for many aspects of the response, ATF6 cleavage is required for UPR transcriptional induction and appears to be the most significant of these effectors in mammalian cells. BiP negatively regulates these pathways. BiP interacts with ATF6 to prevent its transport to the Golgi compartment (a). BiP binds to the lumenal domains of IRE1 (b) and PERK (c) to prevent their dimerization. As unfolded proteins accumulate, they bind BiP and reduce the amount of BiP available to bind and inhibit activation of IRE1, PERK, and ATF6. (a) BiP release from ATF6 permits transport to the Golgi compartment. In the Golgi, ATF6 is cleaved by S1P and S2P proteases to yield a cytosolic fragment that migrates to the nucleus to activate transcription of responsive genes, including XBP1. (b) BiP release from IRE1 permits dimerization to activate its kinase and RNase activities to initiate XBP1 mRNA splicing. XBP1 splicing removes a 26-base intron, creating a translational frameshift to yield a more potent transcriptional activator. (c) BiP release permits PERK dimerization and activation to phosphorylate Ser51 on eIF2α to reduce the frequency of AUG initiation codon recognition. As eIF2α phosphorylation reduces the functional level of eIF2, the general rate of translation initiation is reduced. However, selective mRNAs, such as ATF4 mRNA, are preferentially translated under these conditions, possibly by the presence of open reading frames within the 5′ untranslated region of the mRNA. Upon recovery from the UPR, GADD34 targets PP1 to dephosphorylate eIF2α and increase protein translation.

The UPR as a mediator of programmed cell death In contrast to UPR-signaling adaptation in response to ER stress, prolonged UPR activation leads to apoptotic cell death (Figure 2). The roles of several death-promoting signaling pathways have been shown by analysis of specific gene-deleted cells. Activated IRE1 recruits c-Jun-N-terminal inhibitory kinase (JIK) and the cytosolic adaptor TRAF2 to the ER membrane (22, 40). TRAF2 activates the apoptosis-signaling kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase (MAPKKK) (41). Activated ASK1 leads to activation of the JNK protein kinase and mitochondriadependent caspase activation (40–42).

ER insults lead to caspase activation by mitochondria/APAF-1–dependent and –independent pathways. ER stress promotes cytochrome c release from mitochondria, possibly by c-ABL kinase (43) or calcium (44). However, APAF1–/– cells are susceptible to ER stress–induced apoptosis, indicating that the mitochondrial pathway is not essential (45). Caspase-12 is an ER-associated proximal effector in the caspase activation cascade, and cells lacking this enzyme are partially resistant to inducers of ER stress (46). ER stress induces TRAF2 release from procaspase 12, allowing it to bind activated IRE1. As shown in Figure 2, release of TRAF2 permits clustering of procaspase-12 at the ER membrane, leading to its activation (40). Caspase-12 can activate caspase-9, which in turn activates caspase- 3 (47). Procaspase-12 can also be activated by m-calpain in response to calcium release from the ER, although the physiological significance of this pathway is not known (48). In addition, upon ER stress, procaspase-7 is activated and recruited to the ER membrane (49). These findings support the notion that ER stress leads to several redundant pathways for caspase activation.

A second death-signaling pathway activated by ER stress is mediated by transcriptional activation of genes encoding proapoptotic functions. Activation of UPR sensor IRE1, PERK, or ATF6 leads to transcriptional activation of CHOP/GADD153, a bZIP transcription factor that potentiates apoptosis (see Ron, this Perspective series, ref. 2).

The UPR in health and disease Primary amino acid sequence contains all the information for a protein to attain its final folded conformation. However, many folding intermediates exist along the folding pathway (see Horwich, this Perspective series, ref. 1), and some of these intermediates can become irreversibly trapped in low-energy states and activate the UPR. Clearance of such misfolded species requires a functional ER-associated degradation (ERAD) pathway, which is regulated by the UPR. Proteasomal degradation of ER-associated misfolded proteins is required to protect from UPR activation. Proteasomal inhibition is sufficient to activate the UPR, and, in turn, genes encoding several components of ERAD are transcriptionally induced by the UPR (7). Therefore, it is to be expected that UPR activation and impaired ERAD function might contribute to a variety of diseases and that polymorphisms affecting the UPR and ERAD responses could modify disease progression. The following examples provide the best available evidence linking the UPR pathway to the natural history of human diseases and animal models of these diseases.

The UPR and ERAD in genetic disease Many recessive inherited genetic diseases are due to loss  of-function mutations that disturb productive folding and that produce proteins that are either not secreted or not functional. In other cases, protein-folding mutations can interfere with cellular processes, resulting in a gain of function and a dominant pattern of inheritance. In several instances, UPR activation by the accumulation of unfolded proteins in the ER is known to contribute to disease progression. The distinction between these two classes of genetic disease is important, because gain-of-function protein-misfolding mutations will be less amenable to treatment by gene therapy to deliver a wild-type copy of the mutant gene.

One well-characterized protein-folding defect results from a mutation that leads to type 1 diabetes. The Akita mouse has a gain-of-function Cys96Tyr mutation in the proinsulin 2 (Ins2) gene; this mutation disrupts proinsulin folding. The mutant protein is retained in the ER of the pancreatic β cell and activates the UPR. Crucially, the progressive development of diabetes in this model is not solely due to the lack of insulin but is rather a consequence of the misfolded protein accumulation, UPR activation, and β cell death. When bred into a Chop–/–background, the Akita mutation causes a lesser degree of β cell death and delayed onset of diabetes (50), indicating that the loss of at least one downstream signaling component of the UPR can ameliorate pathogenesis in this setting.

Signaling UPR-mediated cell death

Signaling UPR-mediated cell death

Figure 2 Signaling UPR-mediated cell death.


The activation of procaspase-12 is likely the major pathway that induces apoptosis in response to ER stress. Upon activation of the UPR, c-Jun-N-terminal inhibitory kinase (JIK) release from procaspase-12 permits clustering and activation of procaspase-12. Caspase-12 activates procaspase-9 to activate procaspase-3, the executioner of cell death. In addition, activated IRE1 binds JIK and recruits TRAF2, which signals through apoptosis-signaling kinase 1 (ASK1) and JNK to promote mitochondria-dependent apoptosis. In addition, in vitro studies suggest that localized calcium release from the ER activates m-calpain to cleave and activate procaspase-12. Upon UPR activation, procaspase-7 is activated and recruited to the ER membrane. Finally, IRE1, PERK, and ATF6 induce transcription of several genes encoding apoptotic functions, including CHOP/GADD153. CSP, caspase; pCSP, procaspase.

Deficiency in α1-proteinase inhibitor (α1-PI, also known as α1-antitrypsin) results in emphysema and destructive lung disease in one out of 1,800 births. However, a subgroup of affected individuals develop chronic liver disease and hepatocellular carcinoma as a consequence of a secretion defect in the misfolded protein at the site of synthesis, the hepatocyte. This is the most common genetic cause of liver disease in children. The Z allele of the α1 gene PI (Glu342Lys mutation) produces a protein that polymerizes and is retained in the ER for degradation by the proteasome (see Lomas and Mahadeva, this Perspective series, ref. 51; and Perlmutter, this series, ref. 52). While α1-PI Z neither binds BiP nor activates the UPR, analysis of fibroblasts obtained from these patients demonstrates that individuals susceptible to liver disease have inherited a second trait that slows degradation of the misfolded protein in the ER (53), consistent with the idea that polymorphisms that reduce ERAD function can exacerbate pathogenesis of certain diseases.

There are numerous additional genetic misfolding diseases that are also likely influenced by UPR signaling. Because BiP release from IRE1, PERK, or ATF6 can activate the UPR, the expression of any wild-type or mutant protein that binds BiP can have a similar effect. In contrast, misfolded proteins that do not bind BiP are unlikely to activate the UPR. For example, cystic fibrosis is due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Approximately 70% of patients with this disease carry a common mutation, deletion of Phe508, that results in a molecule that is retained in the ER and eventually degraded by the proteosome (see Gelman and Kopito, this Perspective series, ref. 54). Although expression of ∆508 CFTR does not activate the UPR in cultured cells, the protein does interact with calnexin, as well as HSP70, and requires ERAD function for cell survival.

Osteogenesis imperfecta (OI) results from misfolding mutations in procollagen that produce molecules that bind BiP and activate the UPR (55). Interestingly, Wolcott-Rallison syndrome is due to inactivating mutations in the PERK gene. Affected individuals, as well as mice with deletions in Perk, display osteoporosis and deficient mineralization throughout the skeletal system (56, 57), the same defects that are observed in OI. Procollagen type I accumulates to high levels and mature collagen is not detected in bone and osteoblasts from PERKnull mice. Osteoblasts from PERK-null humans and mice display fragmented and distended ER that is filled with electron-dense material (56, 57). These observations suggest that procollagen type 1 uniquely requires PERK function to maintain its transport out of the ER, processing, and secretion In this case, PERK may be required to limit procollagen synthesis so that it does not saturate the ER protein-folding capacity.

The UPR and ERAD in conformational diseases Diseases caused by expansion of polyglutamine repeats and neurodegenerative diseases, such as Alzheimer disease and Parkinson disease, represent a large class of conformational diseases associated with accumulation of abnormal protein aggregates in and around affected neurons. Recent evidence indicates that the pathogenesis of these diseases is due to a defect in proteasomal function that results in UPR activation, leading to cell death. The protein aggregates in these diseases are localized to the nucleus or the cytoplasm and would not be predicted to disturb ER function directly. Nevertheless, they have been found in some cases to activate the UPR and to promote cell death. Analysis of the polyglutamine repeat associated with the spinocerebrocellular atrophy protein (SCA3) in Machado-Joseph disease suggests that cytoplasmic accumulation of the SCA3 aggregate can inhibit proteasome function, thereby interfering with ERAD to induce the UPR and elicit caspase-12 activation (41, 58). These findings support the idea that the UPR can signal the accumulation of unfolded proteins in the cytosol via proteasomal inhibition and disruption of ERAD function.

Parkinson disease is the most common movement disorder, affecting about 1% of individuals 65 years of age or older. Autosomal recessive juvenile parkinsonism (AR-JP) results from defects in the Parkin gene (59), which encodes a ubiquitin protein ligase (E3) that functions with ubiquitin-conjugating enzyme UbcH7 or UbcH8 to tag proteins for degradation. Overexpression of Parkin suppresses cell death associated with ER stress (60). Inherited Parkinson disease is associated with the accumulation in the ER of dopaminergic neurons of PAEL-R, a putative transmembrane receptor protein that is detected in an insoluble form in the brains of AR-JP patients (61). The accumulation of PAEL-R results from defective Parkin that does not maintain the proteasome-degrading activity necessary to maintain ER function (62). Other, still-unidentified substrates of the Parkin E3 ligase may also be relevant to the pathogenesis of AR-JP.

The UPR in diabetes The metabolism of glucose is tightly controlled at the levels of synthesis and utilization through hormonal regulation. The most dramatic phenotype in Wolcott-Rallison syndrome is pancreatic β cell death with infancy onset diabetes (56). A similar defect is observed in PERK-null mice; this defect also correlated with increased apoptosis of β cells (57, 63). In addition, mice with a homozygous Ser51Ala mutation at the PERK phosphorylation site in eIF2α display an even greater β cell loss that appears in utero (36). Therefore, translational control through PERK-mediated phosphorylation of eIF2α is required to maintain β cell survival (see Ron, this Perspective series, ref. 2). The more severe β cell loss in mice harboring the Ser51Ala eIF2α mutation suggests that additional eIF2α kinases partially complement the requirement for PERK in β cell function (36)

Glucose not only promotes the secretion of insulin but also stimulates insulin transcription and translation (64–66). Our group has proposed that glucose stimulated proinsulin mRNA translation is regulated by PERK-mediated phosphorylation of eIF2α in response to UPR activation 36). As blood glucose declines, energy may become limiting for protein folding in the ER and therefore activate the UPR to promote PERK-mediated phosphorylation of eIF2α. Conversely, a rise in blood glucose would turn off the UPR so that translation would accelerate, allowing entry of new preproinsulin into the ER. In this manner, PERK mediated phosphorylation of eIF2α provides a brake on protein synthesis, including proinsulin translation. Continual elevation of blood glucose may also prolong elevated proinsulin translation, eventually activating the UPR as the secretion capacity of the ER is overwhelmed. Therefore, a delicate balance between glucose levels and eIF2α phosphorylation needs to be maintained: Disturbances in either direction may lead to excessive UPR activation, with eventual β cell death.

The insulin resistance and hyperglycemia associated with type 2 diabetes is accommodated by an increase in proinsulin translation. Under these conditions the UPR is activated to compensate for the increased protein-folding requirement in the ER. Prolonged activation of the UPR could contribute to the β cell death associated with insulin resistance. Thus, the signaling mechanisms that β cells use for sensing glucose levels, triggering insulin secretion, and rapidly controlling insulin biosynthesis may have coevolved with ER signaling pathways to support these specialized functions. Pancreatic β cells are exquisitely sensitive to physiological fluctuations in blood glucose, because, in contrast to other cell types, they lack hexokinase, an enzyme with a low affinity but a high capacity for binding glucose. Therefore, in β cells, the production of glucose 6-phosphate and the production of ATP through glycolysis are controlled by glucokinase (67), and the ratio of ATP to ADP correlates directly with the blood glucose level. Periodic decreases in blood glucose level (as occurs between meals) would decrease the ATP/ADP ratio and compromise protein folding in the ER so that the UPR may be frequently activated in these cells. Hence, when glucose levels vary within the normal physiological range, the ER compartment of the β cell may be exposed to greater energy fluctuations than is the ER of other cell types, making the β cell uniquely dependent on the UPR for survival during intermittent decreases in blood glucose levels, as happens between meals. Additionally, the high-level expression of PERK and IRE1α in the pancreas may predispose these kinases to dimerization and activation in response to intermittent stress.

The UPR in organelle expansion The UPR is required for ER expansion that occurs upon differentiation of highly specialized secretory cells, but ER membrane expansion can also proceed independently of UPR activation. Overexpression of membrane proteins, such as HMG CoA reductase or the peroxisomal protein Pex15, promotes the expansion of smooth membranes without UPR activation (68, 69), as does overexpression of the p180 ribosome acceptor in the rough ER membrane (70). Conversely, protein overexpression, even under circumstances in which secretory capacity is unchanged (as occurs following the induction of high levels of cytochrome p450), can activate the UPR to induce ER chaperone levels to match the expanded membrane area (71, 72).

During the terminal differentiation of certain secretory cells, such as those in the pancreas or liver, membrane expansion is accompanied by a dramatic increase in protein secretion. Likewise, upon B cell maturation into high-level antibody-secreting plasma cells, the ER compartment expands approximately fivefold to accommodate the large increase in Ig synthesis. The requirement for the UPR in this latter process has been demonstrated in XBP1–/– cells. Since deletion of XBP1 produces an embryonic-lethal phenotype at day E14.5, the role of XBP1 in B and T cell development had to be studied in immunoincompetent RAG1–/– mice reconstituted with XBP1–/– embryonic stem cells (73). Work in these chimeric mice demonstrated that XBP1 is required for high-level Ig production. Interestingly, the induction of Ig heavy-chain and light-chain gene rearrangement and the assembly and transport of Igµ to the surface of the B cells occurred normally. However, plasma cells were not detected, suggesting a role for XBP1 in plasma cell differentiation or survival.

These findings support the hypothesis that induction of Ig synthesis activates the UPR to induce ER expansion to accommodate the high-level antibody expression. Alternatively, activation of the UPR may be part of the differentiation program that occurs prior to induction of high-level antibody synthesis. Plasma cell differentiation is stimulated in vivo by treatment with LPS or by ligation of CD40 receptors, treatments that activate the innate immune response and have been shown to induce XBP1 mRNA splicing (19). Thus, the UPR may contribute to a programmed response to signals that increase a cell’s protein-secretory demand.

The UPR in hyperhomocysteinemia. The association between high levels of serum homocysteine and the development of ischemic heart disease and stroke is supported by substantial epidemiological data. Unfortunately, it is not known whether homocysteine is the underlying cause of atherosclerosis and thrombosis. Severe hyperhomocysteinemia is caused by mutation in the cystathionine β-synthase (CBS) gene, whose product is a vitamin B6–dependent enzyme required for the conversion of homocysteine to cysteine. Elevated homocysteine is also associated with vitamin B deficiency. In cultured vascular endothelial cells, homocysteine induces protein misfolding in the ER by interfering with disulfide bond formation, and it activates the UPR to induce expression of several ER stress response proteins, such as BiP, GRP94, CHOP, and HERP (74–76). Homocysteine also activates apoptosis in a manner that requires an intact IRE1-signaling pathway (76).

These findings suggest that homocysteine acts intracellularly to disrupt ER homoeostasis. Indeed, recent studies confirm that induction of hyperhomocysteinemia elicits UPR activation in the livers of normal or Cbs+/– mice (77). In addition, hyperhomocysteinemia activates SREBP cleavage, leading to intracellular accumulation of cholesterol (77). Increased cholesterol biosynthesis may explain the hepatic steatosis and possibly the atherosclerotic lesions associated with hyperhomocysteinemia. Finally, hyperhomocysteinemia accelerates atherosclerosis in ApoE–/– mice (78, 79), although the molecular mechanisms remain to be elucidated.

Hyperhomocysteinemia is also associated with increased amyloid production and increased amyloid-mediated neuronal death in animal models of Alzheimer disease (80). These observations suggest that the UPR may link the disease etiologies of hyperhomocysteinemia and Alzheimer disease. HERP, a homocysteine-induced ER stress–responsive gene, appears to be involved in amyloid β-protein (Aβ) accumulation, including the formation of senile plaques and vascular Aβ deposits (81), and that it interacts with both presenilin-1 (PS1) and presenilin-2 (PS2), thus regulating presenilin-mediated Aβ generation. Immunohistochemical analysis of brains from patients with Alzheimer disease reveals intense HERP staining in activated microglia in senile plaques.

The UPR in cancer Hypoxia is a common feature of solid tumors that display increased malignancy, resistance to therapy, and poor prognosis. Hypoxia in the tumor results from increased demand due to dysregulated cell growth and from vascular abnormalities associated with cancerous tissue. The importance of hypoxia has been seen in the clinic, since it predicts for poor outcome of treatments, independent of treatment modality. Hypoxia activates the UPR, whose downstream signaling events can undermine the efficacy of treatment. Tumor cells need to adapt to the increasingly hypoxic environment that surrounds them as they grow, and the induction of the UPR is key to this response. Induction of the ER stress response genes, for example BiP and GRP94, in cancerous tissue correlates with malignancy, consistent with their antiapoptotic function (82). In addition, the UPR confers resistance to topoisomerase inhibitors, such as etoposide, and some UPR-induced genes directly mediate drug resistance via the multi-drug-resistance gene MDR. Therefore, approaches to prevent UPR activation in cancerous cells may significantly improve treatment outcome.

The proteasome inhibitor PS-341 is now in earlyphase clinical evaluation for the treatment of multiple myeloma, a clonal B cell tumor of differentiated plasma cells (83). The mechanism of PS-341 function is thought to be inhibition of IκB degradation, which prevents activation of the antiapoptotic transcription factor NF-κB. However, proteasomal inhibition would also prevent ERAD. As high-level heavy- or light-chain Ig production is likely associated with a certain degree of protein misfolding, it is possible that inhibition of ERAD function may be selectively toxic to B cell myelomas through activation of the UPR and apoptosis.

The UPR and viral pathogenesis The two major mediators of the IFN-induced arm of the innate immune response are evolutionarily related to IRE1 and PERK. The kinase/endoribonuclease domain of IRE1 is homologous to RNaseL, and the protein kinase domain of PERK is related to the double-stranded RNA–activated (dsRNA-activated) eIF2α protein kinase PKR. RNase L and PKR mediate the IFN induced antiviral response of the host, which is required to limit viral protein synthesis and pathogenesis. As part of the innate immune response to viral infection, RNase L and PKR are activated by dsRNAs produced as intermediates in viral replication. In contrast to activation by dsRNA, IRE1 and PERK are activated by ER stress, which can be induced by high-level viral glycoprotein expression. All enveloped viruses produce excess glycoproteins that could elicit PERK and IRE1 activation to meet the need for increased folding and secretory capacity. More studies will be required to elucidate the role of the UPR in various viral diseases.

Hepatitis C virus (HCV) is a positive-stranded RNA virus encoding a single polyprotein. Polyprotein cleavage generates at least ten polypeptides, including two glycoproteins, E1 and E2. A large amount of E1 forms disulfide–cross-linked aggregates with E2 in the ER (84). Since the accumulation of misfolded α1-PI elicits UPR activation, with subsequent hepatocyte death and hepatocellular carcinoma, it is possible that the aggregated E1/E2 complexes in the HCV-infected hepatocyte also contribute to hepatitis and hepatocellular carcinoma. Future studies should identify whether these glycoprotein aggregates activate the UPR to mediate the hepatocyte cell death and transformation associated with the pathogenesis of HCV infection.

The UPR in tissue ischemia Finally, neuronal death due to reperfusion after ischemic injury is associated with activation of the UPR (85, 86). Immediately after reperfusion, protein synthesis is inhibited, due at least in part to phosphorylation of eIF2α; this inhibition may represent a protective mechanism to prevent further neuron damage. Recent studies support the idea that eIF2α phosphorylation in response to reperfusion injury is mediated by PERK and hence that it depends on the UPR (87). If so, UPR activation prior to ischemic injury might protect the brain and other tissues from cell death during periods of reperfusion.

Summary A variety of approaches have been employed to identify the UPR signaling components, their function, and their physiological role. Yeast genetics allowed the definition of the basic ER stress–signaling pathway. The identification of homologous and parallel signaling pathways in higher eukaryotes has produced a mechanistic framework the cell uses to sense and compensate for ER over-load and stress. The high-level tissue-specific expression patterns of several ER stress–signaling molecules indicated the pancreas and intestine as organs that require UPR for physiological function. Analysis of UPR-induced gene expression established that protein degradation is required to reduce the stress of unfolded protein accumulation in the ER. Major advances in identifying UPR function and rele vance to disease were derived from mutation of UPR signaling components in model organisms and the identification of mutations in humans.

Despite tremendous progress, our knowledge of the UPR pathway remains incomplete. Further studies promise to expand our understanding of how ER stress impacts the other cellular signaling pathways. It will be very exciting and informative to understand how the UPR varies when critical components are genetically manipulated by deletion or other types of mutations. In addition, although the accumulation of unfolded protein in the ER is now known to contribute to pathogenesis in a variety of diseases, there are still few therapeutic approaches that target these events. With a greater understanding of protein-folding processes, pharmacological intervention with chemical chaperones to promote proper folding becomes feasible, as observed with sodium phenylbutyrate for ∆508 CFTR (see Gelman and Kopito, this Perspective series, ref. 53). Future intervention should consider activation of different subpathways of the UPR or overexpression of appropriate protein chaperones, as in the case of overexpression of the J domain of cytosolic HSP70, which suppresses polyglutamine toxicity in flies (88). Treatments that activate the ERAD response may also ameliorate pathogenesis in a number of the conformational diseases.

Over the past ten years, tremendous progress has been made in understanding the mechanisms and physiological significance of the UPR. The processes of protein folding and secretion, transcriptional and translational activation, and protein degradation are intimately interconnected to maintain homeostasis in the ER. A variety of environmental insults, genetic disease, and underlying genetic modifiers of UPR function contribute to the pathogenesis of different disease states. As we gain a greater understanding of the mechanisms that control UPR activation, it should be possible to discover methods to activate or inhibit the UPR as desired for therapeutic benefit.

  1. Horwich, A. 2002. Protein aggregation in disease: a role for folding intermediates forming specific multimeric interactions. J. Clin. Invest. 110:1221–1232. doi:10.1172/JCI200216781.
  2. Ron, D. 2002. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110:1383–1388. doi:10.1172/JCI200216784.
  3. Lee, A.S. 1992. Mammalian stress response: induction of the glucose-regulated protein family. Curr. Opin. Cell Biol. 4:267–273.
  4. Kaufman, R.J., et al. 2002. The unfolded protein response in nutrient sensing and differentiation. Nat. Rev. Mol. Cell Biol. 3:411–421.
  5. Ellgaard, L., and Helenius, A. 2001. ER quality control: towards an understanding at the molecular level. Curr. Opin. Cell Biol. 13:431–437.
  6. Jakob, C.A., Burda, P., Te Heesen, S., Aebi, M., and Roth, J. 1998. Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo. Glycobiology. 8:155–164.
  7. Travers, K.J., et al. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101:249–258.


Citation: Cell Death and Disease (2014) 5, e1578; doi:10.1038/cddis.2014.539
Published online 18 December 2014

An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina

T Rana1, V M Shinde1, C R Starr1, A A Kruglov1, E R Boitet1, P Kotla1, S Zolotukhin2, A K Gross1 and M S Gorbatyuk1

  1. 1Department of Vision Sciences, University of Alabama at Birmingham, AL, USA
  2. 2Department of Pediatrics, University of Florida, FL, USA

Correspondence: M Gorbatyuk, Department of Vision Sciences, University of Alabama at Birmingham, 1670 University Boulevard, Birmingham, 35233 AL, USA. Tel: +1 205 934 6762; Fax: +1 205 934 3425; E-mail:mgortk@uab.edu

Received 20 July 2014; Revised 23 October 2014; Accepted 27 November 2014

Edited by P Ekert

Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19%reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1β.


ERG, electroretinography; SD-OCT, spectral domain optical coherence tomography; UPR, unfolded protein response; IL-1β, Interleukin-1β; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; NF-kB, ; nuclear factor kappa B, ; ER, endoplasmic reticulum; ADRP, autosomal dominant retinitis pigmentosa; RHO, rhodopsin; ERAI, ER stress activated indicator; Tn, tunicamycin; ONL, outer nuclear layer; H&E, hematoxylin and eosin; ONH, optic nerve head


ER stress and neuroinflammation: connecting the unfolded protein response to JAK/STAT signaling (P5196)

Gordon Meares,1 and Etty Benveniste1

1Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL

J Immunol May 2013 190 (Meeting Abstract Supplement) 198.5


Neuroinflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by misfolded proteins. In turn, cells respond with activation of the unfolded protein response (UPR). The UPR is a highly conserved pathway that transmits both adaptive and apoptotic signals to restore homeostasis or eliminate the irreparably damaged cell. Recent evidence indicates that ER stress and inflammation are linked. In this study, we have examined the interaction between ER stress and JAK/STAT-dependent inflammation in astrocytes. The JAK/STAT pathway mediates the biological actions of many cytokines and growth factors. We have found that ER stress leads to the activation of STAT3 in a JAK1-dependent fashion. ER stress-induced activation of the JAK1/STAT3 axis leads to expression of IL-6 and several chemokines. The activation of STAT3 signaling is dependent on the protein kinase PERK, a central component of the UPR. Knockdown of PERK abrogates ER stress-induced activation of STAT3 and overexpression of PERK is sufficient to activate STAT3. Additionally, ER stressed astrocytes, via paracrine signaling, can stimulate activation of microglia leading to production of oncostatin M (OSM). OSM can then synergize with ER stress in astrocytes to drive inflammation. Together, this work describes a new PERK-JAK1-STAT3 signaling pathway that may elicit a feed-forward inflammatory loop involving astrocytes and microglia to drive neuroinflammation.


Neural Plasticity
Volume 2014 (2014), Article ID 610343, 15 pages

Review Article

Surveillance, Phagocytosis, and Inflammation: How Never-Resting Microglia Influence Adult Hippocampal Neurogenesis

Amanda Sierra,1,2,3 Sol Beccari,2,3 Irune Diaz-Aparicio,2,3 Juan M. Encinas,1,2,3 Samuel Comeau,4,5 and Marie-Ève Tremblay4,5

1Ikerbasque Foundation, 48011 Bilbao, Spain
2Achucarro Basque Center for Neuroscience, Bizkaia Science and Technology Park, 48170 Zamudio, Spain
3Department of Neurosciences, University of the Basque Country, 48940 Leioa, Spain
4Centre de Recherche du CHU de Québec, Axe Neurosciences, Canada G1P 4C7
5Département de Médecine Moléculaire, Université Laval, Canada G1V 4G2

Received 10 December 2013; Accepted 11 February 2014; Published 19 March 2014

Academic Editor: Carlos Fitzsimons


Microglia cells are the major orchestrator of the brain inflammatory response. As such, they are traditionally studied in various contexts of trauma, injury, and disease, where they are well-known for regulating a wide range of physiological processes by their release of proinflammatory cytokines, reactive oxygen species, and trophic factors, among other crucial mediators. In the last few years, however, this classical view of microglia was challenged by a series of discoveries showing their active and positive contribution to normal brain functions. In light of these discoveries, surveillant microglia are now emerging as an important effector of cellular plasticity in the healthy brain, alongside astrocytes and other types of inflammatory cells. Here, we will review the roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory.

  1. Microglia: The Resident Immune Cells of the Brain

Microglia were first described in 1919 by the Spanish neuroanatomist Pío del Río Hortega, a disciple of the renowned Santiago Ramón y Cajal, almost half a century later than neurons and astrocytes and just before oligodendrocytes [1]. This delayed appearance into the neuroscience arena is still apparent today, as microglia remain one of the least understood cell types of the brain. Traditionally, microglia were simply considered as “brain macrophages” controlling the inflammatory response during acute insults and neurodegenerative conditions, and only recently was their unique origin revealed. Indeed, microglia were shown to derive from primitive myeloid progenitors of the yolk sac that invade the central nervous system (CNS) during early embryonic development (reviewed in [2]). In contrast, circulating monocytes and lymphocytes, as well as most tissue macrophages, derive from hematopoietic stem cells located initially in the foetal liver and later in the bone marrow [3]. In the adult brain, the microglial population is maintained exclusively by self-renewal during normal physiological conditions [2]. As a consequence, microglia are the only immune cells which permanently reside in the CNS parenchyma, alongside neural tube-derived neurons, astrocytes, and oligodendrocytes.

These past few years, unprecedented insights were also provided into their extreme dynamism and functional behaviour, in health as much as in disease. Indeed, microglia were revealed to be exceptional sensors of their environment, responding on a time scale of minutes to even subtle variations of their milieu, by undergoing concerted changes in morphology and gene expression [45]. During pathological insults, “activated” microglia were particularly shown to thicken and retract their processes, extend filopodia, proliferate and migrate, release factors and compounds influencing neuronal survival (such as proinflammatory cytokines, trophic factors, reactive oxygen species (ROS), etc.), and phagocytose pathogens, degenerating cells and debris, thus providing better understanding of their roles in orchestrating the inflammatory response [6]. These abilities as immune cells are also recruited during normal physiological conditions, where “surveillant” microglia further participate in the remodeling of neuronal circuits by their phagocytic elimination of synapses and their regulation of glutamatergic receptors maturation and synaptic transmission, among other previously unexpected roles [79], in addition to their crucial involvement in the phagocytic elimination of newborn cells in the context of adult neurogenesis [10].

Our review will discuss the emerging roles of microglia in adult hippocampal neurogenesis and their regulation by inflammation during chronic stress, aging, and neurodegenerative diseases, with a particular emphasis on their underlying molecular mechanisms and their functional consequences for learning and memory (Figure 1).



Figure 1: The effects of surveillant and inflammatory microglia on the adult hippocampal neurogenic cascade. During physiological conditions, surveillant microglia effectively phagocytose the excess of apoptotic newborn cells and may release antineurogenic factors such as TGF. This anti-inflammatory state is maintained by neuronal (tethered or released) fractalkine. Enriched environment drives microglia towards a phenotype supportive of neurogenesis, via the production of IGF-1. In contrast, inflammatory challenge triggered by LPS, irradiation, aging, or AD induces the production of proinflammatory cytokines such as IL-1, TNF, and IL-6 by microglia as well as resident astrocytes and infiltrating monocytes, neutrophils, and lymphocytes. These cytokines have profound detrimental effects on adult neurogenesis by reducing the proliferation, survival, integration, and differentiation of the newborn neurons and decreasing their recall during learning and memory paradigms.

  1. A Brief Overview of Adult Hippocampal Neurogenesis

Adult hippocampal neurogenesis is continuously maintained by the proliferation of neural stem cells located in the subgranular zone (SGZ) [1113]. These neuroprogenitors have been named “radial glia-like cells” (rNSCs), or type 1 cells, since they morphologically and functionally resemble the embryonic radial glia. They have also been defined as “quiescent neuroprogenitors” because only a small percentage of the population is actively dividing during normal physiological conditions. The lineage of these cells is frequently traced by using analogs of the nucleotide thymidine, such as bromodeoxyuridine (BrdU) which gets incorporated into the DNA of dividing cells during the S phase and can be detected by immunofluorescence. Alternatively, their lineage can be traced by labeling with fluorescent reporters which are delivered to dividing cells by retroviral vectors or expressed by specific cell type promoters via inducible transgenic mice (for a review of the methods commonly used to study adult neurogenesis, see [14]). The daughter cells of rNSCs, also called type 2 cells or amplifying neuroprogenitors (ANPs), rapidly expand their pool by proliferating before becoming postmitotic neuroblasts. Within a month, these neuroblasts differentiate and integrate as mature neurons into the hippocampal circuitry [15]. They however display unique electrophysiological characteristics during several months, being more excitable than mature neurons [16], and constitute a special cell population that is particularly inclined to undergo synaptic remodeling and activity-dependent plasticity [17].

These unique properties of the newborn neurons and the neurogenic cascade in general suggested that adult hippocampal neurogenesis could play an important role in hippocampal-dependent functions that require extensive neuroplasticity such as learning and memory. Indeed, activity-dependent plasticity and learning are long known for modulating adult neurogenesis in a complex, yet specific manner, with adult hippocampal neurogenesis being influenced by learning tasks which depend on the hippocampus [4445]. For instance, hippocampal-dependent learning paradigms were found to regulate the survival of newborn neurons, in a positive manner that depends on the timing between their birth and the phases of learning [4647]. Young (1.5–2 months old) newborn neurons were also shown to be preferentially activated during memory recall in a water maze task, compared to mature neurons, as determined by colabeling of BrdU with immediate early genes such as c-Fos and Arc, in which expression correlates with neuronal firing [48]. Nonetheless, it has only been in the last few years that loss-of-function and gain-of-function approaches with inducible transgenic mice were able to confirm that adult hippocampal neurogenesis is necessary for synaptic transmission and plasticity, including the induction of long-term potentiation (LTP) and long-term depression [49], as well as trace learning in conditioned protocols [50], memory retention in spatial learning tasks [5152], and encoding of overlapping input patterns, that is, pattern separation [53].

Adult hippocampal neurogenesis and its functional implications for learning and memory are however influenced negatively by a variety of conditions that are commonly associated with microglial activation and inflammation in the brain, such as chronic stress, aging, and neurodegenerative diseases, as we will review herein. Indeed, inflammation caused by irradiation produces a sustained inhibition of neurogenesis, notably by decreasing the proliferation and neuronal differentiation of the progenitors, and therefore, exposure to therapeutic doses of cranial irradiation has been widely used for modulating neurogenesis experimentally before the development of more specific approaches [54].

  1. Regulation of Adult Hippocampal Neurogenesis by Inflammation

Inflammation is a natural bodily response to damage or infection that is generally mediated by proinflammatory cytokines such as interleukin 1 beta (IL-1), interleukin 6 (IL-6), and tumour necrosis factor alpha (TNF), in addition to lipidic mediators such as prostaglandins and leukotrienes. Oftentimes, it is associated with an increased production of ROS, as well as nitric oxide (NO). Together, these proinflammatory mediators lead to an increase in local blood flow, adhesion, and extravasation of circulating monocytes, neutrophils, and lymphocytes [55]. In the brain, microglia are the main orchestrator of the neuroinflammatory response, but other resident cell types, including astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and even neurons, can produce proinflammatory mediators, though perhaps not to the same extent as microglia [56]. In addition, peripheral immune cells invading the CNS during inflammation can further produce proinflammatory mediators, but the respective contribution of microglia versus other cell types in the inflammatory response of the brain is poorly understood.

The harmful effects of inflammation are also widely determined by the actual levels of proinflammatory mediators released, rather than the occurrence or absence of an inflammatory response in itself. For instance, TNF regulates synaptic plasticity by potentiating the cell surface expression of AMPA glutamatergic receptors, thus resulting in a homeostatic scaling following prolonged blockage of neuronal activity during visual system development [57]. However, TNF also produces differential effects at higher concentrations,ranging from an inhibition of long-term potentiation to an enhancement of glutamate-mediated excitotoxicityin vitro [58]. Inflammation induced by chronic ventricular infusion of bacterial lipopolysaccharides (LPS; a main component of the outer membrane of Gram-negative bacteria), that is, the most widely used method for inducing an inflammatory challenge, also increases ex vivo the hippocampal levels of TNF and IL-1, thereby impairing novel place recognition, spatial learning, and memory formation, but all these cognitive deficits can be restored by pharmacological treatment with a TNF protein synthesis inhibitor, a novel analog of thalidomide, 3,6′-dithiothalidomide [59].

The impact of inflammation on adult hippocampal neurogenesis was originally discovered by Olle Lindvall and Theo Palmer’s groups in 2003, showing that systemic or intrahippocampal administration of LPS reduces the formation of newborn neurons in the adult hippocampus, an effect that is prevented by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID) which inhibits the synthesis of proinflammatory prostaglandins [6061]. Similarly, inflammation can determine the increase in neurogenesis that is driven by seizures, a context in which neurogenesis can be prevented by LPS and increased by the anti-inflammatory antibiotic minocycline [60]. In these studies, hippocampal proliferation remained unaffected by LPS or minocycline and thus it is likely that inflammation targeted the survival of newborn cells [6061], as LPS is known to increase SGZ apoptosis [62]. Inflammation also has further downstream effects on the neurogenic cascade. For instance, LPS increases the number of thin dendritic spines and the expression of the excitatory synapses marker “postsynaptic density protein of 95kDa” (PSD95) in newborn neurons. LPS in addition increases the expression of GABAA receptors at early stages of synapse formation, leading to suggesting a possible imbalance of excitatory and inhibitory neurotransmission in these young neurons [63]. Finally, LPS also prevents the integration of newborn neurons into behaviourally relevant networks, including most notably their activation during spatial exploration, as determined by the percentage of BrdU cells colabeled with the immediate early gene Arc [64].

Importantly, none of these manipulations is specific to microglia and may directly or indirectly affect other brain cells involved in the inflammatory response of the brain. For instance, both LPS and minocycline affect astrocytic function in vitro and in vivo [6569]. Furthermore, LPS is known to drive infiltration of monocytes and neutrophils into the brain parenchyma [70]. Monocytes and neutrophils produce major proinflammatory mediators and could therefore act on the neurogenic cascade as well. The implication of microglia in LPS-induced decrease in neurogenesis is nonetheless supported in vivo by the negative correlation between the number of newborn neurons (BrdU+, NeuN+ cells) and the number of “activated” microglia (i.e., expressing ED1) [60]. ED1, also called CD68 or macrosialin, is a lysosomal protein which is overexpressed during inflammatory challenge. While the location of ED1 previously suggested its involvement in phagocytosis, its loss of function did not result in phagocytosis deficits and thus, its function still remains unknown (reviewed in [10]). The number of ED1-positive microglia also negatively correlates with neurogenesis during inflammation provoked by cranial irradiation [61]. While correlation does not involve causation, nor can pinpoint to the underlying mechanism, these experiments were the first to reveal a potential role for “activated” microglia in the regulation of adult hippocampal neurogenesis. More direct evidence of microglial mediation in LPS deleterious effects was obtained from in vitro experiments, as it was shown that conditioned media from LPS-challenged microglia contained IL-6, which in turn caused apoptosis of neuroblasts [61]. Nonetheless, astrocytes can also release IL-6 when stimulated with TNF or IL-1 [71] and chronic astrocytic release of IL-6 in transgenic mice reduced proliferation, survival, and differentiation of newborn cells, thus resulting in a net decrease in neurogenesis [72]. In summary, while the detrimental impact of inflammation on neurogenesis is well established, more work is needed to define the specific roles played by the various inflammatory cells populating the brain.

  1. Inflammation Associated with Chronic Stress

Across health and disease, the most prevalent condition that is associated with neuroinflammation is “chronic stress,” which commonly refers to the repeated or sustained inability to cope with stressful environmental, social, and psychological constraints. Chronic stress is characterized by an imbalanced secretion of glucocorticoids by the hypothalamic-pituitary-adrenal (HPA) axis (most notably cortisol in humans and corticosterone in rodents), which leads to an altered brain remodeling, massive loss of synapses, and compromised cognitive function [73]. In particular, an impairment of spatial learning, working memory, novelty seeking, and decision making has been associated with chronic stress [74]. Glucocorticoids are well known for their anti-inflammatory properties, as they interfere with NF-B-mediated cytokine transcription, ultimately delaying wound healing [75]. They are also potent anti-inflammatory mediators in vivo [76] and in purified microglia cultures [77]. Recently, repeated administration of high doses of glucocorticoids by intraperitoneal injection, to mimic their release by chronic stress, was also shown to induce a loss of dendritic spines in the motor cortex, while impairing learning of a motor task. A transcription-dependent pathway acting downstream of the glucocorticoid receptor GR was proposed [7879] but the particular cell types involved were not identified.

Microglia are considered to be a direct target of the glucocorticoids, as they were shown to express GR during normal physiological conditions in vivo [77]. In fact, transgenic mice lacking GR in microglia and macrophages show an increased production of proinflammatory mediators (including TNF and IL-1) and greater neuronal damage in response to an intraparenchymal injection of LPS, compared to wild-type mice [80]. In contrast, glucocorticoids are considered to be proinflammatory in the chronically stressed brain [81], where among other changes they can promote inflammation, oxidative stress, neurodegeneration, and microglial activation [82]. For example, repeated restraint stress induces microglial proliferation and morphological changes, including a hyperramification of their processes in the adult hippocampus following restraint stress [83], but a nearly complete loss of processes in the context of social defeat [84]. Prenatal restraint stress also causes an increase in the basal levels of TNF and IL-1, while increasing the proportion of microglia showing a reactive morphology in the adult hippocampus [85]. Similarly, social defeat leads to an enhanced response to the inflammatory challenge induced by intraperitoneal injection of LPS, including an increased production of TNF and IL-1, and expression of inducible NO synthase (iNOS) by microglia, accompanied by an increased infiltration of circulating monocytes [8486]. Therefore, microglia are a strong candidate for mediating some of the effects of stress on adult neurogenesis, as will be discussed below, in synergy with other types of inflammatory cells.

Chronic stress is well known for its negative effects on hippocampal neurogenesis (reviewed in [8788]), although not all stress paradigms are equally effective [89]. Several stress paradigms can decrease neuroprogenitors proliferation in the tree shrew [90] and in mice [9192], although this effect seems to be compensated by an increased survival of newborn neurons [92] and whether stress results in a net increase or decrease in neurogenesis remains controversial (reviewed in [8788]). The effects of stress on adult neurogenesis seem to be mediated at least partially by glucocorticoids, because mice lacking a single copy of the GR gene show behavioural symptoms of depression including learned helplessness, neuroendocrine alterations of the HPA axis, and impaired neurogenesis [93]. In parallel, chronic stress is associated with an increased inflammatory response, which may inhibit neurogenesis as well. For instance, serum levels of IL-1and IL-6 are significantly increased in depressed patients [94]. In mice, restraint stress leads to a widespread activation of NF-B in the hippocampus, including at the level of neuroprogenitors [95] and increased protein levels of IL-1 [96]. In addition to the direct role of glucocorticoids, IL-1 also seems to mediate some of the effects of mild chronic stress, because in vivo manipulations that block IL-1 (either pharmacologically or in null transgenic mice) prevent the anhedonic stress response and the antineurogenic effect of stress [9196]. Moreover, the corticoids and IL-1 pathways may regulate each other in a bidirectional manner because the administration of a GR antagonist can blunt the LPS-induced production of hippocampal IL-1 in stressed mice [97], whereas mice knockout for the IL-1 receptor (IL-1R1) fail to display the characteristic elevation of corticosterone induced by mild chronic stress [96]. Another stress-related cytokine, IL-6, induces depressive phenotypes and prevents the antidepressant actions of fluoxetine when administered to mice in vivo [98]. So far the effects of stress on neurogenesis via corticosteroids and inflammation have been assumed to be cell autonomous, as neuroprogenitors express both GR [99] and IL-1R1 [95]. The potential participation of microglia is yet to be determined, but there are some reports of a direct effect of stress on microglial activation. For instance, microglia acutely isolated from mice subjected to acute stress (by inescapable tail shock) showed a primed response to LPS challenge by producing higher levels of IL-1 mRNA ex vivo [100], and the specific loss of expression of GR in microglia leads to a blunted inflammatory response in vitro and to a decreased neuronal damage in vivo in response to LPS [80]. In stress paradigms, these enhanced responses of microglia to inflammatory challenges are similar to their age-related “priming” which has been associated with and is possibly due to an increased basal production of proinflammatory mediators. However, whether microglia express increased levels of IL-1 and other proinflammatory cytokines in response to stressful events is presently unclear [101]. It is thus possible that some of the antineurogenic effects of stress are exerted by means of microglial-dependent inflammation, but this hypothesis remains to be experimentally tested.

  1. Inflammation Associated with Aging and Neurodegenerative Diseases

Inflammation is also commonly associated with normal aging and neurodegenerative diseases and, therefore, could represent a putative underlying mechanism that explains their decrease in hippocampal neurogenesis. Nonetheless, inflammation is also associated with neurological diseases, such as epilepsy or stroke, where neurogenesis is thought to be increased, although the data from rodents and humans is somewhat conflictive [102]. Neurogenesis is well known to decline throughout adulthood and normal aging in rodents and humans [103104], but the decay is more pronounced and occurs later in life in mice than in humans [105]. The aging-associated decrease in neurogenesis has been shown to occur mainly as a consequence of exhaustion of the rNSC population which, after being recruited and activated, undergo three rounds of mitosis in average and then terminally differentiate into astrocytes [12106]. In addition, a reduced mitotic capacity of the neuroprogenitors could further contribute to decreasing neurogenesis [106], and moreover, an age-related increase in the levels of proinflammatory cytokines could also hinder neurogenesis in the aging brain. Serum levels of IL-1, IL-6, and TNF are elevated in elderly patients [107108]. Aged microglia express higher levels of these proinflammatory cytokines and show a greater response to LPS inflammatory challenge, that is, a “primed” response, than their younger counterparts [109]. The origin of this low-grade age-related inflammation (“inflamm-aging” [110]) remains unknown and may be related to both aging and damage to the surrounding neurons, as well as aging of the immune system per se.

At the cellular level, stress to the endoplasmic reticulum (ER) caused by various perturbations, such as nutrient depletion, disturbances in calcium or redox status, or increased levels of misfolded proteins, can induce a cell-autonomous inflammatory response to neurons. Stress to the ER, a multifunctional organelle which is involved in protein folding, lipid biosynthesis, and calcium storage triggers a homeostatic response mechanism named the unfolding protein response (UPR), aiming to clear the unfolded proteins in order to restore normal ER homeostasis [111]. However, if the ER stress cannot be resolved, the UPR also initiates inflammatory and apoptotic pathways via activation of the transcription factor NF-B which controls the expression of most proinflammatory cytokines [112]. In the brain, ER stress is often initiated by the formation of abnormal protein aggregates in several neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and prion-related disorders [113]. This neurodegeneration-associated ER stress is assumed to occur mostly in neurons, but there are some examples of microglial protein misfolding as well. For instance, both microglia and neurons overexpress CHOP (C/EBP homologous protein), a transcription factor which is activated during ER stress in human patients and mouse models of ALS [114]. Inflammation has been speculated to be a main negative contributor to the pathology of ALS [115], but a direct microglial involvement in mediating the inflammatory response to abnormal protein aggregation in ALS and other neurodegenerative conditions remains to be tested. Finally, ER stress has been linked to a variety of inflammatory conditions [116117], including chronic stress, diet-induced obesity, and drug abuse, as well as atherosclerosis and arthritis [118120]. During normal aging, a progressive decline in expression and activity of key ER molecular chaperones and folding enzymes could also compromise the adaptive response of the UPR, thereby contributing to the age-associated decline in cellular functions [118]. Therefore, aging is strongly associated with a chronic ER stress which leads to increased activation of NF-B [112]; however, the contribution of the different brain cell types to “inflamm-aging” is still poorly understood. The detrimental effects on neurogenesis of increased proinflammatory cytokines in the aging brain are not necessarily related to microglia, but also to stressed neurons. Furthermore, ER stress may also cause a cell-autonomous response in neural stem cells [121], although its impact on neurogenesis remains to be experimentally determined.

In addition, aging is accompanied by an increased level of mitochondrial oxidative stress, which in turn activates the “Inflammasome” [122], a group of multimeric proteins comprising the interleukin 1 converting enzyme (ICE, caspase 1) which serves to release the active form of the cytokine [123]. IL-1 may act directly on rNSCs (visualised by labeling with the Sox2 marker), as they express IL-1R1 in the adult hippocampus [91]. Treatment with IL-1 decreases hippocampal proliferation in young mice [91] and pharmacological inhibition of ICE partially restores the number of newborn neurons in aged mice without significantly affecting their differentiation rate [124]. Transgenic IL-1 overexpression results in chronic inflammation and depletion of doublecortin-labeled neuroblasts, thus mimicking the aging-associated depletion of neurogenesis [125]. The actual mechanism of action of IL-1 on neurogenesis in aged mice, including decreased proliferation of rNSCs/ANPs and survival of newborn neurons, remains undetermined. Microglia are a main source of IL-1in the aging brain, but the hypothesis that microglia-derived IL-1 is responsible for depleting neurogenesis in the aging brain remains to be directly tested.

The regulation of neurogenesis by IL-1 in the aging brain has been further linked to the activity of another cytokine, the chemokine fractalkine, or CX3CL1. Fractalkine has soluble and membrane-tethered forms and is exclusively expressed by neurons, while the fractalkine receptor (CX3CR1) is expressed in the brain by microglia alone [126]. This module forms a unique neuron-microglia signalling unit that controls the extent of microglial inflammation in several neurodegenerative conditions including PD, ALS [127], or AD [128]. In fact, CX3CR1 blocking antibodies increase the production of hippocampal IL-1 when administered to young adult rats [129]. Importantly, chronic treatment with fractalkine increases hippocampal proliferation and the number of neuroblasts in aged (22 months old) but not young (3 months old) or middle-aged rats (12 months old), whereas an antagonists of CX3CR1 has the opposite effects in young, but not in middle-aged nor old rats [129]. Since fractalkine expression is decreased during aging [129], a reduced neuron-microglia signalling might be releasing the brake on microglial contribution to inflammatory responses, although increased levels of fractalkine were instead reported in aged rat hippocampus by other studies [68]. Additional insights into the role of fractalkine signalling come from knock-in mice in which the endogenous CX3CR1 locus is replaced by the fluorescent reporter GFP [126]. The initial studies suggested that  (i.e., ) mice have no significant differences in brain development and functions [130], but more systematic investigations recently revealed a long list of hippocampal-dependent changes in young (3 months old)  and  mice compared to wild-type mice. These changes notably included decreased neuroprogenitors proliferation and neuroblasts number, impaired LTP, performance in contextual fear conditioning and water maze spatial learning and memory, and, importantly, increased IL-1 protein levels [131]. The signalling pathway of fractalkine-IL-1 is functionally relevant, because IL-1R1 antagonists rescued LTP and cognitive function in  mice [131]. In sum, even though neuronal fractalkine seems to be sufficient for restraining the inflammatory activity of microglia in young rats, its downregulation during aging could activate the microglial inflammatory response and thereby subsequently reduce the proliferation of remaining neuroprogenitors.

In AD, inflammatory cytokines such as IL-1 are overexpressed in the microglia associated with the amyloid beta (A) plaques of postmortem samples [132] and in transgenic mice modeling the disease [133]. The loss of synapses (from hippocampus to frontal cortex) is one of the main pathological substrates in this disease, but adult neurogenesis is also severely reduced in most mouse models of AD, possibly due to a decreased proliferation of neuroprogenitors and a decreased survival of newborn cells, even though the putative changes in the neurogenic cascade in postmortem samples remain controversial (reviewed in [102]). This lack of agreement is possibly explained by the fact that the vast majority of AD cases have a late onset over 65 years of age, when little neurogenesis remains. In contrast, in most transgenic AD mouse models, the Aaccumulation, cognitive deficits, and changes in neurogenesis are already detectable in young animals (2-3 months old). The study of AD is further hindered by the difficulty in comparing the time course and pathology across different mouse models. For instance, early treatment with minocycline can improve cognition and reduce A burden in mice expressing the human amyloid precursor protein (APP) [134]. In contrast, in mice expressing APP and a mutated form of presenilin 1 (PS1), which is part of the  secretase pathway that cleaves A, inflammation is reduced without any detectable changes in A plaques deposition [135]. Concomitantly with a decrease in tissue inflammatory cytokines and number of microglial cells, minocycline restores neurogenesis and hippocampus-dependent memory deficits in these APP/PS1 mice [135], indirectly suggesting that cognitive decay in AD may be at least in part related to a detrimental effect of inflammation on hippocampal neurogenesis. Direct evidence that neurogenesis is associated with the cognitive performance in AD is still lacking. Further research is also necessary to determine the neurogenic targets of AD-related inflammation. One central open question for future therapies aiming at increasing neurogenesis and cognition in AD is whether neuroprogenitors are spared or whether their age-induced loss becomes accelerated. Rather than increasing the proliferation and neurogenic output of the few rNSCs remaining in an old AD brain, it may be more relevant to develop strategies that prevent the age-related loss of neuroprogenitors in presymptomatic patients.

In summary, inflammation associated with a wide variety of experimental models of disease produces strong detrimental effects on hippocampal neurogenesis. These effects on human neurogenesis are however not so well described and, in vitro, IL-1 increases the proliferation of hippocampal embryonic neuroprogenitors but decreases their differentiation into neurons [136]. Novel methods to assess hippocampal neurogenesis in the living human brain, from metabolomics of neuroprogenitors to hippocampal blood brain volume (reviewed in [102]), will help to determine the contribution of inflammation to adult neurogenesis in the healthy and diseased human brain during aging.

  1. Normal Physiological Conditions

In the healthy mature brain, microglia are an essential component of the neurogenic SGZ niche, where they physically intermingle with neuroprogenitors, neuroblasts, and newborn neurons [62]. Here, surveillant microglia effectively and rapidly phagocytose the excess of newborn cells undergoing apoptosis [62]. Importantly, microglial phagocytosis in the adult SGZ is not disturbed by inflammation associated with aging or by LPS challenge, as the phagocytic index (i.e., the proportion of apoptotic cells completely engulfed by microglia) is maintained over 90% in these conditions [62]. Nonetheless, the consequences of microglial phagocytosis on adult hippocampal neurogenesis remain elusive. Treatment of mice with annexin V, which binds to the phosphatidylserine (PS) receptor and prevents the recognition of PS on the surface of apoptotic cells, presumably blocking phagocytosis, increases the number of apoptotic cells in the SGZ [40]. Concomitantly, annexin V reduces neurogenesis by decreasing the survival of neuroblasts without affecting neuroprogenitors proliferation [40]. Similar results were obtained in transgenic mice knock-out for ELMO1, a cytoplasm protein which promotes the internalization of apoptotic cells, although the effects on neurogenesis were ascribed to a decreased phagocytic activity of neuroblasts [40]. The actual phagocytic target of the neuroblasts remains undetermined, but the newborn apoptotic cells in the adult SGZ are exclusively phagocytosed by microglia, at least in physiological conditions [62]. Nevertheless, none of the above manipulations has specifically tested the role of microglial phagocytosis in hippocampal-dependent learning and memory and thus, the functional impact of microglial phagocytosis in adult neurogenic niches during normal physiological conditions remains to be elucidated.

Microglial phagocytosis of apoptotic cells is actively anti-inflammatory, at least in vitro, and thus it has been hypothesized that anti-inflammatory cytokines produced by phagocytic microglia may further regulate neurogenesis [10]. For instance, transforming growth factor beta (TGF), which is produced by phagocytic microglia in vitro [137], inhibits the proliferation of SGZ neuroprogenitors [138]. Microglia are further able to produce proneurogenic factors in vitro [139]. When primed with cytokines associated with T helper cells such as interleukin 4 (IL-4) or low doses of interferon gamma (IFN), cultured microglia support neurogenesis and oligodendrogenesis through decreased production of TNF and increased production of insulin-like growth factor 1 (IGF-1) [139], an inducer of neuroprogenitor proliferation [26]. A list of potential factors produced by microglia and known to act on neuroprogenitor proliferation can be found in Table 1. In addition, recent observations suggest that neuroprogenitor cells may not only regulate their own environment, but also influence microglial functions. For instance, vascular endothelial growth factor (VEGF) produced by cultured neuroprecursor cells directly affects microglial proliferation, migration, and phagocytosis [20]. More potential factors produced by neuroprogenitors shown to be influencing microglial activity and function can be found in Table 2. However, it has to be taken into account that most of these observations were obtained in culture and that further research is needed in order to elucidate whether those factors are also secreted and have the same regulatory responses in vivo.

Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.
Microglia secreted
Reference Modulation of neural progenitor cells Reference
BDNF [18] Differentiation [19]
EGF [20] Survival, expansion, proliferation, differentiation [21]
FGF [22] Survival and expansion [23]
GDNF [24] Survival, migration, and differentiation [25]
IGF-1 [21] Proliferation [26]
IL-1 [27] Reduction in migration [27]
IL-6 [28] Inhibition of neurogenesis [29]
IL-7 [20] Differentiation [30]
IL-11 [20] Differentiation [30]
NT-4 [24] Differentiation [31]
PDGF [32] Expansion and differentiation [33]
TGF [34] Inhibition of proliferation [19]


Table 1: Summary of factors secreted by microglia and the potential effect they have on neuroprogenitors in vitro.




Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.

NPC secreted factors Reference Modulation of microglia Reference
BDNF [18] Proliferation and induction of phagocytic activity [35]
Haptoglobin [24] Neuroprotection [36]
IL-1 [37] Intracellular Ca+2 elevation and proliferation [22]
IL-6 [37] Increase in proliferation [38]
M-CSF [20] Mitogen [39]
NGF [40] Decrease in LPS-induced NO [41]
TGF [37] Inhibition of TNF secretion [42]
TNF [37] Upregulation of IL-10 secretion [43]
VEGF [20] Induction of chemotaxis and proliferation [20]


Table 2: Summary of factors secreted by neuroprogenitors and the potential effect they have on microglia in vitro.

In addition, microglial capacity to remodel and eliminate synaptic structures during normal physiological conditions has suggested that microglia could also control the synaptic integration of the newborn neurons generated during adult hippocampal neurogenesis [140]. Three main mechanisms were proposed: (1) the phagocytic elimination of nonapoptotic axon terminals and dendritic spines, (2) the proteolytic remodeling of the perisynaptic environment, and (3) the concomitant structural remodeling of dendritic spines [7140]. Indeed, microglial contacts with synaptic elements are frequently observed in the cortex during normal physiological conditions, sometimes accompanied by their engulfment and phagocytic elimination [141143], as in the developing retinogeniculate system [144]. Microglial cells are distinctively surrounded by pockets of extracellular space, contrarily to all the other cellular elements [142], suggesting that microglia could remodel the volume and geometry of the extracellular space, and thus the concentration of various ions, neurotransmitters, and signalling molecules in the synaptic environment. Whether microglia create the pockets of extracellular space themselves or not remains unknown, but these pockets could result from microglial release of extracellular proteases such as metalloproteinases and cathepsins [145], which are well known for influencing the formation, structural remodeling, and elimination of dendritic spines in situ and also experience-dependent plasticity in vivo [7146]. More recently, microglial phagocytosis of synaptic components was also observed in the developing hippocampus, in the unique time window of synaptogenesis, a process which is notably regulated by fractalkine-CX3CR1 signalling [147]. Therefore, the attractive hypothesis that microglial sculpts the circuitry of newborn cells in the adult hippocampus deserves further attention.

Lastly, microglia were also involved in increasing adult hippocampal neurogenesis in the enriched environment (EE) experimental paradigm. EE is a paradigm mimicking some features of the normal living circumstances of wild animals, as it gives them access to social interactions, toys, running wheels, and edible treats. EE has long been known to enhance neurogenesis by acting on newborn cells survival, resulting ultimately in an enlargement of the dentate gyrus [148]. Functionally, these changes are accompanied by enhanced spatial learning and memory formation with the water maze paradigm [149]. Similar increases in neurogenesis are obtained by subjecting mice to voluntary running paradigms, although in this case the effect is mediated by increased neuroprogenitor proliferation [150]. During inflammatory conditions, EE is antiapoptotic and neuroprotective [151] and it limits the hippocampal response to LPS challenge by decreasing the expression of several cytokines and chemokines, including IL1- and TNF [152]. In fact, EE is believed to counteract the inflammatory environment and rescue the decreased number of neuroblasts in mice compared to wild-type mice [153]. The effects of EE are independent of the IL-1 signalling pathway, as it increases neurogenesis in mice that are null for IL-1R1 [154]. EE also induces microglial proliferation and expression of the proneurogenic IGF-1 [155], but the full phenotype of microglia in EE compared to standard housing and its impact on the neurogenic cascade remains to be determined.

The mechanisms behind the anti-inflammatory actions of EE are unknown, but they were suggested to involve microglial interactions with T lymphocytes through an increased expression of the major histocompatibility complex of class II (MHC-II) during EE [155]. MHC-II is responsible for presenting the phagocytosed and degraded antigens to the antibodies expressed on the surface of a subtype of T lymphocytes (T helper or CD4+ cells), thus initiating their activation and production of antigen-specific antibodies. Severe combined immunodeficient (SCID) mice lacking either T and B lymphocytes or nude mice lacking only T cells have impaired proliferation and neurogenesis in normal and EE housing compared to wild-type mice [155], as well as impaired performance in the water maze [156]. Similarly, antibody-based depletion of T helper lymphocytes impairs basal and exercise-induced proliferation and neurogenesis [157]. Furthermore, a genetic study in heterogeneous stock mice, which descend from eight inbred progenitor strains, has found a significant positive correlation between genetic loci associated to hippocampal proliferation and to the proportion of CD4+ cells among blood CD3+ lymphocytes [158]. Additional experiments are needed to fully determine the possible interactions between microglia and T cells in neurogenesis, because, at least in normal physiological conditions, (1) T cell surveillance of the brain parenchyma is minimal, (2) microglia are poor antigen presenting cells, and (3) antigen presentation by means of MHC-II family of molecules is thought to occur outside the brain, that is, in the meninges and choroid plexus [159]. In fact, during voluntary exercise, there are no significant changes in T cell surveillance of the hippocampus, nor a direct interaction between T cells and microglia, nor any changes in the gene expression profile of microglia, including that of IGF-1, IL-1, and TNF [160]. The number of microglia is also inversely correlated with the number of hippocampal proliferating cells, rNSCs, and neuroblasts in aged (8 months) mice subjected to voluntary running, as well asin vitro cocultures of microglia and neuroprogenitors, which has been interpreted as resulting from an overall inhibitory effect of microglia on adult neurogenesis [161]. Even though EE is clearly a more complex environmental factor than voluntary running, further research is necessary to disregard nonspecific or indirect effects of genetic or antibody-based T cells depletion on microglia and other brain cell populations, including rNSCs. For instance, adoptive transfer of T helper cells treated with glatiramer acetate, a synthetic analog of myelin basic protein (MBP) approved for the treatment of multiple sclerosis, produces a bystander effect on resident astrocytes and microglia by increasing their expression of anti-inflammatory cytokines such as TGF[162]. Alternatively, it has been suggested that T cells may mediate an indirect effect on adult hippocampal neurogenesis by increasing the production of brain-derived neurotrophic factor (BDNF) [157], which is involved in the proneurogenic actions of EE [163]. Whether BDNF can counteract the detrimental effects of T cell depletion on neurogenesis remains unknown. Overall, the roles of microglia in EE and running-induced neurogenesis are unclear and have to be addressed with more precise experimental designs. In summary, surveillant microglia are part of the physical niche surrounding the neural stem cells and newborn neurons of the mature hippocampus, where they continuously phagocytose the excess of newborn cells. Microglia were also linked to the proneurogenic and anti-inflammatory effects of voluntary running and EE, but direct evidence is missing. The overall contribution of microglia to neurogenesis and learning and memory in normal physiological conditions remains largely unexplored at this early stage in the field.

  1. Conclusion

In light of these observations, microglia are now emerging as important effector cells during normal brain development and functions, including adult hippocampal neurogenesis. Microglia can exert a positive or negative influence on the proliferation, survival, or differentiation of newborn cells, depending on the inflammatory context. For instance, microglia can compromise the neurogenic cascade during chronic stress, aging, and neurodegenerative diseases, by their release of proinflammatory cytokines such as IL-1, IL-6, and TNF. A reduced fractalkine signalling between neurons and microglia could also be involved during normal aging. However, microglia are not necessarily the only cell type implicated because astrocytes, endothelial cells, mast cells, perivascular and meningeal macrophages, and to a lesser extent neurons and invading peripheral immune cells could further contribute by releasing proinflammatory mediators.

Additionally, microglia were shown to phagocytose the excess of newborn neurons undergoing apoptosis in the hippocampal neurogenic niche during normal physiological conditions, while a similar role in the synaptic integration of newborn cells was also proposed in light of their capacity to phagocytose synaptic elements. Lastly, microglial interactions with T cells, leading to the release of anti-inflammatory cytokines, neurotrophic factors, and other proneurogenic mediators (notably during EE and voluntary running), could counteract the detrimental effects of inflammation on adult hippocampal neurogenesis and their functional implications for learning and memory.

However, further research is necessary to assess the relative contribution of microglia versus other types of resident and infiltrating inflammatory cells and to determine the nature of the effector cytokines and other inflammatory mediators involved, as well as their cellular and molecular targets in the neurogenic cascade. Such research will undoubtedly help to develop novel strategies aiming at protecting the neurogenic potential and ultimately its essential contribution to learning and memory.


AD: Alzheimer’s disease
ANPs: Amplifying neuroprogenitors
APP: Amyloid precursor protein
A: Amyloid beta
BDNF: Brain-derived neurotrophic factor
BrdU: 5-Bromo-2′-Deoxyuridine
CX3CL1: Fractalkine
CX3CR1: Fractalkine receptor
EAE: Experimental acute encephalomyelitis
EE: Enriched environment
EGF: Epidermal growth factor
FGFb: Basic fibroblast growth factor
GDNF: Glial cell line-derived neurotrophic factor
GFAP: Glial fibrillary acidic protein
GR: Glucocorticoid receptor
HPA: Hypothalamic-pituitary-adrenal axis
ICE: Interleukin 1 converting enzyme
IL-1: Interleukin 1 beta
IL-1R1: Interleukin 1 beta receptor
IL-4: Interleukin 4
IL-6: Interleukin 6
IL-7: Interleukin 7
IL-11: Interleukin 11
IFN: Interferon gamma
IGF-1: Insulin-like growth factor 1
iNOS: Inducible nitric oxide synthase
LPS: Bacterial lipopolysaccharides
LTP: Long term potentiation
M-CSF: Macrophage colony-stimulating factor
MBP: Myelin basic protein
MHC-II: Major histocompatibility complex class II
MOG: Myelin oligodendrocyte glycoprotein
NF-B: Nuclear factor kappa-light-chain-enhancer of activated B cells
NGF: Nerve growth factor
NO: Nitric oxide
NSAID: Nonsteroidal anti-inflammatory drug
NT-4: Neurotrophin-4
PDGF: Platelet-derived growth factor
PS: Phosphatidylserine
PS1: Presenilin 1
ROS: Radical oxygen species
SCID: Severe combined immunodeficiency
SGZ: Subgranular zone
TGF: Transforming growth factor beta
TNF: Tumor necrosis factor alpha
VEGF: Vascular endothelial growth factor.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.


This work was supported by grants from the Spanish Ministry of Economy and Competitiveness to Amanda Sierra (BFU2012-32089) and Juan M. Encinas (SAF2012-40085), from Basque Government (Saiotek S-PC 12UN014) and Ikerbasque start-up funds to Juan M. Encinas and Amanda Sierra, and from The Banting Research Foundation, the Scottish Rite Charitable Foundation of Canada, and start-up funds from Université Laval and Centre de recherche du CHU de Québec to Marie-Ève Tremblay.


  1. P. Rezaie and D. Male, “Mesoglia and microglia—a historical review of the concept of mononuclear phagocytes within the central nervous system,” Journal of the History of the Neurosciences, vol. 11, no. 4, pp. 325–374, 2002. View at Publisher · View at Google Scholar · View at Scopus
  2. F. Ginhoux, S. Lim, G. Hoeffel, D. Low, and T. Huber, “Origin and differentiation of microglia,”Frontiers in Cellular Neuroscience, vol. 7, article 45, 2013. View at Publisher · View at Google Scholar
  3. E. Gomez Perdiguero, C. Schulz, and F. Geissmann, “Development and homeostasis of “resident” myeloid cells: the case of the microglia,” GLIA, vol. 61, no. 1, pp. 112–120, 2013. View at Publisher ·View at Google Scholar
  4. H. Kettenmann, F. Kirchhoff, and A. Verkhratsky, “Microglia: new roles for the synaptic stripper,”Neuron, vol. 77, no. 1, pp. 10–18, 2013. View at Publisher · View at Google Scholar
  5. A. Aguzzi, B. A. Barres, and M. L. Bennett, “Microglia: scapegoat, saboteur, or something else?” Science, vol. 339, no. 6116, pp. 156–161, 2013. View at Publisher · View at Google Scholar
  6. K. Helmut, U. K. Hanisch, M. Noda, and A. Verkhratsky, “Physiology of microglia,” Physiological Reviews, vol. 91, no. 2, pp. 461–553, 2011. View at Publisher · View at Google Scholar · View at Scopus
  7. M. È. Tremblay, B. Stevens, A. Sierra, H. Wake, A. Bessis, and A. Nimmerjahn, “The role of microglia in the healthy brain,” Journal of Neuroscience, vol. 31, no. 45, pp. 16064–16069, 2011. View at Publisher ·View at Google Scholar · View at Scopus
  8. A. Miyamoto, H. Wake, A. J. Moorhouse, and J. Nabekura, “Microglia and synapse interactions: fine tuaning neural circuits and candidate molecules,” Frontiers in Cellular Neuroscience, vol. 7, article 70, 2013. View at Publisher · View at Google Scholar
  9. C. Bechade, Y. Cantaut-Belarif, and A. Bessis, “Microglial control of neuronal activity,” Frontiers in Cellular Neuroscience, vol. 7, article 32, 2013. View at Publisher · View at Google Scholar
  10. A. Sierra, O. Abiega, A. Shahraz, and H. Neumann, “Janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis,” Frontiers in Cellular Neuroscience, vol. 7, article 6, 2013. View at Publisher · View at Google Scholar
  11. G. Kempermann, S. Jessberger, B. Steiner, and G. Kronenberg, “Milestones of neuronal development in the adult hippocampus,” Trends in Neurosciences, vol. 27, no. 8, pp. 447–452, 2004. View at Publisher ·View at Google Scholar · View at Scopus
  12. J. M. Encinas, T. V. Michurina, N. Peunova et al., “Division-coupled astrocytic differentiation and age-related depletion of neural stem cells in the adult hippocampus,” Cell Stem Cell, vol. 8, no. 5, pp. 566–579, 2011. View at Publisher · View at Google Scholar · View at Scopus
  13. M. A. Bonaguidi, M. A. Wheeler, J. S. Shapiro et al., “In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics,” Cell, vol. 145, no. 7, pp. 1142–1155, 2011. View at Publisher · View at Google Scholar · View at Scopus
  14. J. J. Breunig, J. I. Arellano, J. D. Macklis, and P. Rakic, “Everything that glitters isn’t gold: a critical review of postnatal neural precursor analyses,” Cell Stem Cell, vol. 1, no. 6, pp. 612–627, 2007. View at Publisher · View at Google Scholar · View at Scopus
  15. L. S. Overstreet-Wadiche and G. L. Westbrook, “Functional maturation of adult-generated granule cells,” Hippocampus, vol. 16, no. 3, pp. 208–215, 2006. View at Publisher · View at Google Scholar · View at Scopus




Nature Reviews Molecular Cell Biology 8, 519-529 (July 2007) | doi:10.1038/nrm2199

Signal integration in the endoplasmic reticulum unfolded protein response

David Ron & Peter Walter


The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.


Figure 1: The unfolded protein response (UPR) signalling pathways.

FromThe impact of the endoplasmic reticulum protein-folding environment on cancer development

Nature Reviews Cancer 14, 581–597 (2014)  http://dx.doi.org:/10.1038/nrc3800

(UPR) signalling pathways

(UPR) signalling pathways


Upon endoplasmic reticulum (ER) stress, unfolded and misfolded proteins bind and sequester immunoglobulin heavy-chain binding protein (BIP), thereby activating the UPR. The UPR comprises three parallel signalling branches: PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α), inositol-requiring protein 1α (IRE1α)–X-box binding protein 1 (XBP1) and activating transcription factor 6α (ATF6α). The outcome of UPR activation increases protein folding, transport and ER-associated protein degradation (ERAD), while attenuating protein synthesis. If protein misfolding is not resolved, cells enter apoptosis. CHOP, C/EBP homologous protein; GADD34, growth arrest and DNA damage-inducible protein 34; JNK, JUN N-terminal kinase; P, phosphorylation; RIDD, regulated IRE1-dependent decay; ROS, reactive oxygen species; XBP1s, transcriptionally active XBP1; XBP1u, unspliced XBP1.

Figure 3: The unfolded protein response (UPR) and inflammation.

(UPR) and inflammation

(UPR) and inflammation


The three UPR pathways augment the production of reactive oxygen species (ROS) and activate nuclear factor-κB (NF-κB) and activator protein 1 (AP1) pathways, thereby leading to inflammation. NF-κB, which is a master transcriptional regulator of pro-inflammatory pathways, can be activated through binding to the inositol-requiring protein 1α (IRE1α)–TNF receptor-associated factor 2 (TRAF2) complex in response to endoplasmic reticulum (ER) stress, leading to recruitment of the IκB kinase (IKK), IκB phosphorylation (P) and degradation, and nuclear translocation of NF-κB196. Moreover, the IRE1α–TRAF2 complex can recruit apoptosis signal-regulating kinase 1 (ASK1) and activate JUN N-terminal kinase (JNK), increasing the expression of pro-inflammatory genes through enhanced AP1 activity197. The PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α) and activating transcription factor 6α (ATF6α) branches of the UPR activate NF-κB through different mechanisms. Engaging PERK–eIF2α signalling halts overall protein synthesis and increases the ratio of NF-κB to IκB, owing to the short half-life of IκB, thereby freeing NF-κB for nuclear translocation198199. ATF6α activation following exposure to the bacterial subtilase cytotoxin that cleaves immunoglobulin heavy-chain binding protein (BIP) leads to AKT phosphorylation and consequent NF-κB activation109200.



Figure 4

The cancer-supporting role of the unfolded protein response (UPR).


cancer-supporting role of the unfolded protein response

cancer-supporting role of the unfolded protein response

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Protein Energy Malnutrition and Early Child Development

Curator: Larry H. Bernstein, MD, FCAP



In the preceding articles we have seen that poverty and low social class combined with cultural strictures or dependence on a sulfur-poor diet results in childhood stunting and impaired brain development. This is a global health issue.

Protein-Energy Malnutrition

  • Author: Noah S Scheinfeld, JD, MD, FAAD; Chief Editor: Romesh Khardori, MD, PhD, FACP


The World Health Organization (WHO)[1] defines malnutrition as “the cellular imbalance between the supply of nutrients and energy and the body’s demand for them to ensure growth, maintenance, and specific functions.” The term protein-energy malnutrition (PEM) applies to a group of related disorders that includemarasmus, kwashiorkor (see the images below), and intermediate states of marasmus-kwashiorkor. The term marasmus is derived from the Greek wordmarasmos, which means withering or wasting. Marasmus involves inadequate intake of protein and calories and is characterized by emaciation. The term kwashiorkor is taken from the Ga language of Ghana and means “the sickness of the weaning.” Williams first used the term in 1933, and it refers to an inadequate protein intake with reasonable caloric (energy) intake. Edema is characteristic of kwashiorkor but is absent in marasmus.

Studies suggest that marasmus represents an adaptive response to starvation, whereas kwashiorkor represents a maladaptive response to starvation. Children may present with a mixed picture of marasmus and kwashiorkor, and children may present with milder forms of malnutrition. For this reason, Jelliffe suggested the term protein-calorie (energy) malnutrition to include both entities.
Although protein-energy malnutrition affects virtually every organ system, this article primarily focuses on its cutaneous manifestations. Patients with protein-energy malnutrition may also have deficiencies of vitamins, essential fatty acids, and trace elements, all of which may contribute to their dermatosis.

In general, marasmus is an insufficient energy intake to match the body’s requirements. As a result, the body draws on its own stores, resulting in emaciation. In kwashiorkor, adequate carbohydrate consumption and decreased protein intake lead to decreased synthesis of visceral proteins. The resulting hypoalbuminemia contributes to extravascular fluid accumulation. Impaired synthesis of B-lipoprotein produces a fatty liver.

Protein-energy malnutrition also involves an inadequate intake of many essential nutrients. Low serum levels of zinc have been implicated as the cause of skin ulceration in many patients. In a 1979 study of 42 children with marasmus, investigators found that only those children with low serum levels of zinc developed skin ulceration. Serum levels of zinc correlated closely with the presence of edema, stunting of growth, and severe wasting. The classic “mosaic skin” and “flaky paint” dermatosis of kwashiorkor bears considerable resemblance to the skin changes of acrodermatitis enteropathica, the dermatosis of zinc deficiency.

In 2007, Lin et al[2] stated that “a prospective assessment of food and nutrient intake in a population of Malawian children at risk for kwashiorkor” found “no association between the development of kwashiorkor and the consumption of any food or nutrient.”

Marasmus and kwashiorkor can both be associated with impaired glucose clearance that relates to dysfunction of pancreatic beta-cells.[3] In utero, plastic mechanisms appear to operate, adjusting metabolic physiology and adapting postnatal undernutrition and malnutrition to define whether marasmus and kwashiorkor will develop.[4]

In 2012, a report from Texas noted an 18-month-old infant with type 1 glutaric acidemia who had extensive desquamative plaques, generalized nonpitting edema, and red-tinged sparse hair, with low levels of zinc, alkaline phosphatase, albumin, and iron. This patient has a variation on kwashiorkor, and the authors suggest that it be termed acrodermatitis dysmetabolica.[5] On the same note, a boy aged 18 months with type 1 glutaric acidemia suffered from zinc deficiency and acquired protein energy malnutrition.[6]

For complex reasons, sickle cell anemia can predispose suffers to protein malnutrition.[7]

Protein energy malnutrition ramps up arginase activity in macrophages and monocytes.[8]

Protein energy malnutrition (PEM), brain and various facets of child development.

Protein energy malnutrition (PEM) is a global problem. Nearly 150 million children under 5 years in the world and 70-80 million in India suffer from PEM, nearly 20 million in the world and 4 million in India suffer from severe forms of PEM, viz., marasmus, kwashiorkor and marasmic kwashiorkor. The studies in experimental animals in the west and children in developing countries have revealed the adverse effects of PEM on the biochemistry of developing brain which leads to tissue damage and tissue contents, growth arrest, developmental differentiation, myelination, reduction of synapses, synaptic transmitters and overall development of dendritic activity. Many of these adverse effects have been described in children in clinical data, biochemical studies, reduction in brain size, histology of the spinal cord, quantitative studies and electron microscopy of sural nerve, neuro -CT scan, magnetic resonance imaging (MRI) and morphological changes in the cerebellar cells. Longer the PEM, younger the child, poorer the maternal health and literacy, more adverse are the effects of PEM on the nervous system. Just like the importance of nutrients on the developing brain, so are the adverse effects on the child development of lack of environmental stimulation, emotional support and love and affection to the child. When both the adverse factors are combined, the impact is severe. Hence prevention of PEM in pregnant and lactating mothers, breast feeding, adequate home based supplements, family support and love will improve the physical growth, mental development, social competence and academic performance of the child. Hence nutritional rehabilitation, psychosocial and psychomotor development of the child should begin in infancy and continue throughout. It should be at all levels, most important being in family, school, community and various intervention programmes, local, regional and national. Moreover medical students, health personnel, all medical disciplines concerned with total health care and school teachers should learn and concentrate on the developmental stimulation and enrichment of the child.

Cognitive development in children with chronic protein energy malnutrition

Behav Brain Funct. 2008; 4: 31.  http://dx.doi.org:/10.1186/1744-9081-4-31 
Background: Malnutrition is associated with both structural and functional pathology of the brain. A wide range of cognitive deficits has been reported in malnourished children. Effect of chronic protein energy malnutrition (PEM) causing stunting and wasting in children could also affect the ongoing development of higher cognitive processes during childhood (>5 years of age). The present study examined the effect of stunted growth on the rate of development of cognitive processes using neuropsychological measures.
Methods: Twenty children identified as malnourished and twenty as adequately nourished in the age groups of 5–7 years and 8–10 years were examined. NIMHANS neuropsychological battery for children sensitive to the effects of brain dysfunction and age related improvement was employed. The battery consisted of tests of motor speed, attention, visuospatial ability, executive functions, comprehension and learning and memory
Results: Development of cognitive processes appeared to be governed by both age and nutritional status. Malnourished children performed poor on tests of attention, working memory, learning and memory and visuospatial ability except on the test of motor speed and coordination. Age related improvement was not observed on tests of design fluency, working memory, visual construction, learning and memory in malnourished children. However, age related improvement was observed on tests of attention, visual perception, and verbal comprehension in malnourished children even though the performance was deficient as compared to the performance level of adequately nourished children.
Conclusion: Chronic protein energy malnutrition (stunting) affects the ongoing development of higher cognitive processes during childhood years rather than merely showing a generalized cognitive impairment. Stunting could result in slowing in the age related improvement in certain and not all higher order cognitive processes and may also result in long lasting cognitive impairments.
Malnutrition is the consequence of a combination of inadequate intake of protein, carbohydrates, micronutrients and frequent infections [1]. In India malnutrition is rampant. WHO report states that for the years 1990–1997 52% of Indian children less than 5 years of age suffer from severe to moderate under nutrition [2]. About 35% of preschool children in sub-Saharan Africa are reported to be stunted [3]. Malnutrition is associated with both structural and functional pathology of the brain. Structurally malnutrition results in tissue damage, growth retardation, disorderly differentiation, reduction in synapses and synaptic neurotransmitters, delayed myelination and reduced overall development of dendritic arborization of the developing brain. There are deviations in the temporal sequences of brain maturation, which in turn disturb the formation of neuronal circuits [1]. Long term alterations in brain function have been reported which could be related to long lasting cognitive impairments associated with malnutrition [4]. A wide range of cognitive deficits has been observed in malnourished children in India. In a study, malnourished children were assessed on the Gessell’s developmental schedule from 4 to 52 weeks of age. Children with grades II and III malnutrition had poor development in all areas of behaviour i.e., motor, adaptive, language and personal social [5]. Rural children studying in primary school between the ages of 6–8 years were assessed on measures of social maturity (Vineland social maturity scale), visuomotor co-ordination (Bender gestalt test), and memory (free recall of words, pictures and objects). Malnutrition was associated with deficits of social competence, visuomotor coordination and memory. Malnutrition had a greater effect on the immediate memory of boys as compared with those of girls. Malnourished boys had greater impairment of immediate memory for words, pictures and objects, while malnourished girls had greater impairment of immediate memory for only pictures. Delayed recall of words and pictures of malnourished boys was impaired. Malnourished girls had an impairment of delayed recall of only words. The same authors measured the intelligence of malnourished children using Malin’s Indian adaptation of the Wechsler’s intelligence scale for children. IQ scores decreased with the severity of malnutrition. Significant decreases were observed in performance IQ, as well as on the subtests of information and digit span among the verbal subtests [6]. The above study has shown that though there is decrease in full scale IQ, yet performance on all the subtests was not affected. This suggests that malnutrition may affect different neuropsychological functions to different degrees. Studies done in Africa and South America have focused on the effect of stunted growth on cognitive abilities using verbal intelligence tests based on assessment of reasoning [7]. Such an assessment does not provide a comprehensive and specific assessment of cognitive processes like attention, memory, executive functions, visuo-spatial functions, comprehension as conducted in the present study. Information about the functional status of specific cognitive processes has implications for developing a cognitive rehabilitation program for malnourished children. A neuropsychological assessment would throw light on functional status of brain behaviour relationships affected by malnutrition. Deficits of cognitive, emotional and behavioural functioning are linked to structural abnormalities of different regions of the brain. Brain structures and brain circuits compute different components of cognitive processes [8]. Malnutrition has long lasting effects in the realm of cognition and behaviour, although the cognitive processes like executive functions have not been fully assessed [9]. The differential nature of cognitive deficits associated with malnutrition suggests that different areas of the brain are compromised to different degrees. A neuropsychological assessment would be able to delineate the pattern of brain dysfunction. Malnutrition is a grave problem in our country as 52% of our children are malnourished. Effects of protein-calorie malnutrition are inextricably blended with the effects of social cultural disadvantage; even within the disadvantaged class, literacy environment at home and parental expectation regarding children’s education are powerful variables. Perhaps membership in a higher caste confers some advantage in regard to home literacy, and parental expectation. Short and tall children do differ in some cognitive tests, but not in all as demonstrated in a study done in Orissa, India [10]. But whether or not stunted growth alone is the causative variable for cognitive weakness is not determined as yet. Moreover, the functional integrity of specific cognitive processes is less clear. Chronic PEM resulting in stunting and wasting could result in delay in the development of cognitive processes or in permanent cognitive impairments. Neuropsychological measures can demonstrate delay in normally developing cognitive processes as well as permanent cognitive deficits.
Children in the age range of 5–10 years attending a corporation school in the city of Bangalore participated in the study. Corporation schools in India are government schools with minimal fee attended by children from lowmiddle class. There were 20 children in adequately nourished group and 20 in the malnourished group. The gender distribution was equal. Children in both the groups were from the same ethnic/language background. They were natives of Karnataka living in Bangalore.
After identifying the malnourished and adequately nourished children the coloured progressive matrices test [12] was administered to rule out mental retardation. Children falling at or below the fifth percentile were excluded from the sample, as the 5th percentile is suggestive of intellectually defective range. The percentile points were calculated from the raw scores using Indian norms [13]. Mental retardation was ruled out as otherwise scores on neuropsychological tests would be uniformly depressed and a differentiation of deficits might not occur. Intelligence was not treated as a covariate in the study. The groups did not differ significantly in their scores on CPM (a screening instrument to rule out intellectual impairment in both the groups).
Table 1: Demographic details of the participants
                            Adequately nourished N = 20                  Malnourished N = 20
Mean age              5–7 years        8–10 years                     5–7 years      8–10 years
                               5.8 years        8.8 years                          6.3 years      9.3 years
Gender                   Girls:10           Boys: 10                          Girls:10         Boys: 10
Stunted %
(height for age -2 SD from the median) —-                                  70%
Stunted and wasted %
(height for age and
weight for height: -2 SD from the median) —-                               30%
Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.
Exclusion of behaviour problems and history of neurological disorders The children’s behaviour questionnaire form B [14] was administered to the class teachers of the identified children. Children who scored above the cut off score of 9 were not included in the sample. The personal data sheet was filled in consultation with the parents and teachers to rule out any history of any neurological/psychiatric disorders including head injury and epilepsy and one child with epilepsy was excluded. This was one of the exclusion criteria.
The tests have been grouped under specific cognitive domains on the basis of theoretical rationale and factor analysis. Factor analysis has been done for the battery and the grouping of tests under cognitive functions like executive functions, visuospatial functions, comprehension and learning and memory was done on the basis of the clustering observed in factor analysis as well as on theoretical grounds
The neuropsychological battery consisted of the following tests:
1. Motor speed  Finger tapping test [15]
2. Expressive speech  Expressive speech test was administered to rule out speech related deficits
3. Attention  Color trails test [18] is a measure of focused attention and conceptual tracking.
4. Color cancellation test [21] is a measure of visual scanning/selective attention
5. Executive functions FAS phonemic fluency test is a measure of verbal fluency.
6. Design fluency test [24] is a measure of design fluency, cognitive flexibility and imaginative capacity.
7. Visuo-spatial working memory span task [23]: This test is a measure of visuo-spatial working memory (VSWM) span.
8. Visuospatial functions Motor-free visual perception test [29] is a measure of visuoperceptual ability, having 36 items for visual discrimination, visual closure, figure-ground, perceptual matching and visual memory. Since this test has been originally developed for children between 5–8 years of age, it was modified and items in increasing difficulty level were added by the authors to make it applicable for the children above 8 years. Number of correct responses comprises the score.
9. Picture completion test [30] is a measure of visuoconceptual ability, visual organization and visuo-conceptual reasoning.
10. Block design test [30] is a measure of visuoconstructive ability.
11. Comprehension, learning and memory Token test [31] is a measure of verbal comprehension of commands of increasing complexity.
12. Rey’s auditory verbal learning test (RAVLT) [32] is a measure of verbal learning and memory.
13. Memory for designs test [34] is a measure of visual learning and memory.
Comparison between the performance of adequately nourished children and malnourished children Table 2.0 shows that malnourished group differed significantly from the adequately nourished group on tests of phonemic fluency, design fluency, selective attention, visuospatial working memory, visuospatial functions, verbal comprehension and verbal learning and memory showing poor performance. The two groups did not differ on the test of finger tapping. Since expressive speech was a question answer type assessment looking at repetitive speech, nominative speech and narrative speech, which is like an initial screening for aphasia, like symptoms. Since it did not give a quantitative score, hence was not taken for analysis. As a descriptive account of expressive speech it was observed that malnourished children did not have any difficulty with respect to expressive speech.
Comparison of age related differences in cognitive functions between adequately nourished and malnourished children Data was further subjected to post hoc analysis to compare the two groups across the two age groups to study the rate of improvement with age (Table 2). In both the age groups of 5–7 years and 8–10 years the adequately nourished children performed better than the malnourished children. Figures 1, 2, 3, 4, 5, 6 indicate age related improvement in performance across different cognitive functions in adequately nourished children as compared to malnourished children. Motor speed and coordination was not significantly affected in malnourished children as compared to the adequately nourished children (figure 1). The rate of age related improvement across the two age groups was found rapid on certain functions like selective attention (figure 2) and verbal fluency (figure 3) in malnourished children. However, working memory, design fluency, visuospatial functions, comprehension, learning, and memory showed slowing in terms of age related improvement in malnourished children. Most of the cognitive functions like design fluency (figure 3), working memory (figure 3), Visual perception (figure 4), visuoconceptual reasoning (figure 4), visual construction (figure 4), verbal comprehension (figure 5), verbal and visual memory (figures 6) have shown a very slow rate of improvement with respect to the difference in performance between the two age groups of 5–7 and 8–10 years. On the contrary functions like verbal fluency (figure 3), motor speed (figures 1), and selective attention (figure 2) showed similar rates of improvement in adequately nourished children and malnourished children while comparing the two age groups.
Table 2: Mean comparisons for the cognitive functions across the two age groups of adequately nourished and malnourished children (not shown)
Table 3: Post-hoc comparisons between adequately nourished and malnourished groups across the two age groups (not shown)
Figure 1 Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand) Age related comparisons between adequately nourished and malnourished children on motor speed (right and left hand). (not shown)
Figure 2 Age related comparisons between adequately nourished and malnourished children on selective attention (color cancellation test). (not shown)
Post-hoc comparisons were computed with Tukey’s posthoc tests to compare the means across age groups between malnourished and adequately nourished children for those test scores that showed significant effects. Hence, post hoc tests were not computed for the finger tapping test scores assessing motor speed. Table 3 presents the post-hoc results with the significance (probability level) levels of the differences across age groups and between adequately nourished and malnourished children. Post hoc results have been done to support our theoretical claims about the lack of age related improvement in certain cognitive functions on one hand and the nature of cognitive impairments on the other in malnourished children. Four comparisons were interpreted i.e., comparing performance between the two age groups of adequately nourished and malnourished children separately. The other comparison was between the adequately nourished and malnourished children for the age group of 5–7 years and similarly for the age group of 8–10 years. Results indicate age related differences within each group as well as between the two groups. Age related differences were found significant for some of the test scores between 5–7 and 8–10 year old children in the adequately nourished group but not for most of the test scores for malnourished group indicative of a delay in development of certain cognitive functions. Differences were found significant between the adequately nourished and malnourished children for the same age group for most of the test scores indicative of a deficit in a particular cognitive function. In few of the tests, performance was not found to be significantly different between the two age groups for both adequately nourished and malnourished children.
Discussion The findings of the present study could be discussed in terms of the effect of chronic malnutrition on neuropsychological performance and with respect to the rate of development of cognitive processes.
Effect of malnutrition on neuropsychological performance Our study indicates that malnourished children perform poor on most of the neuropsychological tests except that of motor speed as compared to adequately nourished children. Malnourished children showed poor performance on tests of higher cognitive functions like cognitive flexibility, attention, working memory, visual perception, verbal comprehension, and memory. These findings are supported by another study on Indian malnourished children, which reported memory impairments in undernourished children and spared fine motor coordination [36]. Malnourished children showed poor performance on novel tasks like tests of executive functions i.e., working memory spatial locations. Poor performance on the tests of fluency and working memory also coincides with very slow rate of improvement between the age groups of 5–7 years and 8–10 years. Poor performance on most of the neuropsychological tests indicated a diffuse impairment including attention, executive functions, visuospatial functions, comprehension and memory.
Effect of malnutrition on cognitive development Both the groups were tested on a neuropsychological battery, which has been found to be sensitive to age related differences in cognitive functions in children (5–15 years). The age trends reported in the present study are based on the assessment that employed the NIMHANS neuropsychological battery for children [13]. The test battery has been standardized based on the growth curve modeling approach for empirical validation of age-related differences in performance on neuropsychological tests. The tests in the battery were found sensitive to show age related differences.
Malnourished children showed poor performance with respect to age as compared to adequately nourished children. The performance of malnourished children in the 5–7 years age group was poor and much lower than the adequately nourished children and did not seem to show much improvement in the 8–10 years age group. The rate of cognitive development was found to be different for different cognitive functions. The rate of development was affected for some of the cognitive functions showing minimal age related improvement across the age range of 5–7 years and 8–10 years such as design fluency, working memory, visual construction, verbal comprehension, learning and memory for verbal and visual material. On the contrary, age related improvement was observed on certain other cognitive functions in malnourished children, where the level of performance was low for both the age groups but the rate of improvement between the two age groups was similar to adequately nourished children.
Not shown
Figure 3 Age related comparisons between adequately nourished and malnourished children on executive functions.
Note: VF: verbal fluency; DF: design fluency; WM: working memory; AN: adequately nourished; MN: malnourished.

MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p > .05 8–10 years AN vs MN p < .05 Visual memory (memory for designs test) AN 5–7 vs 8–10 p > .05 MN 5–7 vs 8–10 p > .05 5–7 years AN vs MN p < .05 8–10 years AN vs MN p < .05

Figure 4 Age related comparisons between adequately nourished and malnourished children on visuospatial functions.
Figure 5 Age related comparisons between adequately nourished and malnourished children on verbal comprehension and verbal learning.
Motor speed (right and left hand) was not found impaired in malnourished children and the rate of development was also found similar to adequately nourished children.
Executive functions such as design fluency, selective attention and working memory were found deficient in malnourished children also showing poor rate of improvement between the two age groups. All the three tests of executive functions like fluency, selective attention and working memory for spatial locations involved novel stimuli and performance required cognitive flexibility as well as faster information processing which was affected in malnourished children. Results also indicate that malnourished children showed a very slow rate of improvement on these functions.
Visuo-spatial functions like visual perception, visual construction and visuo-conceptual reasoning showed significantly poor performance when compared to the adequately nourished children but showed a steep age related improvement in performance. Performance on functions like visual perception (visual discrimination, perceptual matching, visual closure and visuospatial relationships) and visual construction was severely affected in malnourished children and also showed poor rate of improvement with age.
Verbal comprehension, learning and memory for verbal and visual material was found poor as compared to adequately nourished children but the rate of improvement between 5–7 years age group and 8–10 years age group was similar to that of adequately nourished children. These results suggest that development of comprehension with age might not be affected in malnourished children. However, other than the poor performance on the AVLT test of verbal learning, malnourished children also showed minimal improvement between the two age groups as compared to the greater magnitude of difference between the two age groups in adequately nourished children. Visual memory was most severely affected in malnourished children in terms of the poor performance on delayed recall on design learning test as well as in terms of the difference between the two age groups.
Malnutrition affects brain growth and development and hence future behavioral outcomes [37]. School-age children who suffered from early childhood malnutrition have generally been found to have poorer IQ levels, cognitive function, school achievement and greater behavioral problems than matched controls and, to a lesser extent, siblings. The disadvantages last at least until adolescence. There is no consistent evidence of a specific cognitive deficit [38]. The functional integrity of specific cognitive processes is less clear. Stunting in early childhood is common in developing countries and is associated with poorer cognition and school achievement in later childhood [39]. Deficits in children’s scores have been reported to be smaller at age 11 years than at age 8 years in a longitudinal study on malnourished children stunted children suggesting that adverse effects may decline over time [7]. In our study also all the children in malnourished group were stunted and the cross sectional assessment of age related improvement has shown similar rate of improvement across 5–7 years to 8–10 years age groups as observed in adequately nourished children though the baseline performance was low in malnourished children. These results indicate that the adverse effects of malnutrition (stunting in particular) may decline with age only for certain cognitive functions but the rate of cognitive development for most of the cognitive processes particularly higher cognitive processes including executive processes and visuospatial perception could be severely affected during the childhood years. Decline in the effects of malnutrition overtime has been reported to be independent of differences in educational, socioeconomic and psychosocial resources [7]. Hence, malnutrition (particularly stunting) may result in delayed development of cognitive processes during childhood years rather than a permanent generalized cognitive impairment.
The neuropsychological interpretation of the cognitive processes more severely affected in malnourished children suggests a diffuse cortical involvement. This is with reference to deficits pertaining to functions mediated by dorsolateral prefrontal cortex (poor performance on tests of attention, fluency and working memory), right parietal (poor performance on tests of visuospatial functions) and bilateral temporal cortex (poor performance on tests of comprehension, verbal learning, and memory for verbal and visual material). The prefrontal cortex may be particularly vulnerable to malnutrition [4]. The adverse effects of malnutrition (PEM-stunting) on cognitive development could be related to the delay in certain processes of structural and functional maturation like delayed myelination and reduced overall development of dendritic arborization of the developing brain [1].
The present study highlights two ways in which malnutrition particularly stunting could affect cognitive functions. On one hand age related improvement in cognitive performance is compromised and on the other hand there could be long lasting cognitive impairments as well. However, the effect is nor specific to a particular cognitive domain and is rather more diffuse. Results of the study also indicate that: certain cognitive functions could be vulnerable to the effect of malnutrition in terms of showing impairment but the rate of development of these functions may not be affected. On the other hand, rate of development of certain cognitive functions may be affected and may also show impairment when compared with adequately nourished children.
Conclusion Chronic protein energy malnutrition (stunting) results in cognitive impairments as well as slowing in the rate of the development of cognitive processes. Rate of development of cognitive functions may follow different patterns in children with malnutrition. Chronic protein energy malnutrition affects the development of cognitive processes differently during childhood years rather than merely showing an overall cognitive dysfunction as compared to adequately nourished children. Stunting could result in delay in the development of cognitive functions as well as in permanent cognitive impairments which show minimal improvement with increase in age. Rate of development of attention, executive functions like cognitive flexibility, working memory, visuospatial functions like visual construction is more severely affected by protein energy malnutrition in childhood years, a period that is marked by rapid ongoing development of cognitive functions.
The effects of protein energy malnutrition in early childhood on intellectual and motor abilities in later childhood and adolescence.
Dev Med Child Neurol. 1976 Jun;18(3):330-50.

Three groups of Ugandan children (20 in each group) and one comparison group of 20 children were examined between 11 and 17 years of age. The first three groups had been admitted to hospital for treatment of protein energy malnutrition between the ages of eight to 15, 16 to 21 and 22 to 27 months, respectively. The comparison group had not been clinically malnourished throughout the whole period up to 27 months of age. All the children came from one tribe and were individually matched for sex, age, education and home environment. It was found that the three malnourished groups fell significantly below the comparison group in anthropometric measurements and in tests of intellectual and motor abilities. No evidence was found for a relationship between the deficit and age at admission. Further analysis among the 60 malnourished children revealed that anthropometry and intellectual and motor abilities are the more affected the greater the degree of ‘chronic undernutrition’ at admission, but no correlation was found with the severity of the ‘acute malnutrition’. The results show a general impairment of intellectual abilities, with reasoning and spatial abilities most affected, memory and rote learning intermediately and language ability least, if at all, affected. These findings are discussed in the context of a comprehensive and critical appraisal of the existing literature.

Quake-Hit Nepal Gears up to Tackle Stunting in Children

By Gopal Sharma  July 08, 2015  http://www.medscape.com/viewarticle/847572

HECHO, Nepal (Thomson Reuters Foundation) – Shanti Maharjan, who gave birth to a baby girl 10 days ago, has spent the last two months living under corrugated iron sheets with her husband and five others after two major earthquakes reduced her mud-and-brick home to rubble.

Adequate food, drinking water and aid such as tents and blankets have been hard to come by, she says, though scores of aid agencies rushed to the Himalayan nation to help survivors.
What worries the 26-year-old mother most is her inability to produce breastmilk for her new-born daughter, who she fears is at serious risk of malnutrition in the aftermath of the 7.8 and 7.3 magnitude quakes in April and May.

“The earthquake destroyed everything, including our food reserves,” said Maharjan, sitting under the iron sheeting on farmland on the outskirts of the capital, Kathmandu.

“There is not enough food. Getting meat, oil and fruits to eat is difficult in this situation. I am worried about my daughter’s nourishment,” she said as the baby, wrapped in a green cloth, lay sleeping on a wooden bed.

The government, aware that disruption caused by the quakes could worsen the country’s already high rate of child malnutrition is sending out teams of community nurses to give advice and food supplements to women and children in the affected areas.

A 2011 government study showed that more than 40% of Napel’s under-five-year-olds were stunted, showing that the country’s child malnutrition rate was one of the world’s highest.
Experts say the two quakes, which killed 8,895 people and destroyed half a million houses, could make things worse as survivors have inadequate food, water, shelter, healthcare and sanitation.

United Nations officials warn that the rate of stunting among children in the South Asian nation could return to the 2001 level of 57%, if authorities and aid agencies do not respond effectively.

“The risk of malnutrition is high and requires the nutrition and other sectors like agriculture, health, water, sanitation, education and social protection to respond adequately,” said Stanley Chitekwe, UNICEF’s nutrition chief in Nepal.


Child malnutrition is an underlying cause of death for 3 million children annually around the world – nearly half of all child deaths – most of whom die from preventable illnesses such as diarrhoea due to weak immune systems.

Those lucky enough to survive grow up without enough energy, protein, vitamins and minerals, causing their brains and bodies to be stunted, and they are often unable to fulfill their potential.

Government officials admit the challenges, citing data showing that almost 70% of Nepali children under the age of two suffer from anaemia caused by iron deficiency.

“This shows that (poor) nutrition is a very big problem. The earthquake will further worsen the situation because people simply don’t have enough to eat, let alone have a nutritious diet,” said Health Ministry official Krishna Prasad Paudel.

Supported by UNICEF, authorities have now launched a drive to reach out to more than 500,000 women and children who need supplementary food and medicines.

More than 10,000 female community volunteers will be fanning out across 14 districts affected by the earthquakes, visiting devastated towns and villages and speaking to new and expectant mothers about breast-feeding their infants.

The volunteers will also advise families on eating locally available nutritious foods such as green vegetables and meat and will distribute vitamin A, iron and folic acid, and other micronutrient supplements to pregnant and breastfeeding women.

In Imadole, a prosperous district on the outskirts of the ancient town of Patan, health volunteer Urmila Sharma Dahal found an extremely thin two-year-old boy weighing 7.5 kg (16.5 pounds) last week, suffering from severe acute malnutrition.

Dahal said she provided his family with sachets of ready-to-use therapeutic food – a paste of peanut, sugar, milk powder, vitamin and oil – and the child gained nearly a kilo (2.2 pounds) in weight in just seven days.

“It does not take much. It can be done with small but right interventions,” said Dahal as she sat next to the child in the family’s brick-and-cement home.

Protein-energy malnutrition occurs due to inadequate intake of food and is a major cause of morbidity and mortality in children in developing countries (Grover and Ee 2009).



Protein energy malnutrition (PEM) has significant negative impacts on children’s growth and development (Grover and Ee 2009). Chronic PEM causes children to have stunted growth (low height for age) and to be underweight (low weight for age); it is estimated that among children under age five, one in every four is stunted and one in every six is underweight. PEM also causes two specific conditions in children: marasmus, which is characterized by an emaciated appearance, and kwashiorkor, in which children develop swollen bellies due to edema (abnormal accumulation of fluid) and discoloration of the hair because of pigment loss among other symptoms (UNWFP 2013b, Ahmed et al. 2012). Countries in sub-Saharan Africa and south Asia have the highest proportions of children suffering from PEM (UNWFP 2013a).

PEM causes direct mortality in children and also increases vulnerability to other serious diseases including diarrhea, pneumonia, and malaria. Children suffering from PEM have compromised immune systems, making them particularly susceptible to infectious diseases.  Furthermore, PEM has negative impacts on children’s brain development, resulting in issues with memory and delayed motor function; these children have decreased ability to learn and have lower productivity as adults. PEM also has serious and potentially long-term impacts on other organ systems including the cardiovascular, respiratory, and gastrointestinal systems (Grover and Ee 2009).

Many adults in developing countries also suffer from PEM, with women disproportionately impacted compared with men, particularly in south Asian countries (UNWFP 2013a). Pregnant women who are undernourished can fall even further behind in their nutritional status due to the increased demand for nutrients by the developing fetus. Women who don’t gain sufficient weight during pregnancy are at increased risk for complications including maternal morbidity and mortality, low birth weight, and neonatal mortality. These women can also have difficulty providing sufficient quantities of breast milk, leading to malnutrition among neonates (Ahmed et al. 2012).

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The relationship of stress hypermetabolism to essential protein needs

Curator: Larry H. Bernstein, MD, FCAP



The relationship of stress hypermetabolism to essential protein needs

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

Subtitle: Transthyretin and the Systemic Inflammatory Response

Larry H. Bernstein, MD, FACP, Clinical Pathologist, Biochemist, and Transfusion Physician
President, Triplex, Trumbull, CT 06611, USA


Brief introduction

Transthyretin  (also known as prealbumin) has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompted a review of the  benefit of using this test in acute and chronic care. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  A much better understanding of the significance of this program has emerged from studies of nitrogen and sulfur in health and disease.

Transthyretin protein structure

Transthyretin protein structure (Photo credit: Wikipedia)

Age-standardised disability-adjusted life year...

Age-standardised disability-adjusted life year (DALY) rates from Protein-energy malnutrition by country (per 100,000 inhabitants). (Photo credit: Wikipedia)


The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum.
Larry H Bernstein
Clin Chem Lab Med 2007; 45(11):0
ICID: 939932
Article type: Editorial

The Transthyretin Inflammatory State Conundrum
Larry H. Bernstein
Current Nutrition & Food Science, 2012, 8, 00-00

Keywords: Tranthyretin (TTR), systemic inflammatory response syndrome (SIRS), protein-energy malnutrition (PEM), C- reactive protein, cytokines, hypermetabolism, catabolism, repair.

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases. It is sensitive to the systemic inflammatory response syndrome (SIRS), and needs to be understood in the context of acute illness to be used effectively.

There are a number of physiologic changes associated with SIRS and the injury/repair process that affect TTR and  in the context of an ICU setting, the contribution of TTR is essential.  The only consideration is the timing of initiation since the metabolic burden is sufficiently high that a substantial elevation is expected in the first 3 days post admission, although the level of this biomarker is related to the severity of injury. Despite the complexity of the situation, TTR is not to be considered a test “for all seasons”. In the context of age, prolonged poor meal intake, chronic or acute illness, TTR needs to be viewed in a multivariable lens, along with estimated lean body mass, C-reactive protein, the absolute lymphocyte count, presence of neutrophilia, and perhaps procalcitonin if there is remaining uncertainty. Furthermore, the reduction of risk of associated complication requires a systematized approach to timely identification, communication, and implementation of a suitable treatment plan.

The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.


Title: The Automated Malnutrition Assessment
Accepted 29 April 2012. http://www.nutritionjrnl.com. Nutrition (2012), doi:10.1016/j.nut.2012.04.017.
Authors: Gil David, PhD; Larry Howard Bernstein, MD; Ronald R Coifman, PhD
Article Type: Original Article

Keywords: Network Algorithm; unsupervised classification; malnutrition screening; protein energy malnutrition (PEM); malnutrition risk; characteristic metric; characteristic profile; data characterization; non-linear differential diagnosis

We have proposed an automated nutritional assessment (ANA) algorithm that provides a method for malnutrition risk prediction with high accuracy and reliability.  The problem of rapidly identifying risk and severity of malnutrition is crucial for minimizing medical and surgical complications. These are not easily performed or adequately expedited. We characterized for each patient a unique profile and mapped similar patients into a classification. We also found that the laboratory parameters were sufficient for the automated risk prediction.


Title: The Increasing Role for the Laboratory in Nutritional Assessment
Article Type: Editorial
Section/Category: Clinical Investigation
Accepted 22 May 2012. http://www.elsevier.com/locate/clinbiochem.
Clin Biochem (2012), doi:10.1016/j.clinbiochem.2012.05.024
Keywords: Protein Energy Malnutrition; Nutritional Screening; Laboratory Testing
Author: Dr. Larry Howard Bernstein, MD

The laboratory role in nutritional management of the patient has seen remarkable growth while there have been dramatic changes in technology over the last 25 years, and it is bound to be transformative in the near term. This editorial is an overview of the importance of the laboratory as an active participant in nutritional care.

The discipline emerged divergently along separate paths with unrelated knowledge domains in physiological chemistry, pathology, microbiology, immunology and blood cell recognition, and then cross-linked emerging into clinical biochemistry, hematology-oncology, infectious diseases, toxicology and therapeutics, genetics, pharmacogenomics, translational genomics and clinical diagnostics.

In reality, the more we learn about nutrition, the more we uncover of metabolic diversity of individuals, the family, and societies in adapting and living in many unique environments and the basic reactions, controls, and responses to illness. This course links metabolism to genomics and individual diversity through metabolomics, which will be enlightened by chemical and bioenergetic insights into biology and translated into laboratory profiling.

Vitamin deficiencies were discovered as clinical entities with observed features as a result of industrialization (rickets and vitamin D deficiency) and mercantile trade (scurvy and vitamin C)[2].  Advances in chemistry led to the isolation of each deficient “substance”.  In some cases, a deficiency of a vitamin and what is later known as an “endocrine hormone” later have confusing distinctions (vitamin D, and islet cell insulin).

The accurate measurement and roles of trace elements, enzymes, and pharmacologic agents was to follow within the next two decades with introduction of atomic absorption, kinetic spectrophotometers, column chromatography and gel electrophoresis.  We had fully automated laboratories by the late 1960s, and over the next ten years basic organ panels became routine.   This was a game changer.

Today child malnutrition prevalence is 7 percent of children under the age of 5 in China, 28 percent in sub-Saharan African, and 43 percent in India, while under-nutrition is found mostly in rural areas with 10 percent of villages and districts accounting for 27-28 percent of all Indian underweight children. This may not be surprising, but it is associated with stunting and wasting, and it has not receded with India’s economic growth. It might go unnoticed viewed alongside a growing concurrent problem of worldwide obesity.

The post WWII images of holocaust survivors awakened sensitivity to nutritional deprivation.

In the medical literature, Studley [HO Studley.  Percentage of weight loss. Basic Indicator of surgical risk in patients with chronic peptic ulcer.  JAMA 1936; 106(6):458-460.  doi:10.1001/jama.1936.02770060032009] reported the association between weight loss and poor surgical outcomes in 1936.  Ingenbleek et al [Y Ingenbleek, M De Vissher, PH De Nayer. Measurement of prealbumin as index of protein-calorie malnutrition. Lancet 1972; 300[7768]: 106-109] first reported that prealbumin (transthyretin, TTR) is a biomarker for malnutrition after finding very low TTR levels in African children with Kwashiorkor in 1972, which went unnoticed for years.  This coincided with the demonstration by Stanley Dudrick  [JA Sanchez, JM Daly. Stanley Dudrick, MD. A Paradigm ShiftArch Surg. 2010; 145(6):512-514] that beagle puppies fed totally through a catheter inserted into the superior vena cava grew, which method was then extended to feeding children with short gut.  Soon after Bistrian and Blackburn [BR Bistrian, GL Blackburn, E Hallowell, et al. Protein status of general surgical patients. JAMA 1974; 230:858; BR Bistrian, GL Blackburn, J Vitale, et al. Prevalence of malnutrition in general medicine patients, JAMA, 1976, 235:1567] showed that malnourished hospitalized medical and surgical patients have increased length of stay, increased morbidity, such as wound dehiscence and wound infection, and increased postoperative mortality, later supported by many studies.

Michael Meguid,MD, PhD, founding editor of Nutrition [Elsevier] held a nutrition conference “Skeleton in the Closet – 20 years later” in Los Angeles in 1995, at which a Beckman Prealbumin Roundtable was held, with Thomas Baumgartner and Michael M Meguid as key participants.  A key finding was that to realize the expected benefits of a nutritional screening and monitoring program requires laboratory support. A Ross Roundtable, chaired by Dr. Lawrence Kaplan, resulted in the first Standard of Laboratory Practice Document of the National Academy of Clinical Biochemists on the use of the clinical laboratory in nutritional support and monitoring. Mears then showed a real benefit to a laboratory interactive program in nutrition screening based on TTR [E Mears. Outcomes of continuous process improvement of a nutritional care program incorporating serum prealbumin measurements. Nutrition 1996; 12 (7/8): 479-484].

A later Ross Roundtable on Quality in Nutritional Care included a study of nutrition screening and time to dietitian intervention organized by Brugler and Di Prinzio that showed a decreased length of hospital stay with $1 million savings in the first year (which repeated), which included reduced cost for dietitian evaluations and lower complication rates.

Presentations were made at the 1st International Transthyretin Congress in Strasbourg, France by Mears [E Mears.  The role of visceral protein markers in protein calorie malnutrition. Clin Chem Lab Med 2002; 40:1360-1369] on the impact of TTR in screening for PEM in a public hospital in Louisiana, and by Potter [MA Potter, G Luxton. Prealbumin measurement as a screening tool for patients with protein calorie malnutrition in emergency hospital admissions: a pilot study.  Clin Invest Med. 1999; 22(2):44-52] that indicated a 17% in-hospital mortality rate in a Canadian hospital for patients with PCM compared with 4% without PCM (p < 0.02), while only 42% of patients with PCM received nutritional supplementation. Cost analysis of screening with prealbumin level projected a saving of $414 per patient screened.  Ingenbleek and Young [Y Ingenbleek, VR Young.  Significance of transthyretin in protein metabolism.  Clin Chem Lab Med. 2002; 40(12):1281–1291.  ISSN (Print) 1434-6621, DOI: 10.1515/ CCLM.2002.222, December 2002. published online: 01/06/2005] tied the TTR to basic effects reflected in protein metabolism.


Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H Bernstein
Clin Biochem 2008; 41(14-15):1126-1130
ICID: 939927
Article type: Original article

TTR levels correlate with patient outcomes and are an accurate predictor of patient recovery in non-critically ill patients, but it is uncertain whether or not TTR level correlates with level of nutrition support and outcome in critically ill patients. This issue has been addressed only in critically ill patients on total parenteral nutrition and there was no association reported with standard outcome measures. We revisit this in all patients admitted to a medical intensive care unit.

Serum TTR was measured on the day of admission, day 3 and day 7 of their ICU stay. APACHE II and SOFA score was assessed on the day of admission. A registered dietician for their entire ICU stay assessed the nutritional status and nutritional requirement. Patients were divided into three groups based on initial TTR level and the outcome analysis was performed for APACHE II score, SOFA score, ICU length of stay, hospital length of stay, and mortality.

TTR showed excellent concordance with the univariate or multivariate classification of patients with PEM or at high malnutrition risk, and followed for seven days in the ICU, it is a measure of the metabolic burden.  TTR levels decline from day 1 to day 7 in spite of providing nutritional support. Twenty-five patients had an initial TTR serum concentration more than 17 mg/dL (group 1), forty-eight patients had mild malnutrition with a concentration between 10 and 17 mg/dL (group 2), Forty-five patients had severe malnutrition with a concentration less than 10 mg/dL (group 3).  Initial TTR level had inverse correlation with ICU length of stay, hospital length of stay, and APACHE II score, SOFA score; and predicted mortality, especially in group 3.


A simplified nutrition screen for hospitalized patients using readily available laboratory and patient
Linda Brugler, Ana K Stankovic, Madeleine Schlefer, Larry Bernstein
Nutrition 2005; 21(6):650-658
ICID: 825623
Article type: Review article
The role of visceral protein markers in protein calorie malnutrition.
Linda Brugler, Ana Stankovic, Larry Bernstein, Frederick Scott, Julie O’Sullivan-Maillet
Clin Chem Lab Med 2002; 40(12):1360-1369
ICID: 636207
Article type: Original article

The Automated Nutrition Score is a data-driven extension of continuous quality improvement.

Larry H Bernstein
Nutrition 2009; 25(3):316-317
ICID: 939934

Transthyretin: its response to malnutrition and stress injury. clinical usefulness and economic implications.
LH Bernstein, Y Ingenbleek
Clin Chem Lab Med 2002; 40(12):1344-1348
ICID: 636205
Article type: Original article


Yves Ingenbleek  MD  PhD  and  Larry Bernstein MD
J CLIN LIGAND ASSAY  (out of print)

The acute reaction to stress is characterized by major metabolic, endocrine and immune alterations. According to classical descriptions, these changes clinically present as a succession of 3 adaptive steps – ebb phase, catabolic flow phase, and anabolic flow phase. The ebb phase, shock and resuscitation, is immediate, lasts several hours, and is characterized by hypokinesis, hypothermia, hemodynamic instability and reduced basal metabolic rate. The catabolic flow phase, beginning within 24 hours and lasting several days, is characterized by catabolism with the flow of gluconeogenic substrates and ketone bodies in response to the acute injury. The magnitude of the response depends on the acuity and the severity of the stress. The last, a reparative anabolic flow phase, lasts weeks and is characterized by the accretion of amino acids (AAs) to rebuilding lean body mass.

The current opinion is that the body economy is reset during the course of stress at novel thresholds of metabolic priorities. This is exemplified mainly by proteolysis of muscle, by an effect on proliferating gut mucosa and lymphoid tissue as substrates are channeled to support wound healing, by altered syntheses of liver proteins with preferential production of acute phase proteins (APPs) and local repair in inflamed tissues (3). The first two stages demonstrate body protein breakdown exceeding the rate of protein synthesis, resulting in a negative nitrogen (N) balance, muscle wasting and weight loss. In contrast, the last stage displays reversed patterns, implying progressive recovery of endogenous N pools and body weight.

These adaptive alterations undergo continuing elucidation. The identification of cytokines, secreted by activated macrophages/monocytes or other reacting cells, has provided further insights into the molecular mechanisms controlling energy expenditure, redistribution of protein pools, reprioritization of syntheses and secretory processes.

The free fraction of hormones bound to specific binding-protein(s) [BP(s)] manifests biological activities, and any change in the BP blood level modifies the effect of the hormone on the end target organ.  The efficacy of these adaptive responses may be severely impaired in protein-energy malnourished (PEM) patients. This is especially critical with respect to changes of the circulating levels of transthyretin (TTR), retinol-binding protein (RBP) and corticosteroid-binding globulin (CBG) conveying thyroid hormones (TH), retinol and cortisol, respectively.  This reaction is characterized by cytokine mediated autocrine, paracrine and endocrine changes. Among the many inducing molecules identified, interleukins 1 and 6 (Il-1, Il-6) and tumor necrosis factor a (TNF) are associated with enhanced production of 3 counterregulatory hormonal families (cortisol, catecholamines and glucagon). Growth hormone (GH) and TH also have roles in these metabolic adjustments.

There is overproduction of cortisol mediated by several cytokines acting on both the adrenal cortex (10) and on the pituitary through hypothalamic CRH with loss of feedback regulation of ACTH production (11). Hypercortisolemia is a major finding observed after surgery (12), sepsis (13), and medical insults, usually correlated with severity of insult and of complications. Rising cortisol values parallel hyperglycemic trends, as an effect of both gluconeogenesis and insulin resistance. Working in concert with TNF, glucocorticoids govern the breakdown of muscle mass, which is regarded as the main factor responsible for the negative N balance.

Under normal conditions, GH exerts both lipolytic and anabolic influences in the whole body economy under the dual control of the hypothalamic hormones somatocrinin (GHRH) and somatostatin (SRIH). GH secretion is usually depressed by rising blood concentrations of glucose and free fatty acids (FFAs) but is paradoxicaly elevated despite hyperglycemia in stressed patients.

The oversecretion of counterregulatory hormones working in concert generates subtle equilibria between glycogenolytic/glycolytic/gluconeogenic adaptive processes. The net result is the neutralization of the main hypoglycemic and anabolic activities of insulin and the development of a persisting and controlled hyperglycemic tone in the stressed body. The molecular mechanisms whereby insulin resistance occurs in the course of stress refer to
cytokine-  and  hormone-induced  phosphorylation abnormalities affecting receptor signaling. The insulin-like anabolic processes of GH are mediated by IGF1 working as relay agent. The expected high IGF1 surge associated with GH oversecretion is not observed in severe stress as plasma values are usually found at the lower limit of normal or even in the subnormal range.  The end result of this dissociation between high GH and low IGF1 levels is to favor the proteolysis of muscle mass to release AAs for gluconeogenesis and the breakdown of adipose tissue to provide ketogenic substrates.

The acute stage of stress is associated with the onset of a low T3 syndrome typically delineated by the drop of both total (TT3) and free (FT3) triiodothyronine plasma levels in the subnormal range. In contrast, both total (TT4) and free (FT4) thyroxine values usually remain within normal ranges with declining trends observed for TT4 and rising tendencies for FT4 (44). This last free compound is regarded as the sensor reflecting the actual thyroid status and governing the release of TSH whereas FT3 works as the active hormonal mediator at nuclear receptor level. The maintenance of an euthyroid sick syndrome is compatible with the down-regulation of most metabolic and energetic processes in healthy tissues. These inhibitory effects , negatively affecting all functional steps of the hypothalamo-pituitary-thyroid axis concern TSH production, iodide uptake, transport and organification into iodotyrosyl residues, peroxidase coupling activity as well as thyroglobulin synthesis and TH leakage. Taken together, the above-mentioned data indicate that the development of hyperglycemia and of insulin-resistance in healthy tissues – mainly in the muscle mass – are hallmarks resulting from the coordinated activities of the counterregulatory hormones.

A growing body of recent data suggest that the stressed territory, whatever the causal agent – bacterial or viral sepsis, auto-immune disorder, traumatic or toxic shock, burns, cancer – manifest differentiated metabolic and immune reactions. The amplitude, duration and efficacy of these responses are reportedly impaired along several ways in PEM patients. These last detrimental effects are accompanied by a number of medical, social and economical consequences, such as extended length of hospital stay and increased complication / mortality rates. It is therefore mandatory to correctly identify and follow up the nutritional status of hospitalized patients. Such approaches are prerequisite to timely and scientifically grounded nutritional and pharmacological mediated interventions.

Contrary to the rest of the body, energy requirements of the inflamed territory are primarily fulfilled by anaerobic glycolysis, an effect triggered by the inhibition of key-enzymes of carbohydrate metabolism, notably pyruvate-dehydrogenase. This non-oxidative combustion of glucose reveals low conversion efficiency but offers the major advantage to maintain, in the context of hyperglycemia, fuel provision to poorly irrigated and/or edematous tissues. The depression of the 5’-monodeiodinating activity (5’-DA) plays a pivotal role in these adaptive changes, yielding inactive reverse T3 (rT3) as index of impaired T4 to T3 conversion rates, but at the same time there is an augmented supply of bioactive T3 molecules and local overstimulation of thyro-dependent processes characterized by thyroid down-regulation.  The same differentiated evolutionary pattern applies to IGF1. In spite of lowered plasma total concentrations, the proportion of IGF1 released in free form may be substantially increased owing to the proteolytic degradation of IGFBP-3 in the intravascular compartment. The digestion of  BP-3 results from the surge of several proteases occurring the course of stress, yielding biologically active IGF1 molecules available for the repair of damaged tissues. In contrast, healthy receptors oppose a strong resistance to IGF1 ligands freed in the general circulation, likely induced by an acquired phosphorylation defect very similar in nature to that for the insulin transduction pathway.

PEM is the generic denomination of a broad spectrum of nutritional disorders, commonly found in hospital settings, and whose extreme poles are identified as marasmus and kwashiorkor. The former condition is usually regarded as the result of long-lasting starvation leading to the loss of lean body mass and fat reserves but relatively well-preserved liver function and immune capacities. The latter condition is typically the consequence of (sub)acute deprivation predominantly affecting the protein content of staplefood, an imbalance causing hepatic steatosis, fall of visceral proteins, edema and increased vulnerability to most stressful factors. PEM may be hypometabolic or hypermetabolic, usually coexists with other diseased states and is frequently associated with complications. Identification of PEM calls upon a large set of clinical and analytical disciplines comprising anthropometry, immunology, hematology and biochemistry.

CBG, TTR and RBP share in common the transport of specific ligands exerting their metabolic effects at nuclear receptor level. Released from their specific BPs in free form, cortisol, FT4 and retinol immediately participe to the strenghtening of the positive and negative responses to stressful stimuli. CBG is a relatively weak responder to short-term nutritional influences (73)  although long-lasting PEM is reportedly capable of causing its significant diminution (74). The dramatic drop of CBG in the course of stress appears as the combined effect of Il-6-induced posttranscriptional blockade of its liver synthesis (75) and peripheral overconsumption by activated neutrophils (61). The divergent alterations outlined by CBG and total cortisolemia result in an increased disposal of free ligand reaching proportions considerably higher than the 4 % recorded under physiological conditions.

The appellation of negative APPs that was once given to the visceral group of carrier-proteins. The NDAD concept takes the opposite view, defending the opinion that their suppressed synthesis releases free ligands which positively contribute to strengthen all aspects of the stress reaction, justifying the ABR denomination. This implies that the role played by ABRs should no longer be interpreted in terms of concentrations but in terms of functionality.


Yves Ingenbleek. The Open Clinical Chemistry Journal, 2011, 4, 34-44.

Vegetarian subjects consuming subnormal amounts of methionine (Met) are characterized by subclinical protein malnutrition causing reduction in size of their lean body mass (LBM) best identified by the serial measurement of plasma transthyretin (TTR). As a result, the transsulfuration pathway is depressed at cystathionine-β-synthase (CβS) level triggering the upstream sequestration of homocysteine (Hcy) in biological fluids and promoting its conversion to Met. Maintenance of beneficial Met homeostasis is counterpoised by the drop of cysteine (Cys) and glutathione (GSH) values downstream to CβS causing in turn declining generation of hydrogen sulfide (H2S) from enzymatic sources. The biogenesis of H2S via non-enzymatic reduction is further inhibited in areas where earth’s crust is depleted in elemental sulfur (S8) and sulfate oxyanions. Combination of subclinical malnutrition and S8-deficiency thus maximizes the defective production of Cys, GSH and H2S reductants, explaining persistence of unabated oxidative burden. The clinical entity increases the risk of developing cardiovascular diseases (CVD) and stroke in underprivileged plant-eating populations regardless of Framingham criteria and vitamin-B status. Although unrecognized up to now, the nutritional disorder is one of the commonest worldwide, reaching top prevalence in populated regions of Southeastern Asia. Increased risk of hyperhomocysteinemia and oxidative stress may also affect individuals suffering from intestinal malabsorption or westernized communities having adopted vegan dietary lifestyles.

Metabolic pathways: Met molecules supplied by dietary proteins are submitted to TM processes allowing to release Hcy which may in turn either undergo Hcy – Met RM pathways or be irreversibly committed into TS decay. Impairment of CbS activity, as described in protein malnutrition, entails supranormal accumulation of Hcy in body fluids, stimulation of activity and maintenance of Met homeostasis. This last beneficial effect is counteracted by decreased concentration of most components generated downstream to CbS, explaining the depressed CbS- and CbL-mediated enzymatic production of H2S along the TS cascade. The restricted dietary intake of elemental S further operates as a limiting factor for its non-enzymatic reduction to H2S which contributes to downsizing a common body pool. Combined protein- and S-deficiencies work in concert to deplete Cys, GSH and H2S from their body reserves, hence impeding these reducing molecules to properly face the oxidative stress imposed by hyperhomocysteinemia.

see also …

McCully, K.S. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am. J. Pathol., 1996, 56, 111-128.

Cheng, Z.; Yang, X.; Wang, H. Hyperhomocysteinemia and endothelial dysfunction. Curr. Hypertens. Rev., 2009, 5,158-165.

Loscalzo, J. The oxidant stress of hyperhomocyst(e)inemia. J. Clin.Invest., 1996, 98, 5-7.

Ingenbleek, Y.; Hardillier, E.; Jung, L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition, 2002, 18, 40-46.

Ingenbleek, Y.; Young, V.R. The essentiality of sulfur is closely related to nitrogen metabolism: a clue to hyperhomocysteinemia. Nutr. Res. Rev., 2004, 17, 135-153.

Hosoki, R.; Matsuki, N.; Kimura, H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem. Biophys. Res. Commun., 1997, 237, 527-531.

Tang, B.; Mustafa, A.; Gupta, S.; Melnyk, S.; James S.J.; Kruger, W.D. Methionine-deficient diet induces post-transcriptional downregulation of cystathionine-􀀁-synthase. Nutrition, 2010, 26, 1170-1175.

Yves Ingenbleek. Plasma Transthyretin Reflects the Fluctuations of Lean Body Mass in Health and Disease. Chapter 20. In S.J. Richardson and V. Cody (eds.), Recent Advances in Transthyretin Evolution, Structure and Biological Functions, DOI: 10.1007/978‐3‐642‐00646‐3_20, # Springer‐Verlag Berlin Heidelberg 2009.

Transthyretin (TTR) is a 55-kDa protein secreted mainly by the choroid plexus and the liver. Whereas its intracerebral production appears as a stable secretory process allowing even distribution of intrathecal thyroid hormones, its hepatic synthesis is influenced by nutritional and inflammatory circumstances working concomitantly. Both morbid conditions are governed by distinct pathogenic mechanisms leading to the reduction in size of lean body mass (LBM). The liver production of TTR integrates the dietary and stressful components of any disease spectrum, explaining why it is the sole plasma protein whose evolutionary patterns closely follow the shape outlined by LBM fluctuations. Serial measurement of TTR therefore provides unequalled information on the alterations affecting overall protein nutritional status. Recent advances in TTR physiopathology emphasize the detecting power and preventive role played by the protein in hyperhomocysteinemic states, acquired metabolic disorders currently ascribed to dietary restriction in water-soluble vitamins. Sulfur (S)-deficiency is proposed as an additional causal factor in the sizeable proportion of hyperhomocysteinemic patients characterized by adequate vitamin intake but experiencing varying degrees of nitrogen (N)-depletion. Owing to the fact that N and S coexist in plant and animal tissues within tightly related concentrations, decreasing LBM as an effect of dietary shortage and/or excessive hypercatabolic losses induces proportionate S-losses. Regardless of water-soluble vitamin status, elevation of homocysteine plasma levels is negatively correlated with LBM reduction and declining TTR plasma levels. These findings occur as the result of impaired cystathionine-b-synthase activity, an enzyme initiating the transsulfuration pathway and whose suppression promotes the upstream accumulation and remethylation of homocysteine molecules. Under conditions of N- and S-deficiencies, the maintenance of methionine homeostasis indicates high metabolic priority.

Schematically, the human body may be divided into two major compartments, namely fat mass (FM) and FFM that is obtained by substracting
FM from body weight (BW). The fat cell mass sequesters about 80% of the total body lipids, is poorly hydrated and contains only small quantities of lean tissues and nonfat constituents. FFM comprises the sizeable part of lean tissues and minor mineral compounds among which are Ca, P, Na, and Cl pools totaling about 1.7 kg or 2.5% of BW in a healthy man weighing 70 kg. Subtraction of mineral mass from FFM provides LBM, a composite aggregation of organs and tissues with specific functional properties. LBM is thus nearly but not strictly equivalent to FFM. With extracellular mineral content subtracted, LBM accounts for most of total body proteins (TBP) and of TBN assuming a mean 6.25 ratio between protein and N content.

SM accounts for 45% of TBN whereas the remaining 55% is in nonmuscle lean tissues. The LBM of the reference man contains 98% of total
body potassium (TBK) and the bulk of total body sulfur (TBS). TBK and TBS reach equal intracellular amounts (140 g each) and share distribution patterns (half in SM and half in the rest of cell mass).  The body content of K and S largely exceeds that of magnesium (19 g), iron (4.2 g) and zinc (2.3 g). The average hydration level of LBM in healthy subjects of all age is 73% with the proportion of the intracellular/extracellular fluid spaces being 4:3. SM is of particular relevance in nutritional studies due to its capacity to serve as a major reservoir of amino acids (AAs) and as a dispenser of gluconeogenic substrates. An indirect estimate of SM size consists in the measurement of urinary creatinine, end-product of the nonenzymatic hydrolysis of phosphocreatine which is limited to muscle cells.

During ageing, all the protein components of the human body decrease regularly. This shrinking tendency is especially well documented for SM  whose absolute amount is preserved until the end of the fifth decade, consistent with studies showing unmodified muscle structure, intracellular K content and working capacit. TBN and TBK are highly correlated in healthy subjects and both parameters manifest an age-dependent curvilinear decline
with an accelerated decrease after 65 years.  The trend toward sarcopenia is more marked and rapid in elderly men than in elderly women decreasing strength and functional capacity. The downward SM slope may be somewhat prevented by physical training or accelerated by supranormal cytokine status as reported in apparently healthy aged persons suffering low-grade inflammation. 2002) or in critically ill patients whose muscle mass undergoes proteolysis and contractile dysfunction.

The serial measurement of plasma TTR in healthy children shows that BP values are low in the neonatal period and rise linearly with superimposable concentrations in both sexes during infant growth consistent with superimposable N accretion and protein synthesis rates. Starting from the sixties, TTR values progressively decline showing steeper slopes in elderly males. The lowering trend seems to be initiated by the attenuation of androgen influences and trophic stimuli with increasing age. The normal human TTR trajectory from birth to death has been well documented by scientists belonging to the Foundation for Blood Research. TTR is the first plasma protein to decline in response to marginal protein restricion, thus working as an early signal warning that adaptive mechanisms maintaining homeostasis are undergoing decompensation.

TTR was proposed as a marker of protein nutritional status following a clinical investigation undertaken in 1972 on protein-energy malnourished (PEM) Senegalese children (Ingenbleek et al. 1972). By comparison with ALB and transferrin (TF) plasma values, TTR revealed a much higher degree of sensitivity to changes in protein status that has been attributed to its shorter biological half-life (2 days) and to its unusual Trp richness (Ingenbleek et al. 1972, 1975a). Transcription of the TTR gene in the liver is directed by CCAAT/enhancer binding protein (C/EBP) bound to hepatocyte nuclear factor 1 (HNF1) under the control of several other HNFs. The mechanism responsible for the suppressed TTR synthesis in PEM-states is a restricted AA and energy supply working as limiting factors (Ingenbleek and Young 2002). The rapidly turning over TTR protein is highly responsive to any change in protein flux and energy supply, being clearly situated on the cutting edge of the equipoise.

LBM shrinking may be the consequence of either dietary restriction reducing protein syntheses to levels compatible with survival or that of cytokine-induced tissue proteolysis exceeding protein synthesis and resulting in a net body negative N balance. The size of LBM in turn determines plasma TTR concentrations whose liver production similarly depends on both dietary provision and inflammatory conditions. In animal cancer models, reduced TBN pools were correlated with decreasing plasma TTR values and provided the same predictive ability. In kidney patients, LBM is proposed as an excellent predictor of outcome working in the same direction as TTR plasma levels.  High N intake, supposed to preserve LBM reserves, reduces significantly the mortality rate of kidney patients and is positively correlated with the alterations of TTR plasma concentrations appearing as the sole predictor of final outcome. It is noteworthy that most SELDI or MALDI workers interested in defining protein nutritional status have chosen TTR as a biomarker, showing that there exists a large consensus considering the BP as the most reliable indicator of protein depletion in most morbid circumstances.

Total homocysteine (tHcy) is a S-containing AA not found in customary diets but endogenously produced in the body of mammals by the enzymatic transmethylation of methionine (Met), one of the eight IAAs supplied by staplefoods. tHcy may either serve as precursor substrate for the synthesis of new Met molecules along the remethylation (RM) pathway or undergo irreversible kidney leakage through a cascade of derivatives defining the transsulfuration (TS) pathway. Hcy is thus situated at the crossroad of RM and TS pathways that are regulated by three water-soluble vitamins (pyridoxine, B6; folates, B9; cobalamins, B12).

Significant positive correlations are found between tHcy and plasma urea and plasma creatinine, indicating that both visceral and muscular tissues undergo proteolytic degradation throughout the course of rampant inflammatory burden. In healthy individuals, tHcy plasma concentrations maintain positive correlations with LBM and TTR from birth until the end of adulthood. Starting from the onset of normal old age, tHcy values become disconnected from LBM control and reveal diverging trends with TTR values. Of utmost importance is the finding that, contrary to all protein
components which are downregulated in protein-depleted states, tHcy values are upregulated.  Hyperhomocysteinemia is an acquired clinical entity characterized by mild or moderate elevation in tHcy blood values found in apparently healthy individuals (McCully 1969). This distinct morbid condition appears as a public health problem of increasing importance in the general population, being regarded as an independent and graded risk factor for vascular pathogenesis unrelated to hypercholesterolemia, arterial hypertension, diabetes and smoking.

Studies grounded on stepwise multiple regression analysis have concluded that the two main watersoluble vitamins account for only 28% of tHcy variance whereas vitamins B6, B9, and B12, taken together, did not account for more than 30–40% of variance. Moreover, a number of hyperhomocysteinemic conditions are not responsive to folate and pyridoxine supplementation. This situation prompted us to search for other causal factors which might fill the gap between the public health data and the vitamin triad deficiencies currently incriminated. We suggest that S – the forgotten element – plays central roles in nutritional epidemiology (Ingenbleek and Young 2004).

Aminoacidemia studies performed in PEM children, adult patients and elderly subjects have reported that the concentrations of plasma IAAs invariably display lowering trends as the morbid condition worsens. The depressed tendency is especially pronounced in the case of tryptophan and for the so-called branched-chain AAs (BCAAs, isoleucine, leucine, valine) the decreases in which are regarded as a salient PEM feature following the direction outlined by TTR (Ingenbleek et al. 1986). Met constitutes a notable exception to the above described evolutionary profiles, showing unusual stability in chronically protein depleted states.

Maintenance of normal methioninemia is associated with supranormal tHcy blood values in PEMadults (Ingenbleek et al. 1986) and increased tHcy leakage in the urinary output of PEM children. In contrast, most plasma and urinary S-containing compounds produced along the TS pathway downstream to CbSconverting step (Fig. 20.1) display significantly diminished values. This is notably the case for cystathionine (Ingenbleek et al. 1986), glutathione, taurine, and sulfaturia. Such distorted patterns are reminiscent of abnormalities defining homocystinuria, an inborn disease of Met metabolism characterized by CbS refractoriness to pyridoxine stimuli, thereby promoting the upstream retention of tHcy in biological fluids. It
was hypothesized more than 20 years ago (Ingenbleek et al. 1986) that PEM is apparently able to similarly depress CbS activity, suggesting that the enzyme is a N-status sensitive step working as a bidirectional lockgate, overstimulated by high Met intake (Finkelstein and Martin 1986) and downregulated under N-deprivation conditions (Ingenbleek et al. 2002). Confirmation that N dietary deprivation may inhibit CbS activity has recently provided. The tHcy precursor pool is enlarged in biological fluids, boosting Met remethylation processes along the RM pathway, consistent with studies showing overstimulation of Met-synthase activity in conditions of protein restriction. In other words, high tHcy plasma concentrations observed in PEM states are the dark side of adaptive mechanisms for maintaining Met homeostasis. This is consistent with the unique role played by Met in the preservation of N body stores.

The classical interpretation that strict vegans, who consume plenty of folates in their diet and manifest nevertheless higher tHcy plasma concentrations than omnivorous counterparts, needs to be revisited. On the basis of hematological and biochemical criteria, cobalamin deficiency is one of the most prevalent vitamin-deficiencies wordwide, being often incriminated as deficient in vegan subjects. It seems, however, likely that its true causal impact on rising tHcy values is substantially overestimated in most studies owing to the modest contribution played by cobalamins on tHcy
variance analyses. In contrast, there exists a growing body of converging data indicating that the role played by the protein component is largely underscored in vegan studies. It is worth recalling that S is the main intracellular anion coexisting with N within a constant mean S:N ratio (1:14.5) in animal tissues and dietary products of animal origin (Ingenbleek 2006). The mean S:N ratio found in plant items ranges from 1:20 to 1:35, a proportion that does not optimally meet human tissue requirements (Ingenbleek 2006), paving the way for borderline S and N deficiencies.

A recent Taiwanese investigation on hyperhomocysteinemic nuns consuming traditional vegetarian regimens consisting of mainly rice, soy products,
vegetables and fruits with few or no dairy items illustrates such clinical misinterpretation (Hung et al. 2002). The authors reported that folates and cobalamins, taken together, accounted for only 28.6% of tHcy variance in the vegetarian cohort whereas pyridoxine was inoperative (Hung et al. 2002). The daily vegetable N and Met intakes were situated highly significantly (p < 0.001) below the recommended allowances for humans (FAO/WHO/United Nations University 1985), causing a stage of unrecognized PEM documented by significantly depressed BCAA plasma
concentrations. Met levels escaped the overall decline in IAAs levels, emphasizing that efficient homeostatic mechanisms operate at the expense of an acquired hyperhomocysteinemic state. The diagnosis of subclinical PEM was missed because the authors ignored the exquisitely sensitive TTR detecting power. A proper PEM identification would have allowed the authors to confirm the previously described TTR–tHcy relationship that was established in Western Africa from comparable field studies involving country dwellers living on plant products.

The concept that acute or chronic stressful conditions may exert similar inhibitory effects on CbS activity and thereby promote hyperhomocysteinemic states is founded on previous studies showing that hypercatabolic states are characterized by increased urinary N and S losses maintaining tightly correlated depletion rates (Cuthbertson 1931; Ingenbleek and Young 2004; Sherman and Hawk 1900) which reflect the S:N ratio found in tissues undergoing cytokine induced proteolysis. This has been documented in coronary infarction and in acute pancreatitis where tHcy elevation evolves too rapidly to allow for a nutritional vitamin B-deficit explanation.  tHcy is considered stable in plasma and the two investigations report unaltered folate and cobalamin plasma concentrations.

The clinical usefulness of TTR as a nutritional biomarker, described in the early seventies (Ingenbleek et al. 1972) has been substantially disregarded by the scientific community for nearly four decades. This long-lasting reluctance expressed by many investigators is largely due to the fact that protein malnutrition and stressful disorders of various causes have combined inhibitory effects on hepatic TTR synthesis. Declining TTR plasma concentrations may result from either dietary protein and energy restrictions or from cytokine-induced transcriptional blockade (Murakami et al. 1988) of its hepatic synthesis. The proposed marker was therefore seen as having high sensitivity but poor specificity. Recent advances in protein metabolism settle the controversy by throwing further light on the relationships between TTR and the N-components of body composition.

The developmental patterns of LBM and TTR exhibit striking similarities. Both parameters rise from birth to puberty, manifest gender dimorphism during full sexual maturity then decrease during ageing. Uncomplicated PEM primarily affects both visceral and structural pools of LBM with distinct kinetics, reducing protein synthesis to levels compatible with prolonged survival. In acute or chronic stressful disorders, LBM undergoes muscle proteolysis exceeding the upregulation of protein syntheses in liver and injured areas, yielding a net body negative N balance. These adaptive responses are well identified by the measurement of TTR plasma concentrations which therefore appear as a plasma marker for LBM fluctuations.
Attenuation of stress and/or introduction of nutritional rehabilitation restores both LBM and TTR to normal values following parallel slopes. TTR fulfills, therefore, a unique position in assessing actual protein nutritional status, monitoring the efficacy of dietetic support and predicting the patient’s outcome (Bernstein and Pleban 1996).

see also…

Acosta PB, Yannicelli S, Ryan AS, Arnold G, Marriage BJ, Plewinska M, Bernstein L, Fox J, Lewis V, Miller M, Velazquez A (2005) Nutritional therapy improves growth and protein status of children with a urea cycle enzyme defect. Mol Genet Metab 86:448–455.

Arroyave G, Wilson D, Be´har M, Scrimshaw NS (1961) Serum and urinary creatinine in children with severe protein malnutrition. Am J Clin Nutr 9:176–179.

Bates CJ, Mansoor MA, van der Pols J, Prentice A, Cole TJ, Finch S (1997) Plasma total homocysteine in a representative sample of 972 British men and women aged 65 and over. Eur J Clin Nutr 51:691–697.

Battezzatti A, Bertoli S, San Romerio A, Testolin G (2007) Body composition: An important determinant of homocysteine and methionine concentrations in healthy individuals. Nutr Metab Cardiovasc Dis 17:525–534.

Bernstein LH, Bachman TE, Meguid M, Ament M, Baumgartner T, Kinosian B, Martindale R, Spiekerman M (1995) Prealbumin in nutritional care Consensus Group. Measurement of visceral protein status in assessing protein and energy malnutrition: Standard of care. Nutrition 11:169–171

Bernstein LH, Ingenbleek Y (2002) Transthyretin: Its response to malnutrition and stress injury. Clinical usefulness and economical implications. Clin Chem Lab Med 40:1344–1348.

Boorsook H, Dubnoff JW (1947) The hydrolysis of phosphocreatine and the origin of creatinine. J Biol Chem 168:493–510.

Briend A, Garenne M, Maire B, Fontaine O, Dieng F (1989) Nutritional status, age and survival: The muscle mass hypothesis. Eur J Clin Nutr 43:715–726

Gray GE, Landel AM, Meguid MM (1994) Taurine-supplemented total parenteral nutrition and taurine status of malnourished cancer patients. Nutrition 10:11–15

Heymsfield SB, McManus C, Stevens V, Smith J (1982) Muscle mass: Reliable indicator of protein-energy malnutrition and outcome. Am J Clin Nutr 35:1192–1199

Ingenbleek Y (2006) The nutritional relationship linking sulfur to nitrogen in living organisms. J Nutr 136:S1641–S1651
Ingenbleek Y (2008) Plasma transthyretin indicates the direction of both nitrogen balance and retinoid status in health and disease. Open Clin Chem J 1:1–12
Ingenbleek Y, Bernstein LH (1999a) The stressful condition as a nutritionally dependent adaptive dichotomy. Nutrition 15:305–320
Ingenbleek Y, Bernstein LH (1999b) The nutritionally dependent adaptive dichotomy (NDAD) and stress hypermetabolism. J Clin Ligand Assay 22:259–267
Ingenbleek Y, Carpentier YA (1985) A prognostic inflammatory and nutritional index scoring critically ill patients. Internat J Vitam Nutr Res 55:91–101

Ingenbleek Y, Young VR (1994) Transthyretin (prealbumin) in health and disease: Nutritional implications. Annu Rev Nutr 14:495–533
Ingenbleek Y, Young VR (2002) Significance of transthyretin in protein metabolism. Clin Chem Lab Med 40:1281–1291
Ingenbleek Y, Young VR (2004) The essentiality of sulfur is closely related to nitrogen metabolism. Nutr Res Rev 17:135–151

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Simple representation of the toll-like recepto...

Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control

Curator and Author: Larry H Bernstein, MD, FCAP

What is Septic Shock?
Scripps Research Professor Wolfram Ruf and colleagues have identified a key connection between the signaling pathways and the immune system spiraling out of control involving the coagulation system and vascular endothelium that, if disrupted may be a target for sepsis. (Science Daily, Feb 29, 2008). It may be caused by a bacterial infection that enters the bloodstream, but we now recognize the same cascade not triggered by bacterial invasion. These invading bacteria produce endotoxins and other toxins that trigger a widespread inflammatory response of the innate immune system–a response that is necessary, as it turns out, because without the inflammation, the body cannot fight off the bacterial infection. During sepsis, the inflammation triggers widespread coagulation in the bloodstream. This coagulation can block blood vessels in vital organs, starving the organs of oxygen and damaging them. The organs can be further damaged when the blood starts to flow again because the lining of the blood vessels remain leaky due to inflammatory cytokines and damage by intravascular coagulation.
What is the Pathogenesis of Sepsis?
The acute respiratory distress syndrome (ARDS) has been defined as a severe form of acute lung injury featuring pulmonary inflammation and increased capillary leak. ARDS is associated with a high mortality rate and accounts for 100,000 deaths annually in the United States. ARDS may arise in a number of clinical situations, especially in patients with sepsis. A well-described pathophysiological model of ARDS is one form of the acute lung inflammation mediated by neutrophils, cytokines, and oxidant stress. Neutrophils are major effect cells at the frontier of innate immune responses, and they play a critical role in host defense against invading microorganisms. The tissue injury appears to be related to proteases and toxic reactive oxygen radicals released from activated neutrophils. In addition, neutrophils can produce cytokines and chemokines that enhance the acute inflammatory response. Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP). Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.
Uncontrolled activation of the coagulation cascade following lung injury contributes to the development of lung inflammation and fibrosis in acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and fibrotic lung disease. This article reviews our current understanding of the mechanisms leading to the activation of the coagulation cascade in response to lung injury and the evidence that excessive procoagulant activity is of pathophysiological significance in these disease settings. This is consistent with a pneumonia or lung injury preceding sepsis. On the other hand, it is not surprising that abdominal, cardiac bypass, and post cardiac revascularization may also lead to events resembling sepsis and/or cardiovascular collapse. The tissue factor-dependent extrinsic pathway is the predominant mechanism by which the coagulation cascade is locally activated in the lungs of patients with ALI/ARDS and pulmonary fibrosis. The cellular effects mediated via activation of proteinase-activated receptors (PARs) may be of particular importance in influencing inflammatory and fibroproliferative responses in experimental models involving direct injury to the lung. In this regard, studies in PAR1 knockout mice have shown that this receptor plays a major role in orchestrating the interplay between coagulation, inflammation and lung fibrosis.
The activation of the coagulation cascade is one of the earliest events initiated following tissue injury. The prime function of this complex and highly regulated proteolytic system is to generate insoluble, crosslinked fibrin strands, which bind and stabilize weak platelet hemostatic plugs, formed at sites of tissue injury. The formation of this provisional clot is critically dependent on the action of thrombin, and is generated following the stepwise activation of coagulation proteinases via the extrinsic and intrinsic systems. Under normal circumstances, blood is not exposed to tissue factor (TF). However, upon tissue injury, exposure of plasma to TF expressed on non-vascular cells or on activated endothelial cells results in the formation of the TF-activated factor VII (FVIIa) complex. The TF–FVIIa complex subsequently catalyses the initial activation of FX to activated factor X (FXa) and FIX to activated factor IX. FXa in association with activated factor V catalyses the conversion of prothrombin to thrombin. Sustained coagulation is achieved when thrombin synthesized through the initial TF–FVIIa–FXa complex catalyses the activation of FXI, FIX, FVIII and FX. In this manner, the intrinsic pathway is activated.
The systemic inflammatory response syndrome (SIRS) is the massive inflammatory reaction resulting from systemic mediator release that may lead to multiple organ dysfunction. I introduce an analysis of the roles of cytokines, cytokine production, and the relationship of cytokine production to the development of SIRS. The article postulates a three-stage development of SIRS, in which stage 1 is a local production of cytokines in response to an injury or infection. Stage 2 is the protective release of a small amount of cytokines into the body’s circulation. Stage 3 is the massive systemic reaction where cytokines turn destructive by compromising the integrity of the capillary walls and flooding end organs. While cytokines are generally viewed as a destructive development in the patient that generally leads to multiple organ dysfunction, cytokines also protect the body when localized. It will be necessary to study the positive effects of cytokines while also studying their role in causing SIRS. It will also be important to investigate the relationship between cytokines and their blockers in SIRS.
Monocyte/macrophage- and neutrophil-mediated inflammatory responses can be stimulated through a variety of receptors, including G protein-linked 7-transmembrane receptors (e.g., FPR1; MIM 136537), Fc receptors (see MIM 146790), CD14 (MIM 158120) and Toll-like receptors (e.g., TLR4; MIM 603030), and cytokine receptors (e.g., IFNGR1; MIM 107470). Engagement of these receptors can also prime myeloid cells to respond to other stimuli. Myeloid cells express receptors belonging to the Ig superfamily, such as TREM1, or to the C-type lectin superfamily. Depending on their transmembrane and cytoplasmic sequence structure, these receptors have either activating (e.g., KIR2DS1; MIM 604952) or inhibitory functions (e.g., KIR2DL1; MIM 604936).[supplied by OMIM].
TREM-1 associates with and signals via the adapter protein 12DAP12/12TYROBP, which contains an ITAM. To mediate activation, TREM-1 associates with the transmembrane adapter molecule 12DAP12. In sharp contrast to the effect by Ad-FDAP12, transgene expression in the liver of soluble form of extracellular domain of TREM-1 as an antagonist of 12DAP12 signaling, remarkably inhibited zymosan A-induced granuloma formation at every time point examined.
For signal transduction, 01TREM-1 couples to the ITAM-containing adapter DNAX activation protein of 12 kDa (23DAP12 ). MARV and EBOV activate TREM-1 on human neutrophils, resulting in 12DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. TREM-1 is the best-characterized member of a growing family of 12DAP12-associated receptors that regulate the function of myeloid cells in innate and adaptive responses. TREM-1 (triggering receptor expressed on myeloid cells), a recently discovered receptor of the immunoglobulin superfamily, activates neutrophils and monocytes/macrophages by signaling through the adapter protein 12DAP12. 522Granulocyte TREM-1 expression was high at baseline and immediately down-regulated upon LPS exposure along with an increase in soluble TREM-1.
DIC is primarily a laboratory diagnosis, based on the combination of elevated fibrin-related markers (FRM), with decreased procoagulant factors and platelets. Non-overt DIC is observed in most patients with sepsis, whereas overt DIC is less frequent. Consumption coagulopathy is a bleeding disorder caused by low levels of platelets and procoagulant factors associated with massive coagulation activation. Treatment with drotrecogin alfa (activated) improves survival and other outcome parameters in severe sepsis, including a subgroup of patients fulfilling the laboratory criteria of overt DIC. No randomized trials demonstrating effective therapies in consumption coagulopathy have been published.
Sepsis is a complex syndrome characterized by simultaneous activation of inflammation and coagulation manifested as systemic inflammatory response syndrome (SIRS)/sepsis symptoms through release of proinflammatory cytokines, procoagulants, and adhesion molecules from immune cells and/or damaged endothelium. Conventional treatments have focused on source control, antimicrobials, vasopressors, and fluid resuscitation; however, a new treatment paradigm exists: that of treating the host response to infection with adjunct therapies including early goal-directed therapy, drotrecogin alfa (activated), and immunonutrition. The drotrecogin alfa (activated) has been shown to reduce mortality in the severely septic patient when combined with traditional treatment. Therapies targeting improved oxygen and blood flow and reduction of apoptosis and free radicals are under investigation. Ultimately, intervention timing may be the most important factor in reducing severe sepsis mortality.

Cell Signaling in Sepsis
Recent data have shown stable patterns of activation among peripheral blood mononuclear cells and neutrophils in healthy human subjects. Although polymorphisms in Toll-like receptors play a contributory role in determining cellular activation, other factors are involved as well. In addition, circulating and locally released mediators of inflammation, including cytokines, complement fragments, and components of activated coagulation and fibrinolytic systems, that are generated in increased amounts during severe infection also interact with membrane-based receptors, leading to activation of intracellular path ways capable of further accelerating proinflammatory cascades. Circulating and organ-specific cell populations are activated to produce proinflammatory mediators during sepsis. Neutrophils and PBMCs bear TLR2 and TLR4, as well as other receptors, such as protein —coupled receptor, that induce increased generation of cytokines and other immunoregulatory proteins, as well as enhance release of proinflammatory mediators, including reactive oxygen species.
The expression of cytokines such as TNF-α and IL-1β is increased in sepsis, and engagement of TNF-α with type I(p55) and type II(p75) TNF receptors or IL-1β with IL-1 receptors belonging to the TLR/IL-1 receptor family produces activation of kinases (including Src, p38, extracellular signal—regulated kinase, and phosphoinositide 3–kinase) and transcriptional factors (such as nuclear factor [NF]–κB) important for further up-regulation of inflammatory proteins.
Genetic polymorphisms lead to alterations in TLR conformation (a small percentage of the variability in humans when their cells are exposed to bacterial products) that are accompanied by decreased cellular activation after exposure to bacterial products. The stable variability in cellular activation that is present among the genetically heterogeneous human population, only a limited number of studies have examined how such patterns may correlate with clinical outcome. A number of studies have examined the transcriptional factor NF-κB and kinases, including p38 and Akt, and provide insights into how heterogeneity in cell signaling may contribute to subsequent clinical course.
Increased activation of the mitogen-activated protein kinase protein 38, Akt, and nuclear factor (NF)–κB in neutrophils and other cell populations obtained at early time points in the clinical course of sepsis-induced acute lung injury or after accidental trauma is associated with a more-severe clinical course, suggesting that a proinflammatory cellular phenotype contributes to organ system dysfunction in such settings. Identification of patients with cellular phenotypes characterized by increased activation of NF-κB, Akt, and protein 38, as well as discrete patterns of gene activation, may permit identification of patients with sepsis who are likely to have a worse clinical outcome, thereby permitting early institution of therapies that modulate deleterious signaling pathways before organ system dysfunction develops, reducing morbidity and improving survival.


The transcriptional regulatory factor NF-κB is a central participant in modulating the expression of many immuno regulatory mediators involved in the acute inflammatory response [30–35]. NF-κB/rel transcription factors function as dimers held latently in the cytoplasm of cells by inhibitory IκB proteins. Signaling pathways initiated by engagement of TLRs, such as TLR 2 and TLR 4, by microbial products and other inflammatory mediators lead to nuclear accumulation of NF-κB and enhanced transcription of genes responsible for the expression of cytokines, chemokines, adhesion molecules, and other mediators of the inflammatory response associated with infection. Association of NF-κB with the inhibitory protein κB-α in the cytoplasm blocks the nuclear localization sequence of NF-κB, inhibiting its movement into the nucleus. Phosphorylation events, in addition to those involving IKKα/β and IκB-α, and involving NF-κB subunits (such as p 65) and nuclear coactivator proteins (such as TATA box binding protein or cAMP-responsive element—binding protein) are mediated by p 38, Akt, and other kinases and play an important role in regulating the transcriptional activity of NF-κB.

Studies have shown that greater nuclear accumulation of NF-κB is accompanied by higher mortality and worse clinical course in patients with sepsis. These clinical series demonstrated that persistent activation of NF-κB was found in nonsurvivors, with surviving patients having lower nuclear concentrations of NF-κB at early time points in their septic course than did nonsurvivors as well as more rapid return of nuclear accumulation of NF-κB.  Although studies of patients with sepsis have generally shown that nuclear concentrations of NF-κB are higher in non survivors than in survivors, an unresolved issue is whether such changes occur early and, therefore, define the subsequent course of sepsis or whether pathophysiological changes that result in poor clinical outcome also produce NF-κB activation as a secondary event, so that such changes in NF-κB are simply associated with more severe organ system dysfunction but do not contribute directly to outcome. A study of surgical patients without sepsis supports the hypothesis that neutrophil phenotypes defined by NF-κB activation patterns predict clinical outcome [54]. In that clinical series of patients undergoing repair of aortic aneurysms, higher preoperative levels of NF-κB in peripheral neutrophils were associated with death and with the development of postoperative organ dysfunction.


NF-κB (Photo credit: Wikipedia)

Stable high and low responder phenotypes in the healthy population, implies that the presence of a preexistent high responder neutrophil phenotype, as characterized by increased nuclear translocation of NF-κB after stimulation with TLR 2 or TLR 4 ligands, would be associated with more severe pulmonary inflammatory response and clinical course in response to infection. Conversely, persons whose neutrophils have diminished activation of NF-κB after stimulation would be expected to have less-intense neutrophil-driven inflammation, as well as organ dysfunction. In addition, Nuclear levels of nuclear factor (NF)–κB are significantly increased in neutrophils obtained within 24h of initiation of mechanical ventilation in patients whose clinical course from sepsis-induced acute lung injury is more severe (as defined by death or ventilation for >14 days—that is, ⩽14 ventilator-free days [VFD]), compared with patients with a less-severe course (as defined by mechanical ventilation for <14 days, or >14 VFD).  Baseline nuclear concentrations of NF-κB were lower in healthy volunteers than in patients with sepsis-induced acute lung injury, regardless of subsequent clinical course, demonstrating baseline activation of NF-κB in association with sepsis. *P <.05, vs. volunteers. †P< .05, vs. >14VFD.

Modulation of intracellular signaling cascades involving kinases, such as p 38 or Akt, or transcriptional factors, such as NF-κB, through specific inhibitory approaches has shown their pathophysiological importance in experimental models. However, the role of specific intra cellular pathways in contributing to clinical outcomes in patients with sepsis remains incompletely determined, primarily because such alterations in cellular activation patterns have not been examined at early time points before the onset of multiple organ dysfunction. Recent information shows that alterations in p38, Akt, and NF-κB among neutrophils and other cell populations not only precedes the development of organ system dysfunction but also has predictive value in identifying patients with a more severe subsequent clinical course.

RC Chambers. Procoagulant signalling mechanisms in lung inflammation and fibrosis: novel opportunities for pharmacological intervention? British Journal of Pharmacology 2008; 153, S367–S378; doi:10.1038/sj.bjp.0707603.

RC Bone. Toward a theory regarding the pathogenesis of the systemic inflammatory response syndrome: What we do and do not know about cytokine regulation. Crit Care Med 1996; 24:163-172.

Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature 2001; 410 (6832): 1103-7. doi:/10.1038/35074114. PMID 11323674.

Bleharski JR, Kiessler V, Buonsanti C, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J. Immunol. 2003; 170 (7): 3812-8. PMID 12646648.

Colonna M, Facchetti F. TREM-1 (triggering receptor expressed on myeloid cells): a new player in acute inflammatory responses. J. Infect. Dis 2003; 187 (Suppl 2): S397-401. PMID 12792857.

Dempfle CE. Coagulopathy of Sepsis. Thromb Hemost 2004; 91:213-224.

Cunneen J, Cartwright M. The Puzzle of Sepsis: Fitting the Pieces of the Inflammatory Response with Treatment. AACN Clin Issues 2004;15:18-44.

Ren-Feng Guo, NC Riedemann, Lei Sun, Hongwei Gao, KX Shi, et al. Divergent Signaling Pathways in Phagocytic Cells during Sepsis. The Journal of Immunology, 2006, 177: 1306–1313.

Abraham E.  Alterations in Cell Signaling in Sepsis. Clin Infect Dis 2005: 41 (Supplement 7): S459-S464. doi: 10.1086/431997

Yang KY, Arcaroli JJ, Abraham E. Early alterations in neutrophil activation are associated with outcome in acute lung injury. Am J Respir Crit Care Med 2003; 167:1567-74.

Abraham E. Neutrophils and Acute Lung Injury. Crit Care Med 2003; 31:195-9.

Abraham E, Carmody A, Shenkar R, Arcaroli J. Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2000; 279:1137-45.

Sepsis Bundles

The Institute for Healthcare Improvement (IHI) has highlighted sepsis as an area of focus and has identified several deficiencies that may cause suboptimal care of patients with severe sepsis.

These deficiencies include inconsistency in the early diagnosis of severe sepsis and septic shock, frequent inadequate volume resuscitation without defined endpoints, late or inadequate use of antibiotics, frequent failure to support the cardiac output when depressed, frequent failure to control hyperglycemia adequately, frequent failure to use low tidal volumes and pressures in acute lung injury, and frequent failure to treat adrenal inadequacy in refractory shock.

To address these deficiencies, the Surviving Sepsis Campaign and IHI have revised and added to the Surviving Sepsis Guidelines and created 2 sepsis treatment bundles (resuscitation and management) to guide therapy for patients with severe sepsis.

“Implicit in the use of the bundles is the need to adopt all the elements contained in the bundle,” the authors write. “One cannot choose to apply only selected items from the bundle and expect to achieve comparable benefit. The IHI sepsis website provides tools to screen patients for severe sepsis, as well as to measure success with adherence to implementing the bundles (http://www.ihi.org/IHI/Topics/CriticalCare/Sepsis/).” (The authors are employees of Eli Lilly and Co, the maker of drotrecogin alfa (activated). South Med J. 2007;100:594-600.

The sepsis resuscitation bundle, which should be accomplished as soon as possible and scored during the first 6 hours

Prealbumin (Transthyretin)

Discharge prealbumin and the change in prealbumin were positively correlated with protein and energy intake and inversely correlated with markers of inflammation, particularly CRP and IL-6. When all covariates were included in a multivariable regression analysis, the markers of inflammation predominantly accounted for the variance in prealbumin change (56%), whereas discharge protein intake accounted for 6%.

These authors propose an updated approach that incorporates current understanding of the systemic inflammatory response to help guide assessment, diagnosis, and treatment. An appreciation of a continuum of inflammatory response in relation to malnutrition syndromes is described. This discussion serves to highlight a research agenda to address deficiencies in diagnostics, biomarkers, and therapeutics of inflammation in relation to malnutrition.


The most frequent indication for antibiotic prescriptions in the northwestern hemisphere is lower respiratory tract infections (LRTIs),which range in severity from self-limited acute bronchitis to severe acute exacerbation of chronic obstructive pulmonary disease (COPD), and to life-threatening bacterial community-acquired pneumonia (CAP).4 Clinical signs and symptoms, as well as commonly used laboratory markers, are unreliable in distinguishing viral from bacterial LRTI. As many as 75% of patients with LRTI are treated with antibiotics, despitethe predominantly viral origin of their infection. An approach to estimate the probability of bacterial origin in LRTI is the measurement of serum procalcitonin (PCT).

In patients with LRTIs, a strategy of PCT guidance compared with standard guidelines resulted in similar rates of adverse outcomes, as well as lower rates of antibiotic exposure and antibiotic-associated adverse effects. (Trial Registration isrctn.org Identifier: ISRCTN95122877)

Neutrophil CD64

Despite improvements in the treatment of sepsis in recent years, there have been few diagnostic innovations which improve the sensitivity and specificity of diagnosis or facilitate therapeutic monitoring. The clinical reliance on the CBC and leukocyte differential with associated band count to indicate myeloid left shift of immaturity is not accurate, and it is not comparable to the measurement of the metamyeloctes and myelocytes. Only the introduction of a test which measures procalcitonin (PCT), an acute phase marker which is claimed to be more specific for bacterial infections than for viral infections, can be cited as a new diagnostic for the evaluation of patients with suspected infection. A need still persists for improved diagnostic indictors of infection or sepsis, as well as better tests to facilitate monitoring of therapy in the treatment of infection, so that use of antibiotics might be less empirical.

Studies have indicated that quantitative neutrophil CD64 expression is a sensitive and specific laboratory indicator of sepsis or the presence of a systemic acute inflammatory response.  Neutrophil CD64 is a highly sensitive marker for neonatal sepsis. Prospective studies incorporating CD64 into a sepsis scoring system are warranted. Studies have indicated that quantitative neutrophil CD64 (high affinity Fc receptor) expression is a worth­while candidate for evaluation as a more sensitive and specific laboratory indi­cator of sepsis or the presence of a systemic acute inflammatory response than available diagnostics . Neutrophil (PMN) CD64 is one of many activa­tion-related antigenic changes manifested by neutrophils during the normal pathophysiological acute inflammatory or innate immune response. PMN expression of CD64 is up-regulated under the influence of inflammatory relat­ed cytokines such as interleukin 12 (IL-12), interferon gamma (IFN-y) and granulocyte colony stimulating factor (G-CSF).

The first commercially available assay for PMN CD64, developed by Trillium Diagnostics, LLC is a fluorescence based, no wash flow cytometric assay, namely the Leuko64. The assay kit contains a cocktail of monoclonal antibodies includ­ing two monoclonal antibodies to CD64 and a monoclonal antibody to CD163, red cell lysis buffer, fluorescence quantitation beads, and a software program for automated analysis of the flow cytometric data that reports PMN CD64 as a CD64 index. The PMN CD64 index is designed so that normal inactivated PMNs yield values of < 1.00 and blood samples from individuals with docu­mented infection or sepsis typically show values > 1.50. Using clinical flow cytometers, the assay can be completed within 30 minutes. While this initial assay format was developed for multiparameter flow cytometers, a new version of the assay has been developed to give nearly identical results on the CD4000 and Sapphire (manufactured by Abbott Diagnostics, Santa Clara, CA) blood cell counters, which are equipped with laser light sources and fluorescence detection capabilities. If these blood cell counters are available in diagnostic haematology laboratories, the Leuko64 assay can be utilised on a 24 hour basis, in contrast to the more typical daytime operation hours of flow cytometric diagnostic laboratories.

Leukocare and Trillium Diagnostics entered an agreement to develop and market Leukocare’s method for detecting inflammatory activity using circulating cell-free DNA. Trillium aims to create a cf-DNA test as a “simple and cost effective” tool that healthcare professionals can use to obtain clinically relevant data on patients who are suspected of having sepsis. The companies said that they expect to finish developing the assay and market it in two years.

B Casserly, R Read, MM Levy. Multimarker Panels  in Sepsis. Crit Care Clin 27 (2011) 391–405 doi:10.1016/j.ccc.2010.12.011 criticalcare.theclinics.com

Dennis RA, Johnson LE, Roberson PK, Heif M, Bopp MM, et al.  Changes in prealbumin, nutrient intake, and systemic inflammation in elderly recuperative care patients.  J Am Geriatr Soc. 2008; 56(7):1270-5. Epub 2008 Jun 10. PMID: 18547360

Jensen GL, Bistrian B, Roubenoff R, Heimburger DC.  Malnutrition Syndromes: A Conundrum vs Continuum.

Bernstein LH. The systemic inflammatory response syndrome C-reactive protein and transthyretin conundrum. Clinical Chemistry Laboratory Medicine 2007; 45(11):1566–1567, ISSN (Online) 14374331, ISSN (Print) 14346621, DOI: 10.1515/CCLM.2007.334.

Schuetz P, Christ-Crain M, Thomann R, Falconnier C, Wolbers M, et al.  for the ProHOSP Study Group. Effect of Procalcitonin-Based Guidelines vs Standard Guidelines on Antibiotic Use in Lower Respiratory Tract Infections: The ProHOSP Randomized Controlled Trial.  JAMA  2009; 302(10): 1059

Bhandari V, Wang C, Rinder C, Rinder H. Hematologic Profile of Sepsis in Neonates: Neutrophil CD64 as a Diagnostic Marker. Pediatrics 2007; 31:4005.   (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). doi:10.1542/peds.2007-1308

Davis BH.  Neutrophil CD64 expression in infection and sepsis. CLI Ocober 2006.

Chapter 1 Statement of Inferential    Second Opinion

Realtime Clinical Expert Support

Gil David and Larry Bernstein have developed, in consultation with Prof. Ronald Coifman, in the Yale University Applied Mathematics Program, a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options.

Keywords: Entropy, Maximum Likelihood Function, separatory clustering, peripheral smear, automated hemogram, Anomaly, classification by anomaly, multivariable and multisyndromic, automated second opinion

Abbreviations: Akaike Information Criterion, AIC;  Bayes Information Criterion, BIC, Systemic Inflammatory Response Syndrome, SIRS.

Background: The current design of the Electronic Medical Record (EMR) is a linear presentation of portions of the record by services, by diagnostic method, and by date, to cite examples.  This allows perusal through a graphical user interface (GUI) that partitions the information or necessary reports in a workstation entered by keying to icons.  This requires that the medical practitioner finds the history, medications, laboratory reports, cardiac imaging and EKGs, and radiology in different workspaces.  The introduction of a DASHBOARD has allowed a presentation of drug reactions, allergies, primary and secondary diagnoses, and critical information about any patient the care giver needing access to the record.  The advantage of this innovation is obvious.  The startup problem is what information is presented and how it is displayed, which is a source of variability and a key to its success.

Intent: We are proposing an innovation that supercedes the main design elements of a DASHBOARD and utilizes the conjoined syndromic features of the disparate data elements.  So the important determinant of the success of this endeavor is that it facilitates both the workflow and the decision-making process with a reduction of medical error. Continuing work is in progress in extending the capabilities with model datasets, and sufficient data because the extraction of data from disparate sources will, in the long run, further improve this process.  For instance, the finding of  both ST depression on EKG coincident with an elevated cardiac biomarker (troponin), particularly in the absence of substantially reduced renal function. The conversion of hematology based data into useful clinical information requires the establishment of problem-solving constructs based on the measured data.

The most commonly ordered test used for managing patients worldwide is the hemogram that often incorporates the review of a peripheral smear.  While the hemogram has undergone progressive modification of the measured features over time the subsequent expansion of the panel of tests has provided a window into the cellular changes in the production, release or suppression of the formed elements from the blood-forming organ to the circulation.  In the hemogram one can view data reflecting the characteristics of a broad spectrum of medical conditions.

Progressive modification of the measured features of the hemogram has delineated characteristics expressed as measurements of size, density, and concentration, resulting in many characteristic features of classification. In the diagnosis of hematological disorders proliferation of marrow precursors, the domination of a cell line, and features of suppression of hematopoiesis provide a two dimensional model.  Other dimensions are created by considering the maturity of the circulating cells.  The application of rules-based, automated problem solving should provide a valid approach to the classification and interpretation of the data used to determine a knowledge-based clinical opinion. The exponential growth of knowledge since the mapping of the human genome enabled by parallel advances in applied mathematics that have not been a part of traditional clinical problem solving.  As the complexity of statistical models has increased the dependencies have become less clear to the individual.  Contemporary statistical modeling has a primary goal of finding an underlying structure in studied data sets.  The development of an evidence-based inference engine that can substantially interpret the data at hand and convert it in real time to a “knowledge-based opinion” could improve clinical decision-making by incorporating multiple complex clinical features as well as duration of onset into the model.

An example of a difficult area for clinical problem solving is found in the diagnosis of SIRS and associated sepsis.  SIRS (and associated sepsis) is a costly diagnosis in hospitalized patients.   Failure to diagnose sepsis in a timely manner creates a potential financial and safety hazard.  The early diagnosis of SIRS/sepsis is made by the application of defined criteria (temperature, heart rate, respiratory rate and WBC count) by the clinician.   The application of those clinical criteria, however, defines the condition after it has developed and has not provided a reliable method for the early diagnosis of SIRS.  The early diagnosis of SIRS may possibly be enhanced by the measurement of proteomic biomarkers, including transthyretin, C-reactive protein and procalcitonin.  Immature granulocyte (IG) measurement has been proposed as a more readily available indicator of the presence of granulocyte precursors (left shift).  The use of such markers, obtained by automated systems in conjunction with innovative statistical modeling, provides a promising approach to enhance workflow and decision making.   Such a system utilizes the conjoined syndromic features of disparate data elements with an anticipated reduction of medical error.  This study is only an extension of our approach to repairing a longstanding problem in the construction of the many-sided electronic medical record (EMR).  In a classic study carried out at Bell Laboratories, Didner found that information technologies reflect the view of the creators, not the users, and Front-to-Back Design (R Didner) is needed.

Costs would be reduced, and accuracy improved, if the clinical data could be captured directly at the point it is generated, in a form suitable for transmission to insurers, or machine transformable into other formats.  Such data capture, could also be used to improve the form and structure of how this information is viewed by physicians, and form a basis of a more comprehensive database linking clinical protocols to outcomes, that could improve the knowledge of this relationship, hence clinical outcomes.

How we frame our expectations is so important that it determines the data we collect to examine the process.   In the absence of data to support an assumed benefit, there is no proof of validity at whatever cost.   This has meaning for hospital operations, for nonhospital laboratory operations, for companies in the diagnostic business, and for planning of health systems.

In 1983, a vision for creating the EMR was introduced by Lawrence Weed,  expressed by McGowan and Winstead-Fry (J J McGowan and P Winstead-Fry. Problem Knowledge Couplers: reengineering evidence-based medicine through interdisciplinary development, decision support, and research. Bull Med Libr Assoc. 1999 October; 87(4): 462–470.)   PMCID: PMC226622    Copyright notice

They introduce Problem Knowledge Couplers as a clinical decision support software tool that  recognizes that functionality must be predicated upon combining unique patient information, but obtained through relevant structured question sets, with the appropriate knowledge found in the world’s peer-reviewed medical literature.  The premise of this is stated by LL WEED in “Idols of the Mind” (Dec 13, 2006): “ a root cause of a major defect in the health care system is that, while we falsely admire and extol the intellectual powers of highly educated physicians, we do not search for the external aids their minds require”.  HIT use has been focused on information retrieval, leaving the unaided mind burdened with information processing.

The data presented has to be comprehended in context with vital signs, key symptoms, and an accurate medical history.  Consequently, the limits of memory and cognition are tested in medical practice on a daily basis.  We deal with problems in the interpretation of data presented to the physician, and how through better design of the software that presents this data the situation could be improved.  The computer architecture that the physician uses to view the results is more often than not presented as the designer would prefer, and not as the end-user would like.  In order to optimize the interface for physician, the system would have a “front-to-back” design, with the call up for any patient ideally consisting of a dashboard design that presents the crucial information that the physician would likely act on in an easily accessible manner.  The key point is that each item used has to be closely related to a corresponding criterion needed for a decision.  Currently, improved design is heading in that direction.  In removing this limitation the output requirements have to be defined before the database is designed to produce the required output.  The ability to see any other information, or to see a sequential visualization of the patient’s course would be steps to home in on other views.  In addition, the amount of relevant information, even when presented well, is a cognitive challenge unless it is presented in a disease- or organ-system structure.  So the interaction between the user and the electronic medical record has a significant effect on practitioner time, ability to minimize errors of interpretation, facilitate treatment, and manage costs.  The reality is that clinicians are challenged by the need to view a large amount of data, with only a few resources available to know which of these values are relevant, or the need for action on a result, or its urgency. The challenge then becomes how fundamental measurement theory can lead to the creation at the point of care of more meaningful actionable presentations of results.  WP Fisher refers to the creation of a context in which computational resources for meeting the challenges will be incorporated into the electronic medical record.  The one which he chooses is a probabilistic conjoint (Rasch) measurement model, which uses scale-free standard measures and meets data quality standards. He illustrates this by fitting a set of data provided by Bernstein (19)(27 items for the diagnosis of acute myocardial infarction (AMI) to a Rasch multiple rating scale model testing the hypothesis that items work together to delineate a unidimensional measurement continuum. The results indicated that highly improbable observations could be discarded, data volume could be reduced based on internal, and increased ability of the care provider to interpret the data.


Classified data a separate issue from automation

 Feature Extraction. This further breakdown in the modern era is determined by genetically characteristic gene sequences that are transcribed into what we measure.  Eugene Rypka contributed greatly to clarifying the extraction of features in a series of articles, which set the groundwork for the methods used today in clinical microbiology.  The method he describes is termed S-clustering, and will have a significant bearing on how we can view hematology data.  He describes S-clustering as extracting features from endogenous data that amplify or maximize structural information to create distinctive classes.  The method classifies by taking the number of features with sufficient variety to map into a theoretic standard. The mapping is done by a truth table, and each variable is scaled to assign values for each: message choice.  The number of messages and the number of choices forms an N-by N table.  He points out that the message choice in an antibody titer would be converted from 0 + ++ +++ to 0 1 2 3.

Even though there may be a large number of measured values, the variety is reduced by this compression, even though there is risk of loss of information.  Yet the real issue is how a combination of variables falls into a table with meaningful information.  We are concerned with accurate assignment into uniquely variable groups by information in test relationships. One determines the effectiveness of each variable by its contribution to information gain in the system.  The reference or null set is the class having no information.  Uncertainty in assigning to a classification is only relieved by providing sufficient information.  One determines the effectiveness of each variable by its contribution to information gain in the system.  The possibility for realizing a good model for approximating the effects of factors supported by data used for inference owes much to the discovery of Kullback-Liebler distance or “information”, and Akaike found a simple relationship between K-L information and Fisher’s maximized log-likelihood function. A solid foundation in this work was elaborated by Eugene Rypka.  Of course, this was made far less complicated by the genetic complement that defines its function, which made  more accessible the study of biochemical pathways.  In addition, the genetic relationships in plant genetics were accessible to Ronald Fisher for the application of the linear discriminant function.    In the last 60 years the application of entropy comparable to the entropy of physics, information, noise, and signal processing, has been fully developed by Shannon, Kullback, and others,  and has been integrated with modern statistics, as a result of the seminal work of Akaike, Leo Goodman, Magidson and Vermunt, and unrelated work by Coifman. Dr. Magidson writes about Latent Class Model evolution:

The recent increase in interest in latent class models is due to the development of extended algorithms which allow today’s computers to perform LC analyses on data containing more than just a few variables, and the recent realization that the use of such models can yield powerful improvements over traditional approaches to segmentation, as well as to cluster, factor, regression and other kinds of analysis.

Perhaps the application to medical diagnostics had been slowed by limitations of data capture and computer architecture as well as lack of clarity in definition of what are the most distinguishing features needed for diagnostic clarification.  Bernstein and colleagues had a series of studies using Kullback-Liebler Distance  (effective information) for clustering to examine the latent structure of the elements commonly used for diagnosis of myocardial infarction (CK-MB, LD and the isoenzyme-1 of LD),  protein-energy malnutrition (serum albumin, serum transthyretin, condition associated with protein malnutrition (see Jeejeebhoy and subjective global assessment), prolonged period with no oral intake), prediction of respiratory distress syndrome of the newborn (RDS), and prediction of lymph nodal involvement of prostate cancer, among other studies.   The exploration of syndromic classification has made a substantial contribution to the diagnostic literature, but has only been made useful through publication on the web of calculators and nomograms (such as Epocrates and Medcalc) accessible to physicians through an iPhone.  These are not an integral part of the EMR, and the applications require an anticipation of the need for such processing.

Gil David et al. introduced an AUTOMATED processing of the data available to the ordering physician and can anticipate an enormous impact in diagnosis and treatment of perhaps half of the top 20 most common causes of hospital admission that carry a high cost and morbidity.  For example: anemias (iron deficiency, vitamin B12 and folate deficiency, and hemolytic anemia or myelodysplastic syndrome); pneumonia; systemic inflammatory response syndrome (SIRS) with or without bacteremia; multiple organ failure and hemodynamic shock; electrolyte/acid base balance disorders; acute and chronic liver disease; acute and chronic renal disease; diabetes mellitus; protein-energy malnutrition; acute respiratory distress of the newborn; acute coronary syndrome; congestive heart failure; disordered bone mineral metabolism; hemostatic disorders; leukemia and lymphoma; malabsorption syndromes; and cancer(s)[breast, prostate, colorectal, pancreas, stomach, liver, esophagus, thyroid, and parathyroid].

Extension of conditions and presentation to the electronic medical record (EMR)

We have published on the application of an automated inference engine to the Systemic Inflammatory Response (SIRS), a serious infection, or emerging sepsis.  We can report on this without going over previous ground.  Of considerable interest is the morbidity and mortality of sepsis, and the hospital costs from a late diagnosis.  If missed early, it could be problematic, and it could be seen as a hospital complication when it is not. Improving on previous work, we have the opportunity to look at the contribution of a fluorescence labeled flow cytometric measurement of the immature granulocytes (IG), which is now widely used, but has not been adequately evaluated from the perspective of diagnostic usage.  We have done considerable work on protein-energy malnutrition (PEM), to which the automated interpretation is currently in review.  Of course, the

cholesterol, lymphocyte count, serum albumin provide the weight of evidence with the primary diagnosis (emphysema, chronic renal disease, eating disorder), and serum transthyretin would be low and remain low for a week in critical care.  This could be a modifier with age in providing discriminatory power.

Chapter  3           References

The Cost Burden of Disease: U.S. and Michigan. CHRT Brief. January 2010. @www.chrt.org

The National Hospital Bill: The Most Expensive Conditions by Payer, 2006. HCUP Brief #59.

Rudolph RA, Bernstein LH, Babb J: Information-Induction for the diagnosis of

myocardial infarction. Clin Chem 1988;34:2031-2038.

Bernstein LH (Chairman). Prealbumin in Nutritional Care Consensus Group.

Measurement of visceral protein status in assessing protein and energy malnutrition: standard of care. Nutrition 1995; 11:169-171.

Bernstein LH, Qamar A, McPherson C, Zarich S, Rudolph R. Diagnosis of myocardial infarction: integration of serum markers and clinical descriptors using information theory. Yale J Biol Med 1999; 72: 5-13.

Kaplan L.A.; Chapman J.F.; Bock J.L.; Santa Maria E.; Clejan S.; Huddleston D.J.; Reed R.G.; Bernstein L.H.; Gillen-Goldstein J. Prediction of Respiratory Distress Syndrome using the Abbott FLM-II amniotic fluid assay. The National Academy of Clinical Biochemistry (NACB) Fetal Lung Maturity Assessment Project.  Clin Chim Acta 2002; 326(8): 61-68.

Bernstein LH, Qamar A, McPherson C, Zarich S. Evaluating a new graphical ordinal logit method (GOLDminer) in the diagnosis of myocardial infarction utilizing clinical features and laboratory data. Yale J Biol Med 1999; 72:259-268.

Bernstein L, Bradley K, Zarich SA. GOLDmineR: Improving models for classifying patients with chest pain. Yale J Biol Med 2002; 75, pp. 183-198.

Ronald Raphael Coifman and Mladen Victor Wickerhauser. Adapted Waveform Analysis as a Tool for Modeling, Feature Extraction, and Denoising. Optical Engineering, 33(7):2170–2174, July 1994.

R. Coifman and N. Saito. Constructions of local orthonormal bases for classification and regression. C. R. Acad. Sci. Paris, 319 Série I:191-196, 1994.

Chapter 4           Clinical Expert System

Realtime Clinical Expert Support and validation System

We have developed a software system that is the equivalent of an intelligent Electronic Health Records Dashboard that provides empirical medical reference and suggests quantitative diagnostics options. The primary purpose is to gather medical information, generate metrics, analyze them in realtime and provide a differential diagnosis, meeting the highest standard of accuracy. The system builds its unique characterization and provides a list of other patients that share this unique profile, therefore utilizing the vast aggregated knowledge (diagnosis, analysis, treatment, etc.) of the medical community. The main mathematical breakthroughs are provided by accurate patient profiling and inference methodologies in which anomalous subprofiles are extracted and compared to potentially relevant cases. As the model grows and its knowledge database is extended, the diagnostic and the prognostic become more accurate and precise. We anticipate that the effect of implementing this diagnostic amplifier would result in higher physician productivity at a time of great human resource limitations, safer prescribing practices, rapid identification of unusual patients, better assignment of patients to observation, inpatient beds, intensive care, or referral to clinic, shortened length of patients ICU and bed days.

The main benefit is a real time assessment as well as diagnostic options based on comparable cases, flags for risk and potential problems as illustrated in the following case acquired on 04/21/10. The patient was diagnosed by our system with severe SIRS at a grade of 0.61 .

The patient was treated for SIRS and the blood tests were repeated during the following week. The full combined record of our system’s assessment of the patient, were derived from the further Hematology tests.  Following treatment, the SIRS risk as a major concern was eliminated and the system provides a positive feedback for the treatment of the physician.


Method for data organization and classification via characterization metrics.

Our database organized to enable linking a given profile to known profiles. This is achieved by associating a patient to a peer group of patients having an overall similar profile, where the similar profile is obtained through a randomized search for an appropriate weighting of variables. Given the selection of a patients’ peer group, we build a metric that measures the dissimilarity of the patient from its group. This is achieved through a local iterated statistical analysis in the peer group.

We then use this characteristic metric to locate other patients with similar unique profiles, for each of whom we repeat the procedure described above. This leads to a network of patients with similar risk condition. Then, the classification of the patient is inferred from the medical known condition of some of the patients in the linked network. Given a set of points (the database) and a newly arrived sample (point), we characterize the behavior of the newly arrived sample, according to the database. Then, we detect other points in the database that match this unique characterization. This collection of detected points defines the characteristic neighborhood of the newly arrived sample. We use the characteristic neighbor hood in order to classify the newly arrived sample. This process of differential diagnosis is repeated for every newly arrived point.   The medical colossus we have today has become a system out of control and beset by the elephant in the room – an uncharted complexity. We offer a method that addresses the complexity and enables rather than disables the practitioner.  The method identifies outliers and combines data according to commonality of features.

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

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


This summary is the last of a series on the impact of transcriptomics, proteomics, and metabolomics on disease investigation, and the sorting and integration of genomic signatures and metabolic signatures to explain phenotypic relationships in variability and individuality of response to disease expression and how this leads to  pharmaceutical discovery and personalized medicine.  We have unquestionably better tools at our disposal than has ever existed in the history of mankind, and an enormous knowledge-base that has to be accessed.  I shall conclude here these discussions with the powerful contribution to and current knowledge pertaining to biochemistry, metabolism, protein-interactions, signaling, and the application of the -OMICS to diseases and drug discovery at this time.

The Ever-Transcendent Cell

Deriving physiologic first principles By John S. Torday | The Scientist Nov 1, 2014

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.



The switch from swim bladder to lung as vertebrates moved from water to land is proof of principle that stress-induced evolution in metazoans can be understood from changes at the cellular level.


A MECHANISTIC BASIS FOR LUNG DEVELOPMENT: Stress from periodic atmospheric hypoxia (1) during vertebrate adaptation to land enhances positive selection of the stretch-regulated parathyroid hormone-related protein (PTHrP) in the pituitary and adrenal glands. In the pituitary (2), PTHrP signaling upregulates the release of adrenocorticotropic hormone (ACTH) (3), which stimulates the release of glucocorticoids (GC) by the adrenal gland (4). In the adrenal gland, PTHrP signaling also stimulates glucocorticoid production of adrenaline (5), which in turn affects the secretion of lung surfactant, the distension of alveoli, and the perfusion of alveolar capillaries (6). PTHrP signaling integrates the inflation and deflation of the alveoli with surfactant production and capillary perfusion.  THE SCIENTIST STAFF

From a cell-cell signaling perspective, two critical duplications in genes coding for cell-surface receptors occurred during this period of water-to-land transition—in the stretch-regulated parathyroid hormone-related protein (PTHrP) receptor gene and the β adrenergic (βA) receptor gene. These gene duplications can be disassembled by following their effects on vertebrate physiology backwards over phylogeny. PTHrP signaling is necessary for traits specifically relevant to land adaptation: calcification of bone, skin barrier formation, and the inflation and distention of lung alveoli. Microvascular shear stress in PTHrP-expressing organs such as bone, skin, kidney, and lung would have favored duplication of the PTHrP receptor, since sheer stress generates radical oxygen species (ROS) known to have this effect and PTHrP is a potent vasodilator, acting as an epistatic balancing selection for this constraint.

Positive selection for PTHrP signaling also evolved in the pituitary and adrenal cortex (see figure on this page), stimulating the secretion of ACTH and corticoids, respectively, in response to the stress of land adaptation. This cascade amplified adrenaline production by the adrenal medulla, since corticoids passing through it enzymatically stimulate adrenaline synthesis. Positive selection for this functional trait may have resulted from hypoxic stress that arose during global episodes of atmospheric hypoxia over geologic time. Since hypoxia is the most potent physiologic stressor, such transient oxygen deficiencies would have been acutely alleviated by increasing adrenaline levels, which would have stimulated alveolar surfactant production, increasing gas exchange by facilitating the distension of the alveoli. Over time, increased alveolar distension would have generated more alveoli by stimulating PTHrP secretion, impelling evolution of the alveolar bed of the lung.

This scenario similarly explains βA receptor gene duplication, since increased density of the βA receptor within the alveolar walls was necessary for relieving another constraint during the evolution of the lung in adaptation to land: the bottleneck created by the existence of a common mechanism for blood pressure control in both the lung alveoli and the systemic blood pressure. The pulmonary vasculature was constrained by its ability to withstand the swings in pressure caused by the systemic perfusion necessary to sustain all the other vital organs. PTHrP is a potent vasodilator, subserving the blood pressure constraint, but eventually the βA receptors evolved to coordinate blood pressure in both the lung and the periphery.

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The biochemistry of S amino acids

Larry H. Bernstein, MD, FCAP, Curator


Amino Acid and Sulfur Metabolism

Dr. Rainer Höfgen


 Sulfur is together with nitrogen, phosphorous and potassium a plant macronutrient and a crucial element affecting plant growth, plant performance and yield. The group of Dr. Rainer Hoefgen focuses on characterising the regulation of cysteine and methionine as a result of sulfate uptake and assimilation in the model plant Arabidopsis thaliana.

Cysteine and methionine are two essential amino acids which contain sulfur. We are also looking at interconnections between sulfur metabolism and other plant nutrients. Further, we are investigating means of improving the nutritional quality of crops, with a current focus on rice (Oryza sativa) with respect to a balanced amino acid composition.

In our studies of plant sulfur metabolism, we use two mutually supporting approaches as the basis of our research portfolio. The first is a targeted, pathway-oriented approach aimed at understanding pathway architecture and coordination, and the regulation of the sulfur-containing metabolites as such. The second is a non-biased approach in which functional genomics is used to work out how sulfur metabolism is embedded and controlled within the whole plant system.

sulfur uptake and assimilation

sulfur uptake and assimilation

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Sulfur is a required macronutrient, sulfur uptake and assimilation are crucial determinants in how quickly plants grow and cope with various stresses, and therefore, in how well crops yield.

Inorganic sulfate is taken up through plant roots and, via cysteine biosynthesis, incorporated as organic sulfur. Our investigations focus on fundamental questions about cysteine (cys) and methionine (met) biosynthesis and on the possibility of engineering crop plants enriched in these sulfur-containing amino acids. Methionine is essential for non-ruminant mammals (including man) and uptake of cysteine reduces the methionine requirement. We have used transgenic strategies to generate many plant lines affected in cysteine and methionine biosynthesis, and subjected them to detailed molecular and biochemical analyses. Recently, we embarked on a course to study sulfur metabolism in a holistic way, rather than focusing on single pathways as such. By applying functional genomic tools like transcript, metabolite, and protein profiling in our analysis of transgenic potato (Solanum tuberosum) and of the model plant Arabidopsis thaliana, we are heading for a better understanding of the sulfur metabolism network in plants.

To learn about the control mechanisms involved in sulfur-containing amino acid biosynthesis, we are isolating and studying the involved genes and their promoters. The model plant systems of our investigations are potato and Arabidopsis, although a limited amount of work is also dedicated to rice (Oryza sativa), cucumber (Cucumis sativus), and tomato (Lycopersicon esculentum). Various transgenic plants exhibiting reduced or increased expression of relevant genes in the pathway have been produced and analysed. Fundamental knowledge of pathway regulation has been obtained as well as an improvement of the nutritional quality of a crop plant: Nutritional quality is largely determined by methionine, which is often the most limited of the essential amino acids.

The main thrust of our research recently shifted to analysing sulfur metabolism networks. In a systems biology approach, we investigate the response of Arabidopsis to different periods or degrees of sulfur starvation by applying non-biased, multiparallel tools including transcript, protein, and metabolite profiling. Our results are integrated to form working models for further detailed investigations with a focus on regulatory aspects of metabolism. This work entails the detailed analysis of Arabidopsis mutants and pulls many of our earlier results together into biological context (eg. the increased thiol levels seen during SAT over-expression, glutathione involvement in stress response mechanisms towards active oxygen species, etc.). Our long-term goal is to imbed sulfur metabolism in a broader context such as carbohydrate and nitrogen metabolic networks, which will occur through close collaborations with external and in house research groups.


metabolite profiling

metabolite profiling



Plants are sessile organisms; if they are to survive and reproduce, they must adapt to the growth conditions in which they find themselves. We use variations in sulfur levels as a stimulus and analyse the complex response using diverse multiparallel techniques, particularly transcript and metabolite profiling, trying to piece together the total system response. The plant of choice here is, obviously, Arabidopsis thaliana, although results obtained in this model system are likely to be transferable to other plant species and crop plants. The goal is to provide a consistent and holistic description of plant sulfur metabolism and its regulation.

H Hesse and R Höfgen (2001) Application of Genomics in Agriculture. In: Molecular analysis of plant adapatation to the environment. Eds: Malcolm J. Hawkesford, Peter Buchner. Kluwer AP, Dordrecht, The Netherlands, 61-79

V Nikiforova, J Freitag, S Kempa, M Adamik, H Hesse, R Hoefgen (2003) Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: Interlacing of biosynthetic pathways provides response specificity. The Plant Journal, 33, 633-650.



Plants adapt to available sulfur soil levels by regulating gene expression and protein activity to maintain homeostasis. Sulfur availability in the environment is not static, nor is the plant’s dependence on sulfur at various developmental stages. Thus, one can assume not only that the activities of regulatory proteins are dynamic, but also that changes in the expression of transcription factors involved in triggering downstream gene expression change temporally. Sulfur deprivation triggers a slow adaptive process that resets the level of sulfur homeostasis. Using transcript profiling, we have been able to identify a number of transcription factors involved in this process, which are now the target of further investigations.


Metabolome analysis and bioinformatics

system response

system response

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Gene expression, metabolite spectrum and enzyme activities change under sulfur-limiting conditions.

The response of steady state transcription levels to the sulfur stimulus is but the first chapter of the story. To understand the system response, we have to turn the page and look at protein profiles – levels and activities – and before closing the book, at metabolite profiles, which adjust rapidly in response to changes in protein expression. We are now focusing on metabolome analysis: The same samples used for transcriptome analysis are examined using element analysis (ICP-AES) and metabolite analyses (HPLC, CE, GC/MS, GC/TOF, LC/MS), either in house or in collaboration with outside research groups.

Malcolm J. Hawkesford, Rothamsted Research, UK

As these analyses are refined and data accumulates, it will become more and more important to overlay and compare transcript and metabolite profiles in order to try to generate an in silico representation of the plant sulfur regulatory complement. Various approaches are and will be followed here: bioinformatic tools have to be developed and/or adapted to fully mine the data. Otherwise, it will not be possible to fully describe the system: by looking only at the most highly expressed genes in isolation, we would simply be scratching at the surface.


Transcriptome Analysis

gene expression

gene expression

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Scatterplots of gene expression of the ratio -/+ S


Plants and some photoautotrophic bacteria assimilate inorganic sulfur from sulfates into cysteine, the first sulfur-containing organic compound and, effectively, the sole molecular doorway for reduced sulfur in all living beings. This essential process has been as finely tuned through millennia of evolution as photosynthesis. Cysteine is subsequently converted to methionine, and then into a variety of other sulfur-containing organic compounds. Sulfur assimilation is even more spendy in terms of reduction equivalents than nitrogen assimilation. Obviously, such a costly enterprise is highly controlled in juxtaposition with the rest of metabolism.

To elucidate this network of interactions, we stimulate Arabidopsis with sulfur (i.e. sulfate) at its rhizosphere with various concentrations and at different developmental stages to institute periods of starvation and replenishment. The plant tissue samples (roots, shoots) are then subjected to array hybridisation/transcript profiling after RNA extraction using either macro-arrays of 7,200 non-redundant genes on nylon filters and now full genome chips. The expression profiles are processed to select differentially expressed genes. Depending on the duration of treatment, anything between a handful and thousands of genes exhibit altered expression mirroring the gradual response of the system to conditions of altered sulfur availability. Among these responsive genes we expect to find sulfur-regulated genes; genes involved in perception, signalling, and immediate responses; and genes further down the line involved in more pleiotropic mechanisms like general stress responses. Since they arise in response to sulfur stimulation, the latter are still regarded as sulfur-responsive genes.

Sulfur-responsive genes are grouped by functional category or biosynthetic pathway. As expected, genes of the sulfur assimilation pathway are altered in expression. Furthermore, genes involved in the flavonoid, auxin, and jasmonate biosynthesis pathways are up regulated when sulfur is limiting. We focus most of our attention, however, on the regulatory elements, transcription factors.

V Nikiforova, J Freitag, S Kempa, M Adamik, H Hesse, R Hoefgen (2003) Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: Interlacing of biosynthetic pathways provides response specificity. The Plant Journal, 33, 633-650

Further reading

MY Hirai, T Fujiwara, M Awazuhara, T Kimura, M Noji, K Saito (2003) Global expression profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-acetyl-L-serine as a general regulator of gene expression in response to sulfur nutrition. Plant Journal. 33(4)651-663

A Maruyama-Nakashita, E Inoue, A Watanabe-Takahashi, T Yarnaya, and H Takahashi (2003) Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways. Plant Physiology. 132(2)597-605

Sulfur and Other Plant Nutrients

The plant sulfur assimilation pathway is intricately interconnected with various other pathways and regulatory circuits.

Systems Analysis of Plant Sulfur Metabolism

Every organism is a complex, multi-elemental, multi-functional system living in an ever-changing environment. The viability of the system is provided by, and likewise dependent upon, flexible, effective control circuits of multiple informational fluxes, which interconnect in a dense network of metabolic physiological responses.



L-cysteine L-Met

L-cysteine L-Met

Methionine is synthesised from cysteine and phosphohomoserine

Methionine is synthesised from cysteine and phosphohomoserine



Pathway Analysis of Sulfur Containing Amino Acids

To learn about the control mechanisms involved in the biosynthesis of sulfur-containing amino acids, we are isolating and studying genes involved and their promoters. Methionine is synthesised from cysteine and phosphohomoserine via the enzymes cystathionine gamma-synthase (CgS), cystathionine beta-lyase (CbL), and methionine synthase (MS); we have cloned and characterised these three genes in potato.

Biosynthesis of Sulfur-Containing Amino Acids

Biosynthesis of Sulfur-Containing Amino Acids


Genes from Arabidopsis and potato and, when appropriate, E. coli involved in cysteine and methionine biosynthesis have also been cloned, including various isoforms of O-acetylserine (thiol)-lyase, the enzyme that converts O-acetylserine to cys; ATP-sulfurylase, the enzyme activating the inert sulfate through binding to ATP; and serine acetyltransferase (SAT), the enzyme catalysing the activation of serine to O-acetylserine. We manipulated the expression of these genes in an attempt to create conditions in which flux to either cysteine or methionine is increased.

For example, the over-expression of SAT using an E. coli gene targeted to plastids resulted in cysteine and glutathione (a tripeptide containing glutamic acid, cysteine, and glycine) levels almost twice as high as usual. By blocking the competing pathway to threonine using the partial antisense inhibition of threonine synthase in Arabidopsis and potato, we were able to increase leaf and tuber methionine levels significantly. Moreover, analysis of these transformants made it clear that there are species-specific differences in the regulation of methionine biosynthesis.

Our results in Nicotiana plumbaginifolia and potato have established the essential, but not rate-limiting, role of CbL in plant methionine biosynthesis. Furthermore, we found that regulation at the level of CgS differs between the plant species Arabidopsis and potato. Our objective now is to deepen our understanding of the regulation of methionine biosynthesis and to exploit what we learn in order to improve the nutritional quality of crop plants, which is largely determined by methionine content.

Cysteine Biosynthesis

Cysteine biosynthesis represents the essential step in the incorporation of inorganic sulfide to organic sulfur in plants. In order to gain insight into the control mechanisms involved in cysteine biosynthesis, we are isolating and studying the involved genes and their promoters, including genes coding for O-acetylserine(thiol)-lyase (OAS-TL), the enzyme which converts O-acetylserine to cysteine, and serine acetyltransferase (SAT), the enzyme catalysing the activation of serine to O-acetylserine.

Serine acetyltransferase

Serine acetyltransferase

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Serine acetyltransferase


In addition, spatial and developmental aspects of regulation are investigated with respect to gene expression and enzyme activity. We are manipulating the expression of various genes in transgenic potato plants in an attempt to create conditions in which flux to either cysteine or methionine is increased. For example, the heterologous over-expression of an E. coli SAT gene targeted to plastids resulted in a doubling of both cysteine and glutathione (a tripeptide containing glutamic acid, cysteine, and glycine that is involved in stress tolerance) levels. However, these alterations had no effect on outward plant appearance or on the expression and enzymatic activity of OAS-TL. This example demonstrates the importance of SAT in plant cysteine biosynthesis and shows that the accumulation of cysteine and related sulfur-containing compounds is limited by the supply of activated carbon backbones derived from serine. We are currently investigating this and other transgenic plants affected in cysteine and methionine biosynthesis in respect to sulfur assimilation and glutathione-mediated stress tolerance.

Despite the increase of reduced organic sulfur in our potato SAT over-producers, we did not observe an increase in methionine, although other groups reported methionine increases when using a similar approach in maize (Tsakraklides et al., 2002). Again, species specific differences, probably as a result of adaptation to specific environmental or physiological conditions, have to be taken into account, especially when generalising and transferring these data to plant breeding.

V Nikiforova, S Kempa, M Zeh, S Maimann, O Kreft, A P Casazza, K Riedel, E Tauberger, R Hoefgen, H Hesse. (2002) Engineering of cysteine and methionine biosynthesis in potato. Amino Acids 22(259-278).

K Harms, P von Ballmoos, C Brunold, R Höfgen, and H Hesse (2000) Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and glutathione. Plant J. 22, 335-343

Further reading

MJ Hawkesford (2003) Transporter gene families in plants: the sulphate transporter gene family – redundancy or specialization? Physiologia Plantarum, 117,155-163

G Tsakraklides, M Martin, R Chalam,, MC Tarczynski, A Schmidt, and T Leustek (2002) Sulfate reduction is increased in transgenic Arabidopsis thaliana expressing 5′-adenylylsulfate reductase from Pseudomonas aeruginosa. Plant J. 32, 879

Annu Rev Nutr. 1986;6:179-209.
Metabolism of sulfur-containing amino acids.

Met metabolism occurs primarily by activation of Met to AdoMet and further metabolism of AdoMet by either the transmethylation-transsulfuration pathway or the polyamine biosynthetic pathway. The catabolism of the methyl group and sulfur atom of Met ultimately appears to be dependent upon the transmethylation-transsulfuration pathway because the MTA formed as the co-product of polyamine synthesis is efficiently recycled to Met. On the other hand, the fate of the four-carbon chain of Met appears to depend upon the initial fate of the Met molecule. During transsulfuration, the carbon chain is released as alpha-ketobutyrate, which is further metabolized to CO2. In the polyamine pathway, the carboxyl carbon of Met is lost in the formation of dAdoMet, whereas the other three carbons are ultimately excreted as polyamine derivatives and degradation products. The role of the transamination pathway of Met metabolism is not firmly established. Cys (which may be formed from the sulfur of Met and the carbons of serine via the transsulfuration pathway) appears to be converted to taurine and CO2 primarily by the cysteinesulfinate pathway, and to sulfate and pyruvate primarily by desulfuration pathways in which a reduced form of sulfur with a relatively long biological half-life appears to be an intermediate. With the exception of the nitrogen of Met that is incorporated into polyamines, the nitrogen of Met or Cys is incorporated into urea after it is released as ammonium [in the reactions catalyzed by cystathionase with either cystathionine (from Met) or cystine (from Cys) as substrate] or it is transferred to a keto acid (in Cys or Met transamination). Many areas of sulfur-containing amino acid metabolism need further study. The magnitude of AdoMet flux through the polyamine pathway in the intact animal as well as details about the reactions involved in this pathway remain to be determined. Both the pathways and the possible physiological role of alternate (AdoMet-independent) Met metabolism, including the transamination pathway, must be elucidated. Despite the growing interest in taurine, investigation of Cys metabolism has been a relatively inactive area during the past two decades. Apparent discrepancies in the reported data on Cys metabolism need to be resolved. Future work should consider the role of extrahepatic tissues in amino acid metabolism as well as species differences in the relative roles of various pathways in the metabolism of Met and Cys.

The Sulfur-Containing Amino Acids: An Overview1,2

John T. Brosnan3 and Margaret E. Brosnan

J. Nutr. June 2006; 136(6): 1636S-1640S


Methionine and cysteine may be considered to be the principal sulfur-containing amino acids because they are 2 of the canonical 20 amino acids that are incorporated into proteins. However, homocysteine and taurine also play important physiological roles (Fig. 1). Why does nature employ sulfur in her repertoire of amino acids? The other canonical amino acids are comprised only of carbon, hydrogen, oxygen, and nitrogen atoms. Because both sulfur and oxygen belong to the same group (Group 6) of the Periodic Table and, therefore, are capable of making similar covalent linkages, the question may be restated: why would methionine and cysteine analogs, in which the sulfur atom is replaced by oxygen, not serve the same functions? One of the critical differences between oxygen and sulfur is sulfur’s lower electronegativity. Indeed, oxygen is the second most electronegative element in the periodic table. This accounts for the use of sulfur in methionine; replacement of the sulfur with oxygen would result in a much less hydrophobic amino acid. Cysteine readily forms disulfide linkages because of the ease with which it dissociates to form a thiolate anion. Serine, on the other hand, which differs from cysteine only in the substitution of an oxygen for the sulfur, does not readily make dioxide linkages. The difference results from the fact that thiols are much stronger acids than are alcohols, so that the alcohol group in serine does not dissociate at physiological pH. Substitution of oxygen for sulfur inS-adenosylmethionine would produce so powerful a methylating agent that it would promiscuously methylate cellular nucleophiles without the need for an enzyme.


Structures of the sulfur-containing amino acids.

Methionine and cysteine in proteins.

Although both methionine and cysteine play critical roles in cell metabolism, we suggest that, in general, the 20 canonical amino acids were selected for the roles they play in proteins, not their roles in metabolism. It is important, therefore, to review the role played by these amino acids in proteins. Methionine is among the most hydrophobic of the amino acids. This means that most of the methionine residues in globular proteins are found in the interior hydrophobic core; in membrane-spanning protein domains, methionine is often found to interact with the lipid bilayer. In some proteins a fraction of the methionine residues are somewhat surface exposed. These are susceptible to oxidation to methionine sulfoxide residues. Levine et al. (1) regard these methionine residues as endogenous antioxidants in proteins. In E. coli glutamine synthetase, they tend to be arrayed around the active site and may guard access to this site by reactive oxygen species. Oxidation of these methionine residues has little effect on the catalytic activity of the enzyme. These residues may be reduced to methionine by means of the enzyme methionine sulfoxide reductase (2). Thus, an oxidation–reduction cycle occurs in which exposed methionine residues are oxidized (e.g., by H2O2) to methionine sulfoxide residues, which are subsequently reduced:FormulaFormula

It is considered that the impaired activity of methionine sulfoxide reductase and the subsequent accumulation of methionine sulfoxide residues are associated with age-related diseases, neurodegeneration, and shorter lifespan (2).

Methionine is the initiating amino acid in the synthesis of eukaryotic proteins; N-formyl methionine serves the same function in prokaryotes. Because most of these methionine residues are subsequently removed, it is apparent that their role lies in the initiation of translation, not in protein structure. In eukaryotes, translation initiation involves the association of the initiator tRNA (met-tRNAimet) with eIF-2 and the 40S ribosomal subunit together with a molecule of mRNA. Drabkin and Rajbhandary (3) suggest that the hydrophobic nature of methionine is key to the binding of the initiator tRNA to eIF-2. Using appropriate double mutations (in codon and anticodon), they were able to show that the hydrophobic valine could be used for initiation in mammalian cells but that the polar glutamine was very poor.

Cysteine plays a critical role in protein structure by virtue of its ability to form inter- and intrachain disulfide bonds with other cysteine residues. Most disulfide linkages are found in proteins destined for export or residence on the plasma membrane. These disulfide bonds can be formed nonenzymatically; protein disulfide isomerase, an endoplasmic reticulum protein, can reshuffle any mismatched disulfides to ensure the correct protein folding (4).


S-Adenosylmethionine (SAM)4 is a key intermediate in methionine metabolism. Discovered in 1953 by Cantoni (5) as the “active methionine” required for the methylation of guanidioacetate to creatine, it is now evident that SAM is a coenzyme of remarkable versatility (Fig. 2). In addition to its role as a methyl donor, SAM serves as a source of methylene groups (for the synthesis of cyclopropyl fatty acids), amino groups (in biotin synthesis), aminoisopropyl groups (in the synthesis of polyamines and, also, in the synthesis of ethylene, used by plants to promote plant ripening), and 5′-deoxyadenosyl radicals. SAM also serves as a source of sulfur atoms in the synthesis of biotin and lipoic acid (6). In mammals, however, the great bulk of SAM is used in methyltransferase reactions. The key to SAM’s utility as a methyl donor lies in the sulfonium ion and in the electrophilic nature of the carbon atoms that are adjacent to the sulfur atom. The essence of these methyltransferase reactions is that the positively charged sulfonium renders the adjoining methyl group electron-poor, which facilitates its attack on electron-rich acceptors (nucleophiles).

Metabolic versatility of S-adenosylmethionine.

Metabolic versatility of S-adenosylmethionine.


Metabolic versatility of S-adenosylmethionine.

SAM can donate its methyl group to a wide variety of acceptors, including amino acid residues in proteins, DNA, RNA, small molecules, and even to a metal, the methylation of arsenite (7,8). At present, about 60 methyltransferases have been identified in mammals. However, the number is almost certainly much larger. A bioinformatic analysis of a number of genomes, including the human genome, by Katz et al. (9) has suggested that Class-1 SAM-dependent methyltransferases account for 0.6–1.6% of open reading frames in these genomes. This would indicate about 300 Class 1 methyltransferases in humans, in addition to a smaller number of Class 2 and 3 enzymes. In humans, it appears that guanidinoacetate N-methyltransferase (responsible for creatine synthesis) and phosphatidylethanolamine N-methyltransferase (synthesis of phosphatidylcholine) are the major users of SAM (10). In addition, there is substantial flux through the glycine N-methyltransferase (GNMT) when methionine intakes are high (11). An important property of all known SAM-dependent methyltransferases is that they are inhibited by their product, S-adenosylhomocysteine (SAH).

Methionine metabolism.

Methionine metabolism begins with its activation to SAM (Fig. 3) by methionine adenosyltransferase (MAT). The reaction is unusual in that all 3 phosphates are removed from ATP, an indication of the “high-energy” nature of this sulfonium ion. SAM then donates its methyl group to an acceptor to produce SAH. SAH is hydrolyzed to homocysteine and adenosine by SAH hydrolase. This sequence of reactions is referred to as transmethylation and is ubiquitously present in cells. Homocysteine may be methylated back to methionine by the ubiquitously distributed methionine synthase (MS) and, also, in the liver as well as the kidney of some species, by betaine:homocysteine methyltransferase (BHMT). MS employs 5-methyl-THF as its methyl donor, whereas BHMT employs betaine, which is produced during choline oxidation as well as being provided by the diet (10). Both MS and BHMT effect remethylation, and the combination of transmethylation andremethylation comprise the methionine cycle, which occurs in most, if not all, cells.

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

Major pathways of sulfur-containing amino acid metabolism.

The methionine cycle does not result in the catabolism of methionine. This is brought about by the transsulfuration pathway, which converts homocysteine to cysteine by the combined actions of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL). The transsulfuration pathway has a very limited tissue distribution; it is restricted to the liver, kidney, intestine, and pancreas. The conversion of methionine to cysteine is an irreversible process, which accounts for the well-known nutritional principle that cysteine is not a dietary essential amino acid provided that adequate methionine is available, but methionine is a dietary essential amino acid, regardless of cysteine availability. This pathway for methionine catabolism suggests a paradox: is methionine catabolism constrained by the need for methylation reactions? If this were so, the methionine in a methionine-rich diet might exceed the methylation demand so that full catabolism could not occur via this pathway. GNMT methylates glycine to sarcosine, which may, in turn, be metabolized by sarcosine dehydrogenase to regenerate the glycine and oxidize the methyl group to 5,10-methylene-THF.

Application of sophisticated stable isotope tracer methodology to methionine metabolism in humans has yielded estimates of transmethylation, remethylation, and transsulfuration. Such studies reveal important points of regulation. For example, the sparing effect of cysteine on methionine requirements is evident as an increase in the fraction of the homocysteine pool that is remethylated and a decrease in the fraction that undergoes transsulfuration (12). In young adults ingesting a diet containing 1–1.5 g protein·kg−1·d−1, about 43% of the homocysteine pool was remethylated, and 57% was metabolized through the transsulfuration pathway (transmethylation = 9.7, transulfuration = 5.4, remethylation = 4.4 μmol·kg−1·h−1) (13).

Methionine metabolism affords a remarkable example of the role of vitamins in cell chemistry. MS utilizes methylcobalamin as a prosthetic group, 1 of only 2 mammalian enzymes that are known to require Vitamin B-12. The methyl group utilized by MS is provided from the folic acid 1-carbon pool. Methylenetetrahydrofolate reductase (MTHFR), which reduces 5,10-methylene-THF to 5-methyl-THF, contains FAD as a prosthetic group. Both of the enzymes in the transsulfuration pathway (CBS and CGL) contain pyridoxal phosphate. It is hardly surprising, therefore, that deficiencies of each of these vitamins (Vitamin B-12, folic acid, riboflavin, and pyridoxine) are associated with elevated plasma homocysteine levels. The oxidative decarboxylation of the α-ketobutyrate produced by CGL is brought about by pyruvate dehydrogenase so that niacin (NAD), thiamine (thiamine pyrophosphate), and pantothenic acid (coenzyme A) may also be regarded as being required for methionine metabolism.

Not only are vitamins required for methionine metabolism, but methionine metabolism plays a crucial role in the cellular assimilation of folate. MS has 2 principal functions. In addition to its role in methionine conservation, MS converts 5-methyl-THF to THF, thereby making it available to support DNA synthesis and other functions. Because 5-methyl-THF is the dominant circulating form that is taken into cells, MS is essential for cellular folate assimilation. Impaired MS activity (e.g., brought about by cobalamin deficiency) results in the accumulation of the folate coenzymes as 5-methyl-THF, the so-called methyl trap (14). This hypothesis explains the fact that Vitamin B-12 deficiency causes a functional cellular folate deficiency.

The combined transmethylation and transsulfuration pathways are responsible for the catabolism of the great bulk of methionine. However, there is also evidence for the occurrence of a SAM-independent catabolic pathway that begins with a transamination reaction (15). This is a very minor pathway under normal circumstances, but it becomes more significant at very high methionine concentrations. Because it produces powerful toxins such as methane thiol, it has been considered to be responsible for methionine toxicity. The identity of the initiating transaminase is uncertain; the glutamine transaminase can act on methionine, but it is thought to be unlikely to do so under physiological conditions (15). In view of the likelihood that this pathway plays a role in methionine toxicity, more work is warranted on its components, tissue distribution, and physiological function.

Regulation of methionine metabolism.

The major means by which methionine metabolism is regulated are 1) allosteric regulation by SAM and 2) regulation of the expression of key enzymes. In the liver, SAM exerts powerful effects at a variety of loci. The liver-specific MAT has a highKm for methionine and, therefore, is well fitted to remove excess dietary methionine. It exhibits the unusual property of feedback activation; it is activated by its product, SAM (16). This property has been incorporated into a computer model of hepatic methionine metabolism, and it is clear that it renders methionine disposal exquisitely sensitive to the methionine concentration (17). SAM is also an allosteric activator of CBS and an allosteric inhibitor of MTHFR (18). Therefore, elevated SAM promotes transsulfuration (methionine oxidation) and inhibits remethylation (methionine conservation). Many of the enzymes involved in methionine catabolism (MAT 1, GNMT, CBS) are increased in activity on ingestion of a high-protein diet (18).

In addition to its function in methionine catabolism, the transsulfuration pathway also provides cysteine for glutathione synthesis. Cysteine availability is often limiting for glutathione synthesis, and it appears that in a number of cells (e.g., hepatocytes), at least half of the cysteine required is provided by transsulfuration, even in the presence of physiological concentrations of cysteine (19). Transsulfuration is sensitive to the balance of prooxidants and antioxidants; peroxides increase the transsulfuration flux, whereas antioxidants decrease it (20). It is thought that redox regulation of the transsulfuration pathway occurs at the level of CBS, which contains a heme that may serve as a sensor of the oxidative environment (21).


Taurine is remarkable, both for its high concentrations in animal tissues and because of the variety of functions that have been ascribed to it. Taurine is the most abundant free amino acid in animal tissues. Table 1 shows that, although taurine accounts for only 3% of the free amino acid pool in plasma, it accounts for 25%, 50%, 53%, and 19%, respectively, of this pool in liver, kidney, muscle, and brain. The magnitude of the intracellular taurine pool deserves comment. For example, skeletal muscle contains 15.6 μmol of taurine per gram of tissue, which amounts to an intracellular concentration of about 25 mM. In addition to its role in the synthesis of the bile salt taurocholate, taurine has been proposed, inter alia, to act as an antioxidant, an intracellular osmolyte, a membrane stabilizer, and a neurotransmitter. It is an essential nutrient for cats; kittens born to mothers fed taurine-deficient diets exhibit retinal degeneration (24). Taurine is found in mother’s milk, may be conditionally essential for human infants, and is routinely added to most infant formulas. Recent work has begun to reveal taurine’s action in the retina. It appears that taurine, via an effect on a glycine receptor, promotes the generation of rod photoreceptor cells from retinal progenitor cells (25).

View this table:


Taurine concentrations in rat tissues (22,23)


The sulfur-containing amino acids present a fascinating subject to the protein chemist, the nutritionist, and the metabolic scientist, alike. They play critical roles in protein synthesis, structure, and function. Their metabolism is vital for many critical functions. SAM, a remarkably versatile molecule, is said to be second, only to ATP, in the number of enzymes that require it. Vitamins play a crucial role in the metabolism of these amino acids, which, in turn, play a role in folic acid assimilation. Despite the great advances in our knowledge of the sulfur-containing amino acids, there are important areas where further work is required. These include methionine transamination and the molecular basis for the many functions of taurine.

Disorders of Sulfur Amino Acid Metabolism

  • Generoso Andria,  Brian Fowler,  Gianfranco Sebastio

Chapter  Inborn Metabolic Diseases  pp 224-231




Several defects can exist in the conversion of the sulfur-containing amino acid methionine to cysteine and the ultimate oxidation of cysteine to inorganic sulfate (Fig. 18.1). Cystathionine-β-synthase (CBS) deficiency is the most important. It is associated with severe abnormalities of four organs or organ systems: the eye (dislocation of the lens), the skeleton (dolichostenomelia and arachnodactyly), the vascular system (thromboembolism), and the central nervous system (mental retardation, cerebrovascular accidents). A low-methionine, highcystine diet, pyridoxine, folate, and betaine in various combinations, and antithrombotic treatment may halt the otherwise unfavorable course of the disease. Methionine adenosyltransferase deficiency and γ-cystathionase deficiency usually do not require treatment. Isolated sulfite oxidase deficiency leads (in its severe form) to refractory convulsions, lens dislocation, and early death. No effective treatment exists.

  1. 1.

    Rubba P, Faccenda F, Pauciullo P, Carbone L, Mancini M, Strisciuglio P, Carrozzo R, Sartorio R, Del Giudice E, Andria G (1990) Early signs of vascular disease in homocystinuria: a noninvasive study by ultrasound methods in eight families with cystathionine ß-synthase deficiency. Metabolism 39: 1191–1195 PubMedCrossRef

  2. 2.

    Kang S-S, Wong PWK, Malinow MR (1992) Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu Rev Nutr 12: 279–288 PubMedCrossRef

  3. 3.

    Boushey CJ, Beresford SA, Omenn GS, Motulsky AG (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. JAMA 274: 1049–1057

  4. 4.

    Mudd SH, Skovby F, Levy HL, Pettigrew KD, Wilcken B, Pyeritz RE, Andria G, Boers GHJ, Bromberg IL, Cerone R, Fowler B, Grobe H, Schmidt H, Schweitzer L (1985) The natural history of homocystinuria due to cystathionine (3-synthase deficiency. Am J Hum Genet 37: 1–31 PubMed

  5. 5.

    de Franchis R, Sperandeo MP, Sebastio G, Andria G. The Italian Collaborative Study Group on Homocystinuria (1998) Clinical aspects of cystathionine ß-synthase deficiency: how wide is the spectrum? Eur J Pediatr 157: S67–7o

  6. 6.

    Kraus JP (1994) Molecular basis of phenotype expression in homocystinuria. J Inherited Metab Dis 17: 383–390 PubMedCrossRef

  7. more…

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The relationship of S amino acids to marasmic and kwashiorkor PEM

Larry H. Bernstein, MD, FCAP, Curator


Sulfur is perhaps the most abundant element in the human body.  It is found in most proteins in the sulfur-containing amino acids, along with phosphorus and nitrogen.  These three elements form a triad of important elements needed as building blocks or structural components of all animal tissues.  The sulfur gives animal tissues their strength, and their resiliency.

Sulfur and nutritional balancing.  Suflur is not supplemented in pill form because it is plentiful in foods.  However, nutiritonal balancing emphasizes it and we find if one wishes to be healthy then one must eat meat, eggs and cooked vegetables and then your sulfur needs will be taken care of.

In Metabolic Reactions in the Nervous System, 1970; pp 225-287

Medical Research Council, Neuropsychiatric Research Unit, Carshalton, Surrey, England



Several sulfur amino acids and sulfur compounds are found in mammalian tissues. While some find their origin in the diet, other sulfur amino acids are formed in vivo from methionine in the tissues. Thus it is known that methionine is converted into homocysteine, cystathionine, cysteine, hypotaurine, and taurine. These metabolites are formed in the course of transferring a methyl group to other compounds. The mechanism of demethylation and the subsequent metabolism of the demethylated product, homocysteine, is now well established. The enzyme systems in most cases were first studied in liver preparations. The demonstration that 35S-methionine is converted into 35S-cysteine and 35S-taurine by rat brain in vitro and in vivo gave evidence that the sulfur amino acids are metabolized also in the mammalian brain. Several subsequent studies have shown similarities between the metabolism of methionine in liver and in brain, but they have also revealed some characteristic differences in the metabolism of sulfur amino acids in the brain: (1) the cystathionine and taurine concentrations are much higher in the brain than in the liver, (2) the enzyme cysteine sulfinic acid decarboxylase is predominantly a particulate deaminated to form isethionic acid by rat brain and heart and not by liver. An interesting feature of sulfur amino acid metabolism is that many of the enzyme systems involved in the conversion of methionine into its several metabolites require pyridoxal phosphate (vitamin B6) as a cofactor. Whereas in liver this cofactor is tightly bound to some of these enzymes, the corresponding enzymes in the brain are bound loosely to this cofactor, and their activity in the brain can be demonstrated in vitro only by adding the cofactor.

Sulfur-Containing Amino Acids

The sulfur-containing amino acids (cysteine and methionine) are generally considered to be nonpolar and hydrophobic. In fact, methionine is one of the most hydrophobic amino acids and is almost always found on the interior of proteins. Cysteine on the other hand does ionize to yield the thiolate anion. Even so, it is uncommon to find cysteine on the surface of a protein. There are several reasons. First, sulfur has a low propensity to hydrogen bond, unlike oxygen. A consequence of this fact is that H2S is a gas under conditions that H2O is a liquid. Second, the thiol group of cysteine can react with other thiol groups in an oxidation reaction that yields a disulfide bond. Perhaps as a consequence, cysteine residues are most frequently buried inside proteins.


When in its natural L-form, methionine is a proteinogen amino acid. It is classed as an essential amino acid and cannot be synthesized by the body itself. This means that a sufficient supply of methionine in the diet or as a dietary supplement is of particular importance.

Sulphur compounds occur in all living creatures and have a multitude of functions. Besides cysteine, methionine is the only sulphur-containing amino acid. Furthermore methionine plays an important role in the synthesis of other proteins, such as carnitine or melatonine. Methionine has a fat-dissolving effect and reduces the depositing of fat in the liver.

Methionine is an important cartilage-forming substance

The cartilage in the joints requires sulphur for its production. If there is not enough sulphur available in the body, this can have negative effects for the healthy individual over the long term. People who suffer from arthritis can experience negative effects such as a prolonged healing process for the damaged tissue, if there is a sulphur deficiency at the beginning of the illness.

Studies have shown that the cartilage from healthy people contains approximately three times more suphur than in arthritis patients.To make things more complicated, various arthritis medications connect sulphur, which are the salts in the sulphuric acid. The demand for sulphur is increasing to more than average levels.

J Nutr. 2006 Jun;136(6 Suppl):1636S-1640S.

The sulfur-containing amino acids: an overview.

Brosnan JT1Brosnan ME.

Author information


Methionine, cysteine, homocysteine, and taurine are the 4 common sulfur-containing amino acids, but only the first 2 are incorporated into proteins. Sulfur belongs to the same group in the periodic table as oxygen but is much less electronegative. This difference accounts for some of the distinctive properties of the sulfur-containing amino acids. Methionine is the initiating amino acid in the synthesis of virtually all eukaryotic proteins; N-formylmethionine serves the same function in prokaryotes. Within proteins, many of the methionine residues are buried in the hydrophobic core, but some, which are exposed, are susceptible to oxidative damage. Cysteine, by virtue of its ability to form disulfide bonds, plays a crucial role in protein structure and in protein-folding pathways. Methionine metabolism begins with its activation to S-adenosylmethionine. This is a cofactor of extraordinary versatility, playing roles in methyl group transfer, 5′-deoxyadenosyl group transfer, polyamine synthesis, ethylene synthesis in plants, and many others. In animals, the great bulk of S-adenosylmethionine is used in methylation reactions. S-Adenosylhomocysteine, which is a product of these methyltransferases, gives rise to homocysteine. Homocysteine may be remethylated to methionine or converted to cysteine by the transsulfuration pathway. Methionine may also be metabolized by a transamination pathway. This pathway, which is significant only at high methionine concentrations, produces a number of toxic endproducts. Cysteine may be converted to such important products as glutathione and taurine. Taurine is present in many tissues at higher concentrations than any of the other amino acids. It is an essential nutrient for cats.



Sulfur is an essential nutrient (micro-mineral). It is a nonmetallic element that is essential for life. In most animals it represents about 0.25% of the body weight. However, sulfur is normally present as part of larger compounds, and the requirement for pure sulfur has not been determined for most species. In recent years, sulfur toxicity has become more common because of its high concentration in many byproduct feeds. The use of these feeds in ruminant diets is increasing, which in turn may increase the trace mineral requirement. The purpose of this article is to review our current understanding of sulfur nutrition and to look at how sulfur level in the diet may influence the copper requirement.

Essential Functions

Compounds containing sulfur play a variety of essential functions in the body. They act as structural entities (collagen), as catalysts (enzymes), as oxygen carriers (hemoglobin), as hormones (insulin), and as vitamins (thiamine and biotin). Sulfur is present in four amino acids: methionine, cystine, cysteine and taurine. The secondary structure of many proteins is determined by the cross linkage or folding due to covalent disulfide bonds between amino acids.

Sulfur is the element that gives many key compounds their unique functional properties. For example, acetate is linked to coenzyme A by a thioester linkage to form acetyl coenzyme A. This compound is required for the formation of key metabolic intermediates such as citrate, acetoacetate and malonate. The sulfur in thiamine allows it to serve as a molecule which transfers carbonyl groups. Thiamine plays a key role in the formation of pentose sugars which are required for ribonucleic acids synthesis and photosynthesis. Biotin, another sulfur-containing B vitamin, acts a carrier for carbon dioxide in carboxylation reactions.

Inorganic vs. Organic Sulfur

Despite the fact that sulfur is a key mineral in many compounds essential for life, dietary inorganic sulfur is not necessary for the health of most animals. Pigs and poultry can do quite well with only organic sulfur (sulfur amino acids, thiamine, biotin, etc.) sources in their diets. The total absence of inorganic sulfur from the diet may increase the sulfur-amino acid requirement, which suggest that sulfur from the amino acids is used to synthesize other organic compounds containing sulfur.

In contrast, ruminants may respond to inorganic sulfur supplementation, especially if the diet is high in nonprotein nitrogen. Block et al., (1951) showed that ruminal microorganism are capable of synthesizing all organic sulfur containing compounds essential for life from inorganic sulfur. When urea or other nonprotein nitrogen sources are fed, the diet may become deficient in sulfur. Goodrich et. al., (1978) reported that the nitrogen to sulfur ratio in rumen microbial protein averages 14.5:1. The common recommendation for the nitrogen:sulfur ratio is 10:1 in diets containing high levels of urea.

Sulfur Source

The source of sulfur can influence its bioavailability. Goodrich et. al,. (1978) gave the following rankings from the most available to the least available: L-methionine> calcium sulfate >ammonium sulfate> sodium sulfate>molasses sulfur>sodium sulfide>lignin sulfonate>elemental sulfur. The recommend concentration of sulfur in beef cattle diets is 0.15% (NRC, 1996). However, this assumes the sulfur source is highly bioavailable.

The type of forage in the diet may also influence sulfur requirement. For example, Archer and Wheeler, (1978) showed that increasing the sulfur concentration from 0.08% to 0.12% in cattle grazing sorghum sudangrass increased weight gains by 12%. Sulfur requirements may be higher for cattle grazing sorghum sudangrass because sulfur is required in the detoxification of the cyanogenic glucosides found in most sorghum forages. Sulfur bioavailability varies with the type of forage; fescue has a lower sulfur availability than other grasses. Cattle consuming fescue hay will often respond with improved intake and fiber digestion following sulfur supplementation. Forages usually contain between 0.1-0.3% sulfur, except for corn silage which is often lower.

Zinn et al., (1997) reported that when ammonium sulfate was used to produce diets containing 0.15, 0.20 and 0.25% sulfur (DM basis), feedlot performance was reduced with the higher sulfur concentration. The diets were based on steam-flaked corn and fed to heifers weighing 845 pounds initially. Increasing dietary sulfur above 0.20%, caused a strong trend (P <.10) for decreased gains, feed intake and gain per unit of feed intake. The excess sulfur also caused a reduction (P <.05) in the ribeye area which is an important factor determining the yield grade of the carcass. Sulfur intake from the drinking water was not reported.


Another problem that can occur when high dietary sulfur leads to the production of excess sulfides in the rumen is polioencephalomalacia, or PEM (Gould et al., 1991; Lowe et al., 1996). The most defining sign of PEM is the necrosis of the cerebrocortical region of the brain. Animals with PEM will often press their head against a wall or post. In some instances they become “star gazers,” where they stand with their head back over their shoulders looking up at the sky. If not treated with thiamine, most animals with PEM will die within 48 hours.

A thiamine deficiency has been considered the most common cause of PEM in ruminants. However, recent research suggests that sulfur may play a key role in many instances of PEM. PEM has often been seen in animals that have had access to plants containing high amounts of thiaminase such as bracken fern (Merck, 1991). Thiamine is a B vitamin that plays a key role in the tri-carboxcylic acid cycle and pentose shunt. When thiamine is deficient, key tissues that require large amounts of thiamine, such as the brain and heart, are the first to show lesions.

The exact interaction between dietary sulfur, thiaminase production, and PEM is not understood. Kung et al. (1998) postulated that sulfates in the feed or water are converted to hydrogen sulfide in the rumen. When the hydrogen sulfide is eructated with the other rumen gases, it is inhaled and can damage lung and brain tissues. Several researchers (Oliveria et al., 1996; Brent and Bartley, 1994 and Olkowski, et al., 1992) have suggested that high sulfide levels could cause the brain lesions associated with PEM.

Kung et al., (1998) summarized six different reports in the literature where high sulfur intakes were associated with PEM. In these studies thiamine status was within normal ranges and giving thiamine did not prevent the signs in all cases. In these cases sulfur intakes from feed and water would have ranged from 0.40 to over 0.80% of the diet dry matter.

Drinking Water

Sulfates in the water can be a major source of sulfur intake. For example, in one of the cases cited by Kung et al., (1998), sulfates in the drinking water ranged from 2,200 to 2,800 ppm. When the water sulfur intake was expressed as a percent of the dry matter consumed, it averaged 0.67%. Digesti and Weeth (1976) proposed that the maximum safe concentration of sulfates in drinking water for cattle was 2,500 ppm. Water sulfate concentrations as high as 5,000 ppm have been reported (Veenhuizen et al., 1992).

Accurately estimating water intake in these situations can also be a challenge. Water meters can be used for confined livestock to estimate the average intake, but with grazing animals drinking from ponds or streams, one can only estimate the intake. Usually water consumption will be 3-5 times the dry matter intake. Dry matter intake for grazing beef cattle and sheep will normally be between 1.5 and 2.5% of their body weight. Lactating dairy cows may consume over 3.5% of their body weight when grazing high quality forage. Although this is not a precise means of measuring water sulfur intake, it does allow one to estimate the relative contributions of the feed and water.

Excess Sulfur

Excess sulfur can also impair animal performance by reducing the availability of other minerals. For example, hydrogen sulfide in the rumen binds with molybdenum to form thiomolybdates. Thiomolybdates bind with copper in the rumen to form an insoluble complex. Some thiomolybdates are absorbed and impair the metabolism of copper in the body. For example, Gooneratne et al. (1989) reported that certain thiomolybdates cause copper to be bound to blood albumins which renders the copper unavailable for biochemical reactions in the body. Price et al., (1987) showed that tri- and tetrathiomolybdates were the sulfur-molybdenum complexes responsible for reducing copper absorption, while the di- and trithiomolybdates had the greatest effect on copper metabolism in the body.

Sulfur also reduces copper absorption by the formation of insoluble copper sulfide in the rumen, independent of the formation of thiomolybdates. Rumen protozoa degrade sulfur amino acids to sulfide which binds to copper to form an insoluble complex. Smart et al., (1986) reported that decreasing the sulfate concentration of drinking water from 500 to 42 ppm, improved the copper status of cattle. These same researchers reported that the 10 ppm copper recommended by the Beef NRC (1996) was not adequate when cattle drank high-sulfur water, which resulted in a total dietary sulfur intake of 0.35%.

Copper Supplement

The optimum level of copper supplementation required to combat high sulfur intakes has not been determined. The maximum tolerable level of copper for cattle has been estimated at 100 ppm (NRC 1980). Although this level is being fed in diets that are high in sulfur, certain breeds of dairy cattle such as the Jersey and Guernsey are susceptible to copper toxicity at concentrations below 100 ppm.

In these situations, the source of copper is also important. Although copper sulfate is a common copper source, it would not be recommended if the diet is already high in sulfur. Copper oxide would not contribute to the sulfur problem, but because of its poor availability is not recommended. Copper carbonate is probably the best copper source for this situation. It has a bioavailability similar to copper sulfate, with out increasing sulfur intake.

The Importance of Macro Minerals: Sulfur

K.E. Lanka, Ph.D., P.A.S.


Sulfur (S) is one of seven generally recognized macro minerals needed in the diets of dairy cattle and other animals. Sulfur is a mineral that is found in the amino acids methionine, cysteine (cystine), homocysteine and in taurine. It is also in the B-vitamins, thiamin and biotin. It is an important component of healthy cartilage. As a part of the specified amino acids, it is key to the structure of proteins. Heating protein s u p p l e m e n t s can rearrange the structures of proteins, due to the sulfurcontaining amino acids, which can determine whether these nutrients are soluble and rumen degradable or if they will resist rumen degradation in cattle. Heating also affects the essential amino acid, lysine, when carbohydrates are present in a supplement. An example of this change by heating can be observed when an egg is boiled.

Animals have a need for essential sulfur-containing nutrients, such as methionine and cysteine. However, the microbes in the rumens of cattle and other ruminants can use mineral sources of sulfur to produce some of these important nutrients for dairy and beef cattle. Thus, it is important to feed sulfur at recommended dietary levels to meet the needs of the microbes, as well as the animals. In dairy cattle, it is needed in the diet at the level of 0.20%. For beef cattle, the recommended concentration is a minimum of 0.15% of dietary dry matter (DM). Since about 0.15% of the body weight is sulfur, commercial concentrations in typical beef cattle rations range from 0.18 to 0.24%. Sulfur is essential when a nonprotein nitrogen source, such as urea, is fed. The total Nitrogen:Sulfur (N:S) ratio in a diet should range from 10:1 to 12:1, and the rumen soluble N:S ratio should be 4.0:1 to 5.5:1. Common sources of sulfur for livestock include:

• potassium sulfate

• magnesium sulfate

• sodium sulfate

• ammonium sulfate

• calcium sulfate

• corn gluten feed, distillers grains and other corn coproducts.

Sulfate forms of macro and trace minerals are among the most digestible and easily absorbed forms in the digestive tract. Elemental sulfur in water and feed is not a readily available source for animals. A deficiency of sulfur in the diets of animals can have detrimental effects on their performance. Marginal deficiency symptoms include:

• reduced microbial synthesis

• reduced fiber digestion due to slow microbial growth in ruminants

• slow growth

• reduced milk production

• reduced feed efficiency

• reduced intakes

Severe deficiencies can cause the following symptoms:

• unwillingness to eat

• weight loss

• dullness and slow movement

• excessive salivation

• death

For ruminants, the maximum tolerable level of sulfur in diets is .40% of their dry matter intakes. Excess sulfur will interfere with the digestion and absorption of other minerals, particularly the trace minerals, copper and selenium. Even though these minerals may be adequate in the diet, secondary deficiency symptoms can be observed, simply because the trace minerals were made unavailable, due to too much sulfur in the feed. Other toxicity symptoms or problems that can occur from high levels of sulfur include:

• reduced intakes

• overloading the urinary system, leading to kidney failure

• interference with nerve impulses, including blindness, coma, muscle twitches and intestinal inflammation or bleeding

• The breath of cattle may smell like “rotten eggs,” due to the toxic form of sulfur, hydrogen sulfide.

• polioencephalomalacia (PEM)

With recent increased usage of distillers grains in dairy and feedlot diets, the association between sulfur and PEM has been noted and documented. One of the causes of PEM in ruminants is the interference by sulfur with the B-vitamin, thiamin. Supplementation with thiamine may help to alleviate PEM. This is one reason why thiamin is included in Agri-King base mineral products. The symptoms of PEM include: • excessive salivation • nervousness and twitching (hypersensitivity) • poor muscle coordination and dullness • tilting the head to the side and walking in circles (star gazing) • head pressing • blindness • death Sulfur is an important element in the pH balance of the blood of animals. Sulfates are some of the anionic salts that are used to adjust PCI (Pre-Fresh Cow Index) that affects calcium utilization in cows prior to calving. This can be a key factor in the prevention of milk fevers and retained placentae in fresh cows. In summary, sulfur is needed in dairy rations at a minimum level of .20% of dry matter for a TMR. It is a key macro mineral in maintaining life and production in animals, and it is an essential component of some amino acids, vitamins and other nutrients needed by all animals. Like all required nutrients, too much S can become toxic. The maximum level of sulfur is .40% of the dry matter intake for cattle. Agri-King, Inc is a leader and innovator in animal nutrition. AgriKing rations are balanced to meet the nutrient needs of the animals that are fed by clients. For more information about Agri-King nutrition, contact a representative near you or visit the website at www.agriking.com.

Protein Malnutrition

Reporter and Curator: Dr. Sudipta Saha, Ph.D.


A large part of  the world’s population is undernourished by the standards of Western Europe and North America. Scientists and nonscientists alike recognize as one of the major challenges of our time the problem of how to ensure that the production and distribution of food keep pace with the increasing number of mouths to be fed. In the world as a whole the most widespread and serious dietary deficiency is that of protein. This fact emerges clearly from the reports of the expert committees of WHO and FAO (World Health Organization, 1951, 1953). Nevertheless, many protein chemists, even those associated with medical research, may not realize the extent and severity of protein malnutrition, because it occurs chiefly in the technically underdeveloped countries far from where they work.

Dietary histories and response to treatment point to deficiency of total protein as the primary cause of the clinical syndrome kwashiorkor. The level of calorie intake has an important influence on the pattern of the disease. Deficiency of one or more specific amino acids, or amino acid imbalances in the diet, may perhaps be responsible for some of the symptoms and signs, particularly those whose incidence varies from one part of the world to another. All these variations on a theme are covered by the general term protein malnutrition. The onset is often precipitated by the added burden of diarrhea, infection, and parasitic infestation. The nutritional state influences the resistance to infection, and conversely the presence of an infection affects the state of nutrition. A further contributory factor may be the psychological upheaval in the child when the next baby in the family is born. At the root of all these causes lie poverty, ignorance, and disruption of the family life.

The planning of preventive measures cannot be effective unless it is based on some knowledge of the magnitude of the problem to be tackled. At a very rough estimate, in some countries perhaps 10% of the children suffer from severe protein malnutrition at some age between birth and 4 years. The marginal deficiency states must be much more common, Clinical signs and biochemical changes are of little value in diagnosing the early case; a deficit in body weight still seems to be the best criterion. Prevention ideally would be by greater production and consumption of animal protein, and by the increased use of skim milk and of surplus fish at present often wasted. However, animal protein is likely to remain scarce and expensive. Plant sources are being investigated with a view to encouraging not only domestic production, but also the production on an industrial scale of cheap foodstuffs rich in protein. A preventive program that is nutritionally sound may fail if account is not taken of local food habits, traditions, and customs. Protein requirements are affected by the quality of protein, the intake of calories, and by the state of the body (growth, the presence of disease, etc.). The maintenance requirement and the amount required for growth in children can be estimated, but the requirement for health is still unknown. For the time being, the allowances of protein recommended for people in the world as a whole are based empirically on the known physiological requirement with an arbitrarily added wide margin of safety.

The absorption of nitrogen is remarkably efficient even in severely malnourished infants. In general the nitrogen of plant proteins is less well absorbed than that of milk. When a baby receives a diet in which the protein is derived entirely from vegetabIe sources, incomplete absorption of nitrogen may play a significant part in the production of protein malnutrition. The malnourished baby who responds to treatment is able to retain and utilize nitrogen very efficiently; there is no evidence of any impairment in the mechanisms of protein synthesis. It is possible, however, that these mechanisms may be irreversibly damaged in babies who die, and that this may be the cause of death. The level of calorie intake has an important influence on the efficiency of utilization of nitrogen. An adequate calorie intake promotes conservation of nitrogen in the body as a whole when supplies of protein are short, but this protective effect may not be exerted equally in all organs. In this way the level of calorie intake may modify the pattern of protein depletion. A greater than normal calorie intake is needed for the restoration of depleted protein stores.

The discussion of protein metabolism in protein malnutrition has been purposely limited to a narrow field-to studies made on man, and to the few animal experiments that have a direct bearing on those studies. For technical reasons most of the work discussed relates to plasma proteins. There is a conflict of evidence between results obtained in man and animals about the effect of protein depletion or a low protein diet on the rate of catabolism of plasma albumin. It is of great importance to settle this point. A priori there seems no reason why the rate of protein catabolism should be affected by nutritional state. Preliminary studies with radioactive methionine in infants suggest, as working hypotheses, that in protein malnutrition there may be an increase in the reutilization of amino acids liberated by tissue catabolism, and an apparent concentration of protein synthesis in the more essential organs at the expense of the less essential. There is some experimental support for both these ideas, but further work is badly needed. The concept of protein stores or reserve protein is based entirely on dynamic and not on chemical considerations. It is suggested that the essential difference between a “labile” and a “fixed” protein is a difference in turnover rate. An attempt is made to show that the changes produced by protein depletion in the protein content of organs such as liver and muscle are a necessary consequence of the metabolic characteristics of proteins in those organs. There may be no need to invoke the help of homeostatic or compensatory regulations to explain the changes found in protein depletion.

Aging and growth are processes during which some metabolic adjustments must take place. It is believed that it may be better to regard the changes which are found in protein malnutrition in a similar light: as evidence of an alteration in functional pattern, rather than of damage or disease. Protein malnutrition in man has two aspects-a practical and a theoretical one. From the practical point of view it is an extremely common disease with a high mortality, and there is every reason to believe that it will become more common unless urgent preventive measures are taken. Theoretically it raises many questions that are of interest in relation to other branches of medicine and biochemistry. It is believed that the two aspects are linked, and that progress towards prevention is still impeded by our lack of basic knowledge as well as by our failure to apply what is already known. In protein malnutrition there is no sharp line between health and disease. The simple concept of specific deficiency diseases that grew from the discovery of vitamins is not applicable. We have to go back instead to the ideas of an earlier era, when nutrition was regarded as a branch of physiology, concerned with the functions, fate, and metabolic interrelationships of the major nutrients.

It is a characteristic of protein metabolism that nitrogen balance can be maintained at many different levels of protein intake. These different steady states are achieved by adjustments of the amount and distribution of proteins in the body as a whole, in organs, and in cells. It is believed that these changes in amount and distribution of proteins must result in alterations of metabolic pattern, with a gradation of change from an optimum, which cannot be defined, to a state of irreversible breakdown incompatible with life. In the intermediate stages function is modified and efficiency perhaps impaired. It seems possible that variations in diet, and particularly in the amount and quality of the protein, may underlie many of the differences in incidence and symptomatology of disease which are gradually being uncovered in different parts of the world.

Source References:


Voluntary and Involuntary S- Insufficiency

Writer and Curator: Larry H Bernstein, MD, FCAP


Transthyretin and the Stressful Condition


This article is written among a series of articles concerned with stress, obesity, diet and exercise, as well as altitude and deep water diving for extended periods, and their effects.  There is a reason that I focus on transthyretin (TTR), although much can be said about micronutients and vitamins, and fat soluble vitamins in particular, and iron intake during pregnancy.    While the importance of vitamins and iron are well accepted, the metabolic basis for their activities is not fully understood.  In the case of a single amino acid, methionine, it is hugely important because of the role it plays in sulfur metabolism, the sulfhydryl group being essential for coenzyme A, cytochrome c, and for disulfide bonds.  The distribution of sulfur, like the distribution of iodine, is not uniform across geographic regions.  In addition, the content of sulfur found in plant sources is not comparable to that in animal protein.  There have been previous articles at this site on TTR, amyloid and sepsis.

Transthyretin and Lean Body Mass in Stable and Stressed State


A Second Look at the Transthyretin Nutrition Inflammatory Conundrum


Stabilizers that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity


Thyroid Function and Disorders


Proteomics, Metabolomics, Signaling Pathways, and Cell Regulation: a Compilation of Articles in the Journal http://pharmaceuticalintelligence.com


Malnutrition in India, high newborn death rate and stunting of children age under five years


Vegan Diet is Sulfur Deficient and Heart Unhealthy


How Methionine Imbalance with Sulfur-Insufficiency Leads to Hyperhomocysteinemia


Amyloidosis with Cardiomyopathy


Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets


Sepsis, Multi-organ Dysfunction Syndrome, and Septic Shock: A Conundrum of Signaling Pathways Cascading Out of Control


Automated Inferential Diagnosis of SIRS, sepsis, septic shock


Transthyretin and the Systemic Inflammatory Response 

Transthyretin has been widely used as a biomarker for identifying protein-energy malnutrition (PEM) and for monitoring the improvement of nutritional status after implementing a nutritional intervention by enteral feeding or by parenteral infusion. This has occurred because transthyretin (TTR) has a rapid removal from the circulation in 48 hours and it is readily measured by immunometric assay. Nevertheless, concerns have been raised about the use of TTR in the ICU setting, which prompts a review of the actual benefit of using this test in a number of settings. TTR is easily followed in the underweight and the high risk populations in an ambulatory setting, which has a significant background risk of chronic diseases.  It is sensitive to the systemic inflammatory response syndrom (SIRS), and needs to be understood in the context of acute illness to be used effectively. There are a number of physiologic changes associated with SIRS and the injury/repair process that will affect TTR and will be put in context in this review. The most important point is that in the context of an ICU setting, the contribution of TTR is significant in a complex milieu.  copyright @ Bentham Publishers Ltd. 2009.

Transthyretin as a marker to predict outcome in critically ill patients.
Arun Devakonda, Liziamma George, Suhail Raoof, Adebayo Esan, Anthony Saleh, Larry H. Bernstein.
Clin Biochem Oct 2008; 41(14-15): 1126-1130

A determination of TTR level is an objective method od measuring protein catabolic loss of severly ill patients and numerous studies show that TTR levels correlate with patient outcomes of non-critically ill patients. We evaluated whether TTR level correlates with the prevalence of PEM in the ICUand evaluated serum TTR level as an indicator of the effectiveness of nutrition support and the prognosis in critically ill patients.

TTR showed excellent concordance with patients classified with PEM or at high malnutrition risk, and followed for 7 days, it is a measure of the metabolic burden. TTR levels did not respond early to nutrition support because of the delayed return to anabolic status. It is particularly helpful in removing interpretation bias, and it is an excellent measure of the systemic inflammatory response concurrent with a preexisting state of chronic inanition.

 The Stressful Condition as a Nutritionally Dependent Adaptive Dichotomy

Yves Ingenbleek and Larry Bernstein
Nutrition 1999;15(4):305-320 PII S0899-9007(99)00009-X

The injured body manifests a cascade of cytokine-induced metabolic events aimed at developing defense mechanisms and tissue repair. Rising concentrations of counterregulatory hormones work in concert with cytokines to generate overall insulin and insulin-like growth factor 1 (IGF-1), postreceptor resistance and energy requirements grounded on lipid dependency. Dalient features are self-sustained hypercortisolemia persisting as long as cytokines are oversecreted and down-regulation of the hypothalamo-pituitary-thyroid axis stabilized at low basal levels. Inhibition of thyroxine 5’deiodinating activity (5’DA) accounts for the depressed T3 values associated with the sparing of both N and energy-consuming processes. Both the liver and damaged territories adapt to stressful signals along up-regulated pathways disconnected from the central and peripheral control systems. Cytokines stimulate 5’DA and suppress the synthesis of TTR, causing the drop of retinol-binding protein (RBP) and the leakage of increased amounts of T4 and retinol in free form. TTR and RBP thus work as prohormonal reservoirs of precursor molecules which need to be converted into bioactive derivatives (T3 and retinoic acids) to reach transcriptional efficiency. The converting steps (5’DA and cellular retinol-binding protein-1) are activated to T4 and retinol, themselves operating as limiting factors to positive feedback loops. …The suicidal behavior of TBG, CBG, and IGFBP-3 allows the occurrence of peak endocrine and mitogenic influences at the site of inflammation. The production rate of TTR by the liver is the main determinant of both the hepatic release and blood transport of holoRBP, which explains why poor nutritional status concomitantly impairs thyroid- and retinoid-dependent acute phase responses, hindering the stressed body to appropriately face the survival crisis.  …
abbreviations: TBG, thyroxine-binding globulain; CBG, cortisol-binding globulin; IGFBP-3, insulin growth factor binding protein-3; TTR, transthyretin; RBP, retionol-binding protein.

Why Should Plasma Transthyretin Become a Routine Screening Tool in Elderly Persons? 

Yves Ingenbleek.
J Nutrition, Health & Aging 2009.

The homotetrameric TTR molecule (55 kDa as MM) was first identified in cerebrospinal fluid (CSF).  The initial name of prealbumin (PA)  was assigned based on the electrophoretic migration anodal to albumin. PA was soon recognized as a specific binding protein for thyroid hormone. and also of plasma retinol through the mediation of the small retinol-binding protein (RBP, 21 kDa as MM), which has a circulating half-life half that of TTR (24 h vs 48 h).

There exist at least 3 goos reasons why TTR should become a routine medical screening test in elderly persons.  The first id grounded on the assessment of protein nutritional status that is frequently compromized and may become a life threatening condition.  TTR was proposed as a marker of protein-energy malnutrition (PEM) in 1972. As a result of protein and energy deprivation, TTR hepatic synthesis is suppressed whereas all plasma indispensable amino acids (IAAs) manifest declining trends with the sole exception of methionine (Met) whose concentration usually remains unmodified. By comparison with ALB and transferrin (TF) plasma values, TTR did reveal a much higher degree of reactivity to changes in protein status that has been attributed to its shorter biological half-life and to its unusual tryptophan richness. The predictive ability of outcome offered by TTR is independent of that provided by ALB and TF. Uncomplicated PEM primarily affects the size of body nitrogen (N) pools, allowing reduced protein syntheses to levels compatible with survival.  These adaptiver changes are faithfully identified by the serial measurement of TTR whose reliability has never been disputed in protein-depleted states. On the contrary, the nutritional relevance of TTR has been controverted in acute and chronic inflammatory conditions due to the cytokine-induced transcriptional blockade of liver synthesis which is an obligatory step occurring independently from the prevailing nutritional status. Although PEM and stress ful disorders refer to distinct pathogenic mechanisms, their combined inhibitory effects on TTR liber production fueled a long-lasting strife regarding a poor specificity.  Recent body compositional studies have contributed to disentagling these intermingled morbidities, showing that evolutionary patterns displayed by plasma TTR are closely correlated with the fluctuations of lean body mass (LBM).

The second reason follows from advances describing the unexpected relationship established between TTR and homocysteine (Hcy), a S-containing AA not found in customary diets but resulting from the endogenous transmethylation of dietary methionine.  Hcy may be recycled to Met along a remethylation pathway (RM) or irreversibly degraded throughout the transsulfuration (TS) cascade to relase sulfaturia as end-product. Hcy is thus situated at the crossrad of RM and TS pathways which are in equilibrium keeping plasma Met values unaltered.  Three dietary water soluble B viatamins are implicated in the regulation of the Hcy-Met cycle. Folates (vit B9) are the most powerful agent, working as a supplier of the methyl group required for the RM process whereas cobalamines (vit B12) and pyridoxine (vit B6) operate as cofactors of Met-synthase and cystathionine-β-synthase.  Met synthase promotes the RM pathway whereas the rate-limiting CβS governs the TS degradative cascade. Dietary deficiency in any of the 3 vitamins may upregulate Hcy plasma values, an acquied biochemiucal anomaly increasingly encountered in aged populations.

The third reason refers to recent and fascinating data recorded in neurobiology and emphasizing the specific properties of TTR in the prevention of brain deterioration. TTR participates directly in the maintenance of memory and normal cognitive processes during the aging process by acting on the retinoid signaling pathway.  Moreover, TTR may bind amyloid β peptide in vitro, preventing its transformation into toxic amyloid fibrils and amyloid plaques.  TTR works as a limiting factor for the plasma transport of retinoid, which in turn operates as a limiting determinant of both physiologically active retinoic acid (RA) derivatives, implying that any fluctuation in protein status might well entail corresponding  alterations in cellular bioavailability of retinoid compounds.  Under normal aging circumstances, the concentration of retinoid compounds declines in cerebral tissues together with the downregulation of RA receptor expression. In animal models, depletion of RAs causes the deposition of amyloid-β peptides, favoring the formation of amyloid plaques.

Prealbumin and Nutritional Evaluation

Larry Bernstein, Walter Pleban
Nutrition Apr 1996; 12(4):255-259.

We compressed 16-test-pattern classes of albumin (ALB), cholesterol (CHOL), and total protein (TPR) in 545 chemistry profiles to 4 classes by conveerting decision values to a number code to separate malnourished (1 or 2) from nonmalnourished (NM)(0) patients using as cutoff values for NM (0), mild (1), and moderate (2): ALB 35, 27 g/L; TPR 63, 53 g/L; CHOL 3.9, 2.8 mmol/L; and BUN 9.3, 3.6 mmol/L. The BUN was found to have  to have too low an S-value to make a contribution to the compressed classification. The cutoff values for classifying the data were assigned prior to statistical analysis, after examining information in the structured data. The data was obtained by a natural experiment in which the test profiles routinely done by the laboratory were randomly extracted. The analysis identifies the values used that best classify the data and are not dependent on distributional assumptions. The data were converted to 0, 1, or 2 as outcomes, to create a ternary truth table (eaxch row in nnn, the n value is 0 to 2). This allows for 3(81) possible patterns, without the inclusion of prealbumin (TTR). The emerging system has much fewer patterns in the information-rich truth table formed (a purposeful, far from random event). We added TTR, coded, and examined the data from 129 patients. The classes are a compressed truth table of n-coded patterns with outcomes of 0, 1, or 2 with protein-energy malnutrition (PEM) increasing from an all-0 to all-2 pattern.  Pattern class (F=154), PAB (F=35), ALB (F=56), and CHOL (F=18) were different across PEM class and predicted PEM class (R-sq. = 0.7864, F=119, p < E-5). Kruskall-Wallis analysis of class by ranks was significant for pattern class E-18), TTR (6.1E-15) ALB (E-16), CHOL (9E-10), and TPR (5E-13). The medians and standard error (SEM) for TTR, ALB, and CHOL of four TTR classes (NM, mild, mod, severe) are: TTR = 209, 8.7; 159, 9.3; 137, 10.4; 72, 11.1 mg/L. ALB – 36, 0.7; 30.5, 0.8; 25.0, 0.8; 24.5, 0.8 g/L. CHOL = 4.43, 0.17; 4.04, 0.20; 3.11, 0.21; 2.54, 0.22 mmol/L. TTR and CHOL values show the effect of nutrition support on TTR and CHOL in PEM. Moderately malnourished patients receiving nutrition support have TTR values in the normal range at 137 mg/L and at 159 mg/L when the ALB is at 25 g/L or at 30.5 g/L.

An Informational Approach to Likelihood of Malnutrition 

Larry Bernstein, Thomas Shaw-Stiffel, Lisa Zarney, Walter Pleban.
Nutrition Nov 1996;12(11):772-776.  PII: S0899-9007(96)00222-5.

Unidentified protein-energy malnutrition (PEM) is associated with comorbidities and increased hospital length of stay. We developed a model for identifying severe metabolic stress and likelihood of malnutrition using test patterns of albumin (ALB), cholesterol (CHOL), and total protein (TP) in 545 chemistry profiles…They were compressed to four pattern classes. ALB (F=170), CHOL (F = 21), and TP (F = 5.6) predicted PEM class (R-SQ = 0.806, F= 214; p < E^-6), but pattern class was the best predictor (R-SQ = 0.900, F= 1200, p< E^-10). Ktuskal-Wallis analysis of class by ranks was significant for pattern class (E^18), ALB (E^-18), CHOL (E^-14), TP (@E^-16). The means and SEM for tests in the three PEM classes (mild, mod, severe) were; ALB – 35.7, 0.8; 30.9, 0.5; 24.2, 0.5 g/L. CHOL – 3.93, 0.26; 3.98, 0.16; 3.03, 0.18 µmol/L, and TP – 68.8, 1.7; 60.0, 1.0; 50.6, 1.1 g/L. We classified patients at risk of malnutrition using truth table comprehension.

Downsizing of Lean Body Mass is a Key Determinant of Alzheimer’s Disease

Yves Ingenbleek, Larry Bernstein
J Alzheimer’s Dis 2015; 44: 745-754.

Lean body mass (LBM) encompasses all metabolically active organs distributed into visceral and structural tissue compartments and collecting the bulk of N and K stores of the human body. Transthyretin (TTR)  is a plasma protein mainly secreted by the liver within a trimolecular TTR-RBP-retinol complex revealing from birth to old age strikingly similar evolutionary patterns with LBM in health and disease. TTR is also synthesized by the choroid plexus along distinct regulatory pathways. Chronic dietary methionine (Met) deprivation or cytokine-induced inflammatory disorders generates LBM downsizing following differentiated physiopathological processes. Met-restricted regimens downregulate the transsulfuration cascade causing upstream elevation of homocysteine (Hcy) safeguarding Met homeostasis and downstream drop of hydrogen sulfide (H2S) impairing anti-oxidative capacities. Elderly persons constitute a vulnerable population group exposed to increasing Hcy burden and declining H2S protection, notably in plant-eating communities or in the course of inflammatory illnesses. Appropriate correction of defective protein status and eradication of inflammatory processes may restore an appropriate LBM size allowing the hepatic production of the retinol circulating complex to resume, in contrast with the refractory choroidal TTR secretory process. As a result of improved health status, augmented concentrations of plasma-derived TTR and retinol may reach the cerebrospinal fluid and dismantle senile amyloid plaques, contributing to the prevention or the delay of the onset of neurodegenerative events in elderly subjects at risk of Alzheimer’s disease.

Amyloidogenic and non-amyloidogenic transthyretin variants interact differently with human cardiomyocytes: insights into early events of non-fibrillar tissue damage

Pallavi Manral and Natalia Reixach
Biosci.Rep.(2015)/35/art:e00172 http://dx.doi.org:/10.1042/BSR20140155

TTR (transthyretin) amyloidosis are diseases characterized by the aggregation and extracellular deposition of the normally soluble plasma protein TTR. Ex vivo and tissue culture studies suggest that tissue damage precedes TTR fibril deposition, indicating that early events in the amyloidogenic cascade have an impact on disease development. We used a human cardiomyocyte tissue culture model system to define these events. We previously described that the amyloidogenic V122I TTR variant is cytotoxic to human cardiac cells, whereas the naturally occurring, stable and non-amyloidogenic T119M TTR variant is not. We show that most of the V122I TTR interacting with the cells is extracellular and this interaction is mediated by a membraneprotein(s). In contrast, most of the non-amyloidogenic T119M TTR associated with the cells is intracellular where it undergoes lysosomal degradation. The TTR internalization process is highly dependent on membrane cholesterol content. Using a fluorescent labelled V122I TTR variant that has the same aggregation and cytotoxic potential as the native V122I TTR, we determined that its association with human cardiomyocytes is saturable with a KD near 650nM. Only amyloidogenic V122I TTR compete with fluorescent V122I force ll-binding sites. Finally, incubation of the human cardiomyocytes with V122I TTR but not with T119M TTR, generates superoxide species and activates caspase3/7. In summary, our results show that the interaction of the amyloidogenic V122I TTR is distinct from that of a non-amyloidogenic TTR variant and is characterized by its retention at the cell membrane, where it initiates the cytotoxic cascade.

Emerging roles for retinoids in regeneration and differentiation in normal and disease states

Lorraine J. Gudas
Biochimica et Biophysica Acta 1821 (2012) 213–221

The vitamin (retinol) metabolite, all-transretinoic acid (RA), is a signaling molecule that plays key roles in the development of the body plan and induces the differentiation of many types of cells. In this review the physiological and pathophysiological roles of retinoids (retinol and related metabolites) in mature animals are discussed. Both in the developing embryo and in the adult, RA signaling via combinatorial Hoxgene expression is important for cell positional memory. The genes that require RA for the maturation/differentiation of T cells are only beginning to be cataloged, but it is clear that retinoids play a major role in expression of key genes in the immune system. An exciting, recent publication in regeneration research shows that ALDH1a2(RALDH2), which is the rate-limiting enzyme in the production of RA from retinaldehyde, is highly induced shortly after amputation in the regenerating heart, adult fin, and larval fin in zebrafish. Thus, local generation of RA presumably plays a key role in fin formation during both embryogenesis and in fin regeneration. HIV transgenic mice and human patients with HIV-associated kidney disease exhibit a profound reduction in the level of RARβ protein in the glomeruli, and HIV transgenic mice show reduced retinol dehydrogenase levels, concomitant with a greater than 3-fold reduction in endogenous RA levels in the glomeruli. Levels of endogenous retinoids (those synthesized from retinol within cells) are altered in many different diseases in the lung, kidney, and central nervous system, contributing to pathophysiology.

The Membrane Receptor for Plasma Retinol-Binding Protein, A New Type of Cell-Surface Receptor

Hui Sun and Riki Kawaguchi
Intl Review Cell and Molec Biol, 2011; 288:Chap 1. Pp 1:34

Vitamin A is essential for diverse aspects of life ranging from embryogenesis to the proper functioning of most adul torgans. Its derivatives (retinoids) have potent biological activities such as regulating cell growth and differentiation. Plasma retinol-binding protein (RBP) is the specific vitamin A carrier protein in the blood that binds to vitamin A with high affinity and delivers it to target organs. A large amount of evidence has accumulated over the past decades supporting the existence of a cell-surface receptor for RBP that mediates cellular vitamin A uptake. Using an unbiased strategy, this specific cell-surface RBP receptor has been identified as STRA6, a multi-transmembrane domain protein with previously unknown function. STRA6 is not homologous to any protein of known function and represents a new type of cell-surface receptor. Consistent with the diverse functions of vitamin A, STRA6 is widely expressed in embryonic development and in adult organ systems. Mutations in human STRA6 are associated with severe pathological phenotypes in many organs
such as the eye, brain, heart, and lung. STRA6 binds to RBP with high affinity and mediates vitamin A uptake into cells. This review summarizes the history of the RBP receptor research, its expression in the context of known functions of vitamin A in distinct human organs, structure/function analysis of this new type of membrane receptor, pertinent questions regarding its very existence, and its potential implication in treating human diseases.

Choroid plexus dysfunction impairs beta-amyloid clearance in a triple transgenic mouse model of Alzheimer’s disease

Ibrahim González-Marrero, Lydia Giménez-Llort, Conrad E. Johanson, et al.
Front Cell Neurosc  Feb2015; 9(17): 1-10

Compromised secretory function of choroid plexus (CP) and defective cerebrospinal fluid (CSF) production, along with accumulation of beta-amyloid (Aβ) peptides at the blood-CSF barrier (BCSFB), contribute to complications of Alzheimer’s disease (AD). The AD triple transgenic mouse model (3xTg-AD) at 16 month-old mimics critical hallmarks of the human disease: β-amyloid (Aβ) plaques and neurofibrillary tangles (NFT) with a temporal-and regional-specific profile. Currently, little is known about transport and metabolic responses by CP to the disrupted homeostasis of CNS Aβ in AD. This study analyzed the effects of highly-expressed AD-linked human transgenes (APP, PS1 and tau) on lateral ventricle CP function. Confocal imaging and immunohistochemistry revealed an increase only of Aβ42 isoform in epithelial cytosol and in stroma surrounding choroidal capillaries; this buildup may reflect insufficient clearance transport from CSF to blood. Still, there was increased expression, presumably compensatory, of the choroidal Aβ transporters: the low density lipoprotein receptor-related protein1 (LRP1) and the receptor for advanced glycation end product (RAGE). A thickening of the epithelial basal membrane and greater collagen-IV deposition occurred around capillaries in CP, probably curtailing solute exchanges. Moreover, there was attenuated expression of epithelial aquaporin-1 and transthyretin(TTR) protein compared to Non-Tg mice. Collectively these findings indicate CP dysfunction hypothetically linked to increasing Aβ burden resulting in less efficient ion transport, concurrently with reduced production of CSF (less sink action on brain Aβ) and diminished secretion of TTR (less neuroprotection against cortical Aβ toxicity). The putative effects of a disabled CP-CSF system on CNS functions are discussed in the context of AD.

Endoplasmic reticulum: The unfolded protein response is tangled In neurodegeneration

Jeroen J.M. Hoozemans, Wiep Scheper
Intl J Biochem & Cell Biology 44 (2012) 1295–1298

Organelle facts•The ER is involved in the folding and maturation ofmembrane-bound and secreted proteins.•The ER exerts protein quality control to ensure correct folding and to detect and remove misfolded proteins.•Disturbance of ER homeostasis leads to protein misfolding and induces the UPR.•Activation of the UPR is aimed to restore proteostasis via an intricate transcriptional and (post)translational signaling network.•In neurodegenerative diseases classified as tauopathies the activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau.•The involvement of the UPR in tauopathies makes it a potential therapeutic target.

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ERand tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.

Chemical and/or biological therapeutic strategies to ameliorate protein misfolding diseases

Derrick Sek Tong Ong and Jeffery W Kelly
Current Opin Cell Biol 2011; 23:231–238

Inheriting a mutant misfolding-prone protein that cannot be efficiently folded in a given cell type(s) results in a spectrum of human loss-of-function misfolding diseases. The inability of the biological protein maturation pathways to adapt to a specific misfolding-prone protein also contributes to pathology. Chemical and biological therapeutic strategies are presented that restore protein homeostasis, or proteostasis, either by enhancing the biological capacity of the proteostasis network or through small molecule stabilization of a specific misfolding-prone protein. Herein, we review the recent literature on therapeutic strategies to ameliorate protein misfolding diseases that function through either of these mechanisms, or a combination thereof, and provide our perspective on the promise of alleviating protein misfolding diseases by taking advantage of proteostasis adaptation.

Vegan Diet is Sulfur Deficient and Heart Unhealthy

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2013-11-17/larryhbern/Vegan Diet is Sulfur Deficient and Heart Unhealthy

The following is a reblog of “Heart of the Matter: Plant-Based Diets Lead to High Homocysteine, Low Sulfur and Marginal B12 Status”
Posted on September 26, 2011 by Dr Kaayla Daniel in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Yves Ingenbleek

It is a report of a scientific study carried out by Kilmer S. Cully and Yves Ingenbleek, Harvard Pathology and Univ Louis Pasteur.  I have previously written about the conundrum of transthyretin as an accurate marker of malnutrition, but also being lowered by the septic state.  This is accounted for by the catabolic state that sets off autocannabalization of skeletal muscle and lean body mass to provide gluconeogenic precursors to sustain life.  While serum albumin and transthyretin both decline, the former has a half-life of 20 days, while the latter is 48 hours.  Much work has been done to gain a better understand this rapid turnover protein that transports thyroxine, and the immediate result of the decline in concentration is a shift the the hormone protein binding equilibrium increasing the free thyroxine, a euthyroid hyperthyroid effect.  However, much work by Prof. Inglenbleek, some ion collaboration with Vernon Young, at MIT, showed that transthyretin reflects the sulfur stores of animals.  The sulfur to nitrogen ratio of plants is 1:20, but it is 1:12 in man, so the dietary intake would affect an omnivorous animal.  Recall that S is carried on amino acids that take part in disulfide linkage.  A deficiency in S containing amino acids would have a negative health effect.  The story is presented here.

The World Health Organization (WHO) reports that 16.7 million deaths occur worldwide each year due to cardiovascular disease, and more than half of those deaths occur in developing countries where plant-based diets high in legumes and starches are eaten by the vast majority of the people.

Yet “everyone knows” plant-based diets prevent heart disease.  Indeed this myth  is repeated so often that massive numbers of educated, health-conscious individuals in first world countries are consciously adopting third world style diets in the hope of preventing disease, optimizing health and maximizing longevity.   But if the WHO statistics are correct, plant-based diets might not be protective at all.   And today’s fashionable experiment in veganism could end very badly indeed.

A study out August 26 in the journal Nutrition makes a strong case against plant-based diets for prevention of heart disease.  The title alone  –  “Vegetarianism produces subclinical malnutrition, hyperhomocysteinemia and atherogenesis” — sounds a significant warning.   The article establishes  why subjects who eat mostly vegetarian diets develop morbidity and mortality from cardiovascular disease unrelated to vitamin B status and Framingham criteria.

Co-author Kilmer S. McCully, MD, “Father of the Homocysteine Theory of Heart Disease,” is familiar to WAPF members as winner of the Linus Pauling Award, WAPF’s Integrity in Science Award, and author of numerous articles published in peer-reviewed journals as well as the popular books The Homocysteine Revolution and The Heart Revolution.   In 2009 Dr. McCully was one of the signers of the Weston A. Price Foundation’s petition to the FDA in which we asked the agency to retract its unwarranted 1999 soy/heart disease health claim.  (http://www.westonaprice.org/soy-alert/soy-heart-health-claim)

Dr. McCully teamed up with Yves Ingenbleek, MD, of the University Louis Pasteur in Strasbourg, France, which funded the research.   Dr. Ingenbleek is well known for his work on malnutrition, the essential role of sulfur to nitrogen, and sulfur deficiency as a cause of  hyperhomocysteinemia.

The study took place in Chad, and involved 24 rural male subjects age 18 to 30, and 15 urban male controls, age 18-29.   (Women in this region of Chad could not be studied because of their animistic beliefs and proscriptions against collecting their urine.)

The rural men were apparently healthy, physically active farmers with good lipid profiles.  Their staple foods included cassava, sweet potatoes, beans, millet and ground nuts.   Cassava leaves, cabbages and carrots provided good levels of carotenes, folates and pyridoxine (B6).  The diet is plant-based there because of a shortage of grazing lands and livestock, but subjects occasionally consume  some B12-containing foods, mostly poultry and eggs, though very little dairy or meat.   Their diet could be described as high carb, high fiber,  low in both protein and fat, and low in the sulfur containing amino acids.    In brief, the very diet recommended by many of today’s nutritional “experts” for overall good health and heart disease prevention.

The urban controls were likewise healthy and ate a similar diet, but with beef, smoked fish and canned or powdered milk regularly on the menus.  Their diet was thus higher in protein, fat and the sulfur-containing amino acids though roughly equivalent in calories.

Dr. McCully’s research over the past 40 years on the pathogenesis of atherosclerosis has shown the role of homocysteine in free radical damage and the protective effect of  vitamins B6, B12 and folate.   Indeed, many doctors today recommend taking this trio of B vitamins as an inexpensive heart disease “insurance policy.”

In Chad, both groups showed adequate levels of B6 and folate.  The B12 levels of the vegetarian group were lower, but the difference was only of “borderline significance.”   However, as the researchers point out, ”A previous study undertaken in the same Chadian area in a larger group of 60 rural participants did demonstrate a weak inverse correlation between B12 and homocysteine concentrations in the 20 subjects most severely protein depleted .  .  .  It is therefore likely that the hyperhomocysteinemia status of some of our rural subjects in the present survey might have resulted from combined B12 and protein deficiencies.   The correlation of B12 deficiency with hyperhomocysteinemia could well reach statistical significance if a larger groups of subjects were studied.”

Clearly it’s wise for people on plant-based diets to supplement their diets with B12, but protein malnutrition must also be addressed.   And the issue is not just getting enough protein to eat, but the right kind.   Quality, not just quantity.   The bottom line is we must eat  protein rich in bioavailable, sulfur-containing amino acids — and that means animal products.   (Vegans at this point will surely claim the issue is insufficient protein and trot out soy as the solution.   Soy is indeed a  complete plant based protein, but notoriously low in methionine.  It does contain decent levels of cysteine, but the cysteine is bound up in protease inhibitors, making it largely  biounavailable. (For more information, read  my book The Whole Soy Story: The Dark Side of America’s Favorite Health Food, endorsed by Dr. McCully, as well as our petition to the FDA noted above.)

So what did  Drs. Ingenbleek and  McCully find among the study group of protein-deficient people?   Higher levels of homocysteine, of course.  Also significant alterations in body composition,  lean body mass, body mass index and plasma transthyretin levels.  In plain English, the near-vegetarian subjects were thinner, with poorer muscle tone and showed subclinical signs of protein malnutrition.   (So much for popular ideas of extreme thinness being healthy. )

The plant-based diet of the study group was low in all of the sulfur-containing amino acids.   As would be expected, labwork on these men showed lower plasma cysteine and glutathione levels compared to the controls.  Methionine levels, however,  tested comparably.   The explanation for this is  “adaptive response.”   In brief, mammals trying to function with insufficient sulfur-containing amino acids will do whatever’s necessary to survive.   Given the essential role of methionine in metabolic processes, that means deregulating the transsulfuration pathway, increasing homocysteine levels, and methylating homocysteine to make methionine.

Ultimately, it all boils down to our need for sulfur.   As Stephanie Seneff, PhD, and many others have written in Wise Traditions and on this website, sulfur is vital for disease prevention and maintenance of good health.   In terms of heart disease, Drs. Ingenbleek and McCully have shown sulfur deficiency not only leads to high homocysteine levels, but is the likeliest reason some clinical trials using B6, B12 and folate interventions have proved ineffective for the prevention of cardiovascular and cerebrovascular diseases.    Over the past few years, headlines from such studies have led to widespread dismissal of Dr. McCully’s  “Homocysteine Theory of Heart Disease” and renewed media focus on cholesterol, c-reactive protein and other possible culprits that can be treated by statins and other profitable drugs.   In contrast, Drs. McCully and Ingenbleek research suggests we can better prevent heart disease with three inexpensive B vitamins and traditional diets rich in the sulfur-containing amino acids found in animal foods.

In the blaze of publicity surrounding Forks Over Knives and other blasts of vegan propaganda, few people are likely to hear about this study.   That’s sad, for it provides an important missing piece in our knowledge of heart disease development, a strong argument against the plant-based fad, and a bright new chapter in what the New York Times has called “The Fall and Rise of Kilmer McCully.”

*  *  *  *  *

Thanks to Sylvia Onusic PhD who was able to access a full text copy of this article to share with  me.

This entry was posted in WAPF Blog and tagged B12, Forks over Knives, Kaayla T. Daniel, Kilmer S. McCully, Naughty Nutritionist, soy, sulfur, Yves Ingenbleek. Bookmark the permalink.

Food Insecurity in Africa and GMOs

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


In the GMO-free future, farming still looks pretty much the same. Without insect-resistant crops, farmers spray more broad-spectrum insecticides, which do some collateral damage to surrounding food webs. Without herbicide-resistant crops, farmers spray less glyphosate, which slows the spread of glyphosate-resistant weeds and perhaps leads to healthier soil biota. Farmers also till their fields more often, which kills soil biota, and releases a lot more greenhouse gases.

The banning of GMOs hasn’t led to a transformation of agriculture because GM seed was never a linchpin supporting the conventional food system: Farmers could always do fine without it. Eaters no longer worry about the small potential threat of GMO health hazards, but they are subject to new risks: GMOs were neither the first, nor have they been the last, agricultural innovation, and each of these technologies comes with its own potential hazards. Plant scientists will have increased their use of mutagenesis and epigenetic manipulation, perhaps. We no longer have biotech patents, but we still have traditional seed-breeding patents. Life goes on.

In the other alternate future, where the pro-GMO side wins, we see less insecticide, more herbicide, and less tillage. In this world, with regulations lifted, a surge of small business and garage-biotechnologists got to work on creative solutions for the problems of agriculture.

Genetic engineering is just one tool in the tinkerer’s belt. Newer tools are already available, and scientists continue to make breakthroughs with traditional breeding. So in this future, a few more genetically engineered plants and animals get their chance to compete. Some make the world a little better, while others cause unexpected problems. But the science has moved beyond basic genetic engineering, and most of the risks and benefits of progress are coming from other technologies. Life goes on.

In many ways he’s right. GMOs on the market today – and most of the ones planned – are about making agriculture more efficient and profitable for farmers and seed providers. This is not a trivial thing, but would global agriculture collapse without these GMOs? Of course not.

We rarely see transformative technologies coming. And remember that we are still in the very early days of genetic engineering of crops and animals. I suspect that you could go back and look at the early days of almost any new technology and convincingly downplay its transformative potential.

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

Reviewer, Curator: Larry H. Bernstein, MD, FCAP


The new era of nutrition research translates empirical knowledge to evidence-based molecular science (9). Modern nutrition research focuses on

  • promoting health,
  • preventing or delaying the onset of disease,
  • optimizing performance, and
  • assessing risk.

Personalized nutrition is a conceptual analogue to personalized medicine and means adapting food to individual needs. Nutrigenomics and nutrigenetics

  • build the science foundation for understanding human variability in
  • preferences, requirements, and responses to diet and
  • may become the future tools for consumer assessment

motivated by personalized nutritional counseling for health maintenance and disease prevention.

The primary aim of ―omic‖ technologies is

  • the non-targeted identification of all gene products (transcripts, proteins, and metabolites) present in a specific biological sample.

By their nature, these technologies reveal unexpected properties of biological systems.

A second and more challenging aspect of ―omic‖ technologies is

  • the refined analysis of quantitative dynamics in biological systems (10).

For metabolomics, gas and liquid chromatography coupled to mass spectrometry are well suited for coping with

  • high sample numbers in reliable measurement times with respect to
  • both technical accuracy and the identification and quantitation of small-molecular-weight metabolites.

This potential is a prerequisite for the analysis of dynamic systems. Thus, metabolomics is a key technology for systems biology.

In modern nutrition research, mass spectrometry has developed into a tool

  • to assess health, sensory as well as quality and safety aspects of food.

In this review, we focus on health-related benefits of food components and, accordingly,

  • on biomarkers of exposure (bioavailability) and bioefficacy.

Current nutrition research focuses on unraveling the link between

  • dietary patterns,
  • individual foods or
  • food constituents and

the physiological effects at cellular, tissue and whole body level

  • after acute and chronic uptake.

The bioavailability of bioactive food constituents as well as dose-effectcorrelations are key information to understand

  • the impact of food on defined health outcomes.

Both strongly depend on appropriate analytical tools

  • to identify and quantify minute amounts of individual compounds in highly complex matrices–food or biological fluids–and
  • to monitor molecular changes in the body in a highly specific and sensitive manner.

Based on these requirements,

  • mass spectrometry has become the analytical method of choice
  • with broad applications throughout all areas of nutrition research (11).

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The good, the bad and the ugly of sulfur and volcanic activity

Larry H Bernstein, MD, FCAP, Curator



Climate change deniers have promulgated much ignorance about the planet and our life on earth.  Nevertheless, I shall deal with geophysical and geochemical issues and indirectly, climate change in this portion of the discussion.  The good, the bad, and the ugly has everything to due with the elements and to life on earth.  This is the case, regardless of claims propagated by the tobacco and the carbon fuels interests.  I shall proceed as I have done in the previous discussions.

Is a Lack of Water to Blame for the Conflict in Syria?

A 2006 drought pushed Syrian farmers to migrate to urban centers, setting the stage for massive uprisings

By Joshua Hammer




An Iraqi girl stands on former marshland, drained in the 1990s because of politically motivated water policies. (Essam Al-Sudani / AFP / Getty Images)

The world’s earliest documented water war happened 4,500 years ago, when the armies of Lagash and Umma, city-states near the junction of the Tigris and Euphrates rivers, battled with spears and chariots after Umma’s king drained an irrigation canal leading from the Tigris. “Enannatum, ruler of Lagash, went into battle,” reads an account carved into an ancient stone cylinder, and “left behind 60 soldiers [dead] on the bank of the canal.”


Water loss documented by the Gravity Recovery and Climate Experiment (GRACE), a pair of satellites operated by NASA and Germany’s aerospace center, suggests water-related conflict could be brewing on the riverbank again. GRACE measured groundwater usage between 2003 and 2009 and found that the Tigris-Euphrates Basin—comprising Turkey, Syria, Iraq and western Iran—is losing water faster than any other place in the world except northern India . During those six years, 117 million acre-feet of stored freshwater vanished from the region as a result of dwindling rainfall and poor water management policies. That’s equal to all the water in the Dead Sea. GRACE’s director, Jay Famiglietti, a hydrologist at the University of California, Irvine, calls the data “alarming.”

While the scientists captured dropping water levels, political experts have observed rising tensions. In Iraq, the absence of a strong government since 2003, drought and shrinking aquifers have led to a recent spate of assassinations of irrigation department officials and clashes between rural clans. Some experts say that these local feuds could escalate into full-scale armed conflicts .

In Syria, a devastating drought beginning in 2006 forced many farmers to abandon their fields and migrate to urban centers. There’s some evidence that the migration fueled the civil war there, in which 80,000 people have died. “You had a lot of angry, unemployed men helping to trigger a revolution,” says Aaron Wolf, a water management expert at Oregon State University, who frequently visits the Middle East.

Tensions between nations are also high. Since 1975, Turkey’s dam and hydro­power construction has cut water flow to Iraq by 80 percent and to Syria by 40 percent. Syria and Iraq have accused Turkey of hoarding water.

Hydrologists say that the countries need to find alternatives to sucking the aquifers dry—perhaps recycling wastewater or introducing desalination—and develop equitable ways of sharing their rivers. “Water doesn’t know political boundaries. People have to get together and work,” Famiglietti says. One example lies nearby, in an area not known for cross-border cooperation. Israeli and Jordanian officials met last year for the first time in two decades to discuss rehabilitating the nearly dry Jordan River, and Israel has agreed to release freshwater down the river.

“It could be a model” for the Tigris-Euphrates region, says Gidon Bromberg, a co-director of Friends of the Earth Middle East, who helped get the countries together. Wolf, too, remains optimistic, noting that stress can encourage compromise.

History might suggest a way: The world’s first international water treaty, a cuneiform tablet now hanging in the Louvre, ended the war between Lagash and Umma.



“Rebel forces are targeting water installations to cut off supplies to the largely Shia south of Iraq,” says Matthew Machowski, a Middle East security researcher at the UK houses of parliament and Queen Mary University of London.

“It is already being used as an instrument of war by all sides. One could claim that controlling water resources in Iraq is even more important than controlling the oil refineries, especially in summer. Control of the water supply is fundamentally important. Cut it off and you create great sanitation and health crises,” he said

Isis now controls the Samarra barrage west of Baghdad on the River Tigris and areas around the giant Mosul Dam, higher up on the same river. Because much of Kurdistan depends on the dam, it is strongly defended by Kurdish peshmerga forces and is unlikely to fall without a fierce fight, says Machowski.

Iraqi troops were rushed to defend the massive 8km-long Haditha Dam and its hydroelectrical works on the Euphrates to stop it falling into the hands of Isis forces. Were the dam to fall, say analysts, Isis would control much of Iraq’s electricity and the rebels might fatally tighten their grip on Baghdad.

Isis fighters in Fallujah captured the smaller Nuaimiyah Dam on the Euphrates and deliberately diverted its water to “drown” government forces in the surrounding area. Millions of people in the cities of Karbala, Najaf, Babylon and Nasiriyah had their water cut off but the town of Abu Ghraib was catastrophically flooded along with farms and villages over 200 square miles. According to the UN, around 12,000 families lost their homes.

Earlier, Kurdish forces reportedly diverted water supplies from the Mosul Dam. Equally, Turkey has been accused of reducing flows to the giant Lake Assad, Syria’s largest body of fresh water, to cut off supplies to Aleppo, and Isis forces have reportedly targeted water supplies in the refugee camps set up for internally displaced people.

Iraqis fled from Mosul after Isis cut off power and water and only returned when they were restored, says Machowski. “When they restored water supplies to Mosul, the Sunnis saw it as liberation. Control of water resources in the Mosul area is one reason why people returned,” said Machowski.

Both Isis forces and President Assad’s army are said to have used water tactics to control the city of Aleppo. The Tishrin Dam on the Euphrates, 60 miles east of the city, was captured by Isis in November 2012.

“The deliberate targeting of water supply networks … is now a daily occurrence in the conflict. The water pumping station in Al-Khafsah, Aleppo, stopped working on 10 May, cutting off water supply to half of the city.


A satellite view showing the two main rivers running from Turkey through Syria and Iraq. Credits: MODIS/NASA

The Euphrates River, the Middle East’s second longest river, and the Tigris, have historically been at the centre of conflict. In the 1980s, Saddam Hussein drained 90% of the vast Mesopotamian marshes that were fed by the two rivers to punish the Shias who rose up against his regime. Since 1975, Turkey’s dam and hydropower constructions on the two rivers have cut water flow to Iraq by 80% and to Syria by 40%. Both Syria and Iraq have accused Turkey of hoarding water and threatening their water supply.


The Barada River, shown here in Damascus, is the only notable river flowing entirely within Syrian territory. The city’s water supplies are under huge strain

DAMASCUS, 25 March 2010 (IRIN) – Poor planning and management, wasteful irrigation systems, intensive wheat and cotton farming and a rapidly growing population are straining water resources in Syria in a year which has seen unprecedented internal displacement as a result of drought in eastern and northeastern parts of the country.

In 2007 Syria consumed 19.2 billion cubic metres of water – 3.5 billion more than the amount of water replenished naturally, with the deficit coming from groundwater and reservoirs, according to the Ministry of Irrigation.

Agriculture accounts for almost 90 percent of the country’s water consumption, according to government and private sector.

Agricultural policies encourage water-hungry wheat and cotton cultivation, and inefficient irrigation methods mean much water is wasted.


South Asia is a desperately water-insecure region, and India’s shortages are part of a wider continental crisis. According to a recent report authored by UN climate scientists, coastal areas in Asia will be among the worst affected by climate change. Hundreds of millions of people across East, Southeast and South Asia, the report concluded, will be affected by flooding, droughts, famine, increases in the costs of food and energy, and rising sea levels.

Groundwater serves as a vital buffer against the volatility of monsoon rains, and India’s falling water table therefore threatens catastrophe. 60 percent of north India’s irrigated agriculture is dependent on ground water, as is 85 percent of the region’s drinking water. The World Bank predicts that India only has 20 years before its aquifers will reach “critical condition” – when demand for water will outstrip supply – an eventuality that will devastate the region’s food security, economic growth and livelihoods.

Analysts fear that growing competition for rapidly dwindling natural resources will trigger inter-state or intra-state conflict. China and India continue to draw on water sources that supply the wider region, and a particularly concerning flashpoint is the Indus River Valley basin that spans India and Pakistan. The river’s waters are vital to the economies of areas on both sides of the border and a long-standing treaty, agreed by Pakistan and India in 1960, governs rights of access. But during the “dry season,” between October and March, water levels fall to less than half of those seen during the remainder of the year. The fear is that cooperation over access to the Indus River will fray as shortages become more desperate.


Farm worker heading for the paddy fields at Gubinder Singh’s farm

The Indo-Gangetic Basin, which lies at the foothills of the Himalayas, is one of the areas in the world facing a huge water crisis.  The Basin spans from Pakistan, across Northern India into Bangladesh. Apart from runoff from mountainous streams and glaciers, it also holds one of the largest underground bodies of water in the world. But it’s also in one of the most populous regions of the world, with more than a billion people living on the subcontinent.  Still, parts of the region are well-resourced when it comes to water supplies – like the Indian state of Punjab, which has three rivers running through it and a network of canals in some parts.

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NASA Satellites Unlock Secret to Northern India’s Vanishing Water



NASA Hydrologist Matt Rodell discusses vanishing groundwater in India. Credit:NASA
› Watch Video



soil moisture belt

soil moisture belt


Groundwater resides beneath the soil surface in permeable rock, clay and sand as illustrated in this conceptual image. Many aquifers extend hundreds of feet underground and in some instances have filled with water over the course of thousands of years. Credit: NASA

groundwater withdrawals as a percentage of groundwater recharge

groundwater withdrawals as a percentage of groundwater recharge



The map, showing groundwater withdrawals as a percentage of groundwater recharge, is based on state-level estimates of annual withdrawals and recharge reported by India’s Ministry of Water Resources. The three states included in this study are labeled. Credit:NASA/Matt Rodell


The averaging function (spatial weighting) used to estimate terrestrial water storage changes from GRACE data is mapped. Warmer colors indicate greater sensitivity to terrestrial water storage changes. Credit: NASA/Matt Rodell


Beneath northern India’s irrigated fields of wheat, rice, and barley … beneath its densely populated cities of Jaiphur and New Delhi, the groundwater has been disappearing. Halfway around the world, hydrologists, including Matt Rodell of NASA, have been hunting for it.

Where is northern India’s underground water supply going? According to Rodell and colleagues, it is being pumped and consumed by human activities — principally to irrigate cropland — faster than the aquifers can be replenished by natural processes. They based their conclusions — published in the August 20 issue of Nature — on observations from NASA’s Gravity Recovery and Climate Experiment (GRACE).

“If measures are not taken to ensure sustainable groundwater usage, consequences for the 114 million residents of the region may include a collapse of agricultural output and severe shortages of potable water,” said Rodell, who is based at NASA’s Goddard Space Flight Center in Greenbelt, Md.



The Himalayas are representative of a modern and active mountain-building event, called anorogeny in geologic parlance. Both the Himalayas and the Cascade Range are the result of plate-to-plate collision in the Theory of Plate Tectonics.
The difference between the Himalayas and the Cascade Range volcanoes is based on density of the lithospheric plates. Yes. The Cascade Range is caused by subduction of more dense ocean crust into and underneath lighter, lower density continental crust. As the oceanic plate dives deeper and deeper, the ocean crust warms, melts, and rises upward through the overriding continental crust “inland” from the plate collision boundary. As that molten rock punches through the continental crust, a curvilinear series of volcanoes, generally parallel to the plate collision boundary, begins to form.

Cascade Range Subduction

Cascade Range Subduction


Cascade Range Subduction from J. Wiley & Sons – 2010
In the case of the Cascade Range, the name of this type of volcanic formation is unique in process, as well as geochemistry, and has been referred to as an Andesitic-type after the Andes Mountains. Regardless, the Cascade Range is comprised of intermediate igneous rocks, with a fairly high silica content. High silica makes for high siliceous acid. That creates “sticky” igneous extrusions that often have quite dramatic eruptions [May 1980 Mt. St. Helens eruption].


Igneous Rock Classification

Igneous Rock Classification

Igneous Rock Classification Chart – Public Domain

The Himalayas are also a plate-to-plate collision tectonic boundary. In this case, the Indian Plate [of the Indian Subcontinent] is colliding head-on with the Eurasian Plate. Both plates are comprised of continental lithospheric crust, so there is no appreciable distinction in density. Both have a density of approximately 2.7 g/cm³. This as opposed to ocean crust with a mean density of 3.3 g/cm³. The plates try to compete in the plate-to-plate collision but the equal densities of the two plates cannot push one under the other very deep like that in a subduction zone.  The result is large-scale thickening of the continental crust in the region at and surrounding the collision boundary. Other processes occurring in the Himalayas region associated with the orogeny are metamorphism, thrust [compression] faulting, and plateau uplift.

Depiction of Himalayan Collision

Depiction of Himalayan Collision

Generalized Depiction of Himalayan Collision from FHSU – 2010
A perfect analogy is two trucks of the same make and model colliding head-on. The Himalayan Orogeny is the oft mentioned “crumple zone”. Metal does not deform in a brittle sense like competent rock does, so don’t confuse that too much.

With all that being said, there are tremendous temperatures attained at a continental plate-to-plate collision boundary. However, the crust is simply too thick, and too “squashed together” to allow anything to squeeze up and break through to the surface as volcanic eruptions.

FHSU,  2010.  Image of Himalayan Collision.  Fort Hays State University.  Hays, Kansas.  2010.
Wiley & Sons, J.,  2010.  Image of Cascade Range Subduction Zone.  J. Wiley & Sons.  Hoboken, New Jersey.  2010.


Mt. Everest was formed (is forming) by two tectonic plates colliding–the Indo-Australian Plate and the Eurasian plate.

Sometimes, when two tectonic plates collide, volcanoes form (such as the Juan de Fuca plate and the North American Plate forming the Cascades). However, this has to do with one plate–in this case the Juan de Fuca Plate sliding or subducting beneath another–the North American Plate. This happens because the oceanic plate (the Juan de Fuca Plate) is more dense than than the continental plate (the NA Plate). For reasons I won’t get into here, magma forms between the two plates as one subducts beneath the other and volcanoes are formed.

Mt. Everest is formed by two continental plates colliding. Continental plates are generally too buoyant to subduct beneath each other. While some subduction occurred during this collision, most of what happened was crustal shortening. Think about what happens when you have a rug on a wood floor and push two ends toward each other. It buckles and folds up in itself. This is a simplified version of what happened in the Himalaya.

Because little to no subduction is occurring, no magma is forming and Mt. Everest will not become a volcano.

The Himalayas were created by two continental plates colliding. What happens when two masses of rocks with some similarities, like in density, collide? Both of them rise. There is a lot of heat produced. However, there isn’t enough heat to melt rocks completely. For there to be a volcano, there has to be a source of molten rock.

This material can occur if the two masses of rocks have vastly different densities. In this case, the heavier mass will slide above the other. The mass on the bottom will melt. This molten rock material will rise and create a volcano. or two or more. This, however can not happen in the HImalayas. The two masses in action are the Indian Plate and the Eurasian Plate, which have similar rock density.


Volcanic Eruptions and the Role of Sulfur Dioxide in Climate Change

In March and April of this year, a series of severe volcanic eruptions shook Alaska’s Mount Redoubt.1  To date, the largest of the eruptions produced an ash plume that reached 50,000 feet above sea level and released a significant amount of sulfur dioxide (CAS Registry Number® 7446-09-5) into the earth’s atmosphere.  According to the Alaska Volcano Observatory, “The main concerns for human health in volcanic haze consist of ash, sulfur dioxide gas (SO2), and sulfuric acid droplets (H2SO4), which forms when volcanic SO2 oxidizes in the atmosphere.”1

While there is obvious reason for alarm among local populations, sulfur dioxide from the Mount Redoubt eruption could also have more widespread impacts, particularly on the climate.  According to a 1997 article published in the Journal of Geology, “The mechanism by which large eruptions affect climate is generally accepted: injection of sulfur into the stratosphere and conversion to sulfate aerosol, which in turn reduces the solar energy reaching the earth’s surface.”2

In the years following a volcanic eruption, sulfate aerosol that remains in the atmosphere is thought to cause surface cooling by reflecting the sun’s energy back into space.  In fact, sulfate aerosol from the massive eruption of Indonesia’s Mount Tambora in 1815 is blamed, at least in part, for the “year without a summer” reported in Europe and North America in 1816:

  • “Daily temperatures (especially the daily minimums) were in many cases abnormally low from late spring through early fall; frequent northwest winds brought snow and frost to northern New England and Canada, and heavy rains fell in western Europe.  Many crops failed to ripen, and the poor harvests led to famine, disease, and social distress…”3

Supporting this claim, sulfate aerosol-related climate changes were also reported after the 1991 eruption of Mount Pinatubo in the Philippines.4  An article published inScience in 2002 summarizes a decade’s worth of research on Pinatubo’s effects on the global climate, highlighting impacts far more widespread and complex than previously thought:

You can use SciFinder® or STN® to search the CAS databases for additional information about sulfur dioxide from volcanic eruptions.  If your organization is enabled to use the web version of SciFinder, you can click the links in this article to directly access details of the substances and references.


Volcanic ash vs sulfur aerosols

The primary role of volcanic sulfur aerosols in causing short-term changes in the world’s climate following some eruptions, instead of volcanic ash, was hypothesized by scientists in the early 1980’s. They based their hypothesis on the effects of several explosive eruptions in Indonesia and the world’s largest historical effusive eruption in Iceland.

Scientists studied three historical explosive eruptions of different sizes in Indonesia–Tambora (1815), Krakatau (1883), and Agung (1963). They noted that decreases in surface temperatures after the eruptions were of similar magnitude (0.18-1.3 °C). The amount of material injected into the stratosphere, however, differed greatly. By comparing the estimated amount of ash vs. sulfur injected into the stratosphere by each eruption, it was suggested that the longer residence time of sulfate aerosols, not the ash particles which fall out within a few months of an eruption, was the paramount controlling factor (Rampino and Self, 1982).

In contrast to these explosive eruptions, one of the most severe volcano-related climate effects in historical times was associated with a largely nonexplosive eruption that produced very little ash–the 1783 eruption of Laki crater-row in Iceland. The eruption lasted 8-9 months and extruded about 12.3 km3 of basaltic lava over an area of 565 km2. A bluish haze of sulfur aerosols all over Iceland destroyed most summer crops in the country; the crop failure led to the loss of 75% of all livestock and the deaths of 24% of the population (H. Sigurdsson, 1982). The bluish haze drifted east across Europe during the 1783-1784 winter, which was unusually severe.

Clearly, these examples suggested that the explosivity of an eruption and the amount of ash injected into the stratosphere are not the main factors in causing a change in Earth’s climate. Instead, scientists concluded that it must be the amount of sulfur in the erupting magma.

The eruption of El Chichon, Mexico, in 1982 conclusively demonstrated this idea was correct. The explosive eruption injected at least 8 Mt of sulfur aerosols into the atmosphere, and it was followed by a measureable cooling of parts of the Earth’s surface and a warming of the upper atmosphere. A similar-sized eruption at Mount St. Helens in 1980, however, injected only about 1 Mt of sulfur aerosols into the stratosphere. The eruption of Mount St. Helens injected much less sulfur into the atmosphere–it did not result in a noticeable cooling of the Earth’s surface. The newly launched TOMS satellite (in 1978) made it possible to measure these differences in the eruption clouds. Such direct measurements of the eruption clouds combined with surface temperatures make it possible to study the corrleation between volcanic sulfur aerosols (instead of ash) and temporary changes in the world’s climate after some volcanic eruptions.


Hazards Of Volcanic Ash

A multitude of dangerous particals and gases, such as aerosols, are carried in volcanic ash. Some of these include;

  • Carbon dioxide
  • Sulfates (sulfur dioxide)
  • Hydrochloric acid
  • Hydroflouric acid

These each have different but serious effects on human health if exposed, which will be discussed later.

In addition, volcanic ash can cause reduced visibility, and it is recommended that precautions are taken when driving.

Sources: Where Does It Come From?

Figure 1

volcanoes found all over the Earth, particularly at plate boundaries

volcanoes found all over the Earth, particularly at plate boundaries

There are volcanoes found all over the Earth, particularly at plate boundaries (see figure 1). This is due to the collision of plates, which causes uplift in the overlying crust. This uplift results in the formation of mountainous landforms; melting of the crust due to frictional heating is what creates magma, which can erupt out of these mountains when pressure gets too high.

Some of the most notable volcanic eruptions are:

  • the 1783 eruption of Mt. Laki in Iceland
    • released clouds of poisonous flourine and sulfur dioxide which killed off about 50% of the livestock population
    • that summer in Great Britain was known as “sand-summer” due to ash carried over the Atlantic
    • poisonous clouds spread over Europe, and a buildup of aerosols caused a cooling effect in the entire Northern Hemisphere
  • the 1815 eruption of Mt. Tambora in Indonesia
    • gas releases caused the Stratosphere to change drastically
    • noxious ash and poisoned rain clouds killed off vegetation
  • the 1902 eruption of Mt. Pelee in Martinique
    • spewed toxic clouds traveling at speeds of 600mph
    • largest eruption in the 20th century

For further information on volcanoes around the world, visit http://www.mapsofworld.com/major-volcanoes.htm.



  • EEA-33 emissions of sulphur oxides (SOX) have decreased by 74% between 1990 and 2011. In 2011, the most significant sectoral source of SOX emissions was ‘Energy production and distribution’ (58% of total emissions), followed by emissions occurring from ‘Energy use in industry’ (20%) and in the ‘Commercial, institutional and households’ (15%) sector.
  • The reduction in emissions since 1990 has been achieved as a result of a combination of measures, including fuel-switching in energy-related sectors away from high-sulphur solid and liquid fuels to low-sulphur fuels such as natural gas, the fitting of flue gas desulphurisation abatement technology in industrial facilities and the impact of European Union directives relating to the sulphur content of certain liquid fuels.
  • All of the EU-28 Member States have reduced their national SOX emissions below the level of the 2010 emission ceilings set in the National Emission Ceilings Directive (NECD)[1]. Emissions in 2011 for the three EEA countries having emission ceilings set under the UNECE/CLRTAP Gothenburg protocol (Liechtenstein, Norway and Switzerland) were also below the level of their respective 2010 ceilings.
  • Environmental context: Typically, sulphur dioxide is emitted when fuels or other materials containing sulphur are combusted or oxidised. It is a pollutant that contributes to acid deposition, which, in turn, can lead to changes in soil and water quality. The subsequent impacts of acid deposition can be significant, including adverse effects on aquatic ecosystems in rivers and lakes and damage to forests, crops and other vegetation. SO2 emissions also aggravate asthma conditions and can reduce lung function and inflame the respiratory tract. They also contribute, as a secondary particulate pollutant, to the formation of particulate matter in the atmosphere, an important air pollutant in terms of its adverse impact on human health. Furthermore, the formation of sulphate particles in the atmosphere following the release of SO2 results in reflection of solar radiation, which leads to net cooling of the atmosphere.
faults  sn-seafloor

faults sn-seafloor


Glacier - Helheim

Glacier – Helheim


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Making North America





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What caused the Nepal earthquake

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