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Old 09-26-2012, 09:52 PM
gdpawel gdpawel is offline
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Default Molecular Biology vs Cellular Biology

There is a growing litany of observations that call into question molecular biologist's preternatural fixation on genomic analyses. Human biology is not simple and malignantly transformed cells more complex still. Investigators who insist upon using genomic platforms to force disorderly cells into artificially ordered sub-categories, have once again been forced to admit that these oversimplifications fail to provide the needed insights for the advancement of cancer therapeutics. Those laboratories and corporations that offer "high price" genomic analyses for the selection of chemotherapy drugs should read the literature carefully as reports portend a troubling future for their current business model.

The particular sequence of DNA that an organism possesses (genotype) does not determine what bodily or behavioral form (phenotype) the organism will finally display. Among other things, environmental influences can cause the suppression of some gene functions and the activation of others. The knowledge of genomic complexity tells us that genes and parts of genes interact with other genes, as do their protein products, and the whole system is constantly being affected by internal and external environmental factors. The gene may not be central to the phenotype at all, or at least it shares the spotlight with other influences. Environmental tissue and cytoplasmic factors clearly dominate the phenotypic expression processes, which may in turn, be affected by a variety of unpredictable protein-interaction events.

This view is not shared by molecular biologists, who disagree about the precise roles of genes and other factors, but it signals many scientists discomfort with a strictly deterministic view of the role of genes in an organism's functioning. Until such time as cancer patients are selected for therapies predicted upon their own unique biology, we will confront one targeted drug after another. A better solution to this problem is to investigate the targeting agents in each individual patient's tissue culture, alone and in combination with other drugs, to gauge the likelihood that the targeting will favorably influence each patient's outcome. Functionally cytometric profiling these results in patients with a multitude type of cancers suggest this to be a highly productive direction.

Without cell function analysis, gene therapy would be beyond imagination. Tissue culture methods have made gene therapy possible. The ability to transfect cultured cells with DNA gene sequences has allowed scientists to assign functions to different genes and understand the mechanisms that activate or redress their function. The interaction between cell biology and genetics gave birth to molecular biology. The set of all malignant cells that could evolve must apply to "all" pathways of tumor cell evolution and "all" combinations of genetic and epigenetic alterations. It must be independent of any particular pathway of tumor cell evolution. The normal cellular machinery that potentially can carry out malignant behavior is encoded within the normal human genome, essentially the same for all types of cancer.

Functional cytometric profiling has allowed the identification of clinically relevant gene expression patterns which correlate with clinical drug resistance and sensitivity for different drugs in specific diseases. There is no single gene whose expression accurately predicts therapy outcome, emphasizing that cancer is a complex disease and needs to be attacked on many fronts. Functional cytometric profiling assesses the activity of a drug upon combined effect of all cellular processes (cell "population" level rather than at the "single" cell level), using combined metabolic (cell metabolism) and morphologic (structure) endpoints.

Molecular tests, such as those which identify DNA or RNA sequences or expression of individual proteins often examine only one component of a much larger, interactive process. Drug resistance and sensitivity is multifactorial. Functional cytometric profiling can show this at the cell population level, measuring the interaction of the entire genome. It visualizes directly the drug effect upon cancer cells. Photomicrographs of actual tumor cells show the condition of cells as they are received and enriched in the lab, and also the conditions of control cells post-culture.

In this visualization, the microscopic slides sometime show that the exact same identical individual culture well, shows some clusters have taken up vast amounts of the molecular drug, while right next door, clusters of the same size, same appearance, same everything haven't taken up any of the drug. Not only is this an important predictive test but it is also a unique tool that can help to identify newer and better drugs, evaluate promising drug combinations, and serve as a "gold standard" correlative model with which to develop new DNA, RNA, and protein-based tests that better predict for drug activity.

The functional cytometric profiling platform has the capacity to measure genetic and epigenetic events as a functional, real-time adjunct to static genomic and proteomic platforms.

Literature Citation:

BMJ 2007;334(suppl 1):s18 (6 January), doi:10.1136/bmj.39034.719942.94

Functional profiling with cell culture-based assays for kinase and anti-angiogenic agents Eur J Clin Invest 37 (suppl. 1):60, 2007

Functional Profiling of Human Tumors in Primary Culture: A Platform for Drug Discovery and Therapy Selection (AACR: Apr 2008-AB-1546)
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Last edited by gdpawel : 09-26-2012 at 09:54 PM.
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Old 09-29-2012, 12:46 AM
gdpawel gdpawel is offline
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Default Of Helicobacter, Cancer and the Medical Establishment

Robert Nagourney, M.D., PhD.

The 2005 Nobel Prize in physiology was awarded to Barry Marshall and Robin Warren. These two practicing physicians made the discovery that peptic ulcer disease resulted, not from excess acid production, the prevailing theory, but instead from infection with an enteric pathogen – helicobacter pylori. In 1982, Marshall and Warren identified this organism in the stomach of an ulcer patient. When they proposed the causative relationship with ulcers they were virtually laughed off the stage. First, no organism could withstand the high concentration of acid found in the stomach. Second, excess acid, not infections caused ulcers.

These investigators wrote letters to the Lancet describing their early findings, while they continued to accumulate supporting documentation that correlated the presence of these spiral shaped organisms with gastric ulceration. This led other gastroenterologists and pathologists to more closely inspect gastric biopsy specimens for the presence of these pathogens.

What seemed so obvious to Warren and Marshall met with enormous resistance. After all, the acid causation theory had been in place for almost a century. The treatment of peptic ulcer disease had spawned an industry. From Maalox to Mylanta to Tums, sodium bicarbonate and even to Coca Cola and dairy products, soothing patient’s gastric symptoms had become a cause celebré for Western medicine. Ulcer surgery in the form of the vagotomy and pyloroplasties (V&P), Bilroth1 and Bilroth2, even gastrectomies, had come to constitute the most widely practiced surgical procedures in the United States. Gastric ulcers were good for business and no one from the pharmaceutical industry, to the hospitals, or the operating surgeons, were very interested in changing that.

Frustrated by their lack of traction amongst their colleagues, Marshall consumed a flask filled with helicobacter, thereby inflicting himself with an ulcer that was confirmed at the time of an endoscopy 10 days later. Treating the ulcer successfully with antibiotics still left little impact on his doubting Thomas colleagues. But clearly some were listening. By 1987, the first triple therapy cocktail had been developed. The success of this medical treatment became increasingly irrefutable. Slowly, but surely, these two unsung heroes were recognized for their fundamental and practice-altering observations.

These two physicians represent the very best of medical scientists. They began with an observation and painstakingly worked back to an etiology. This is how most medical discoveries are made. Yet, this is not the model for today’s oncologic investigation wherein, scientists conceive of novel theories and then demand that physicians test them, rarely to good effect. These Australian physicians were not highly acclaimed academics, or senior professors. Instead, they practiced their art and unceremoniously made important observations. . Confronting immense inertia in an entrenched medical community, they stood their ground and ultimately carried the day. Aided by the invention of the fiber-optic gastroscope, they were able to prove correlations, repeat experiments and ultimately confirm their results. It took 20 years, but the Nobel committee finally recognized their contribution.

Cancer research today is inhabited by these same entrenched forces, which are convinced of certain principles and unwilling to reconsider their positions. Like the environment in which Warren and Marshall found themselves 30 years ago, the academic community eschew any idea that disrupts their hegemony However, similar paradigm shifts are occurring today in oncology: Yesterday’s gastric acid theory is akin to today’s cell proliferation model. The development of the fiberoptic endoscope in the 1980s is the equivalent of today’s advance in primary culture laboratory platforms. Marshall and Warren changed medical history. Do we really need to wait another 30 years to do the same for cancer patients?
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Old 09-29-2012, 12:48 AM
gdpawel gdpawel is offline
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Default The Discoverer

I remember reading about Drs. Barry J. Marshall and Robin Warren, both Australians, winning the Nobel Prize in medicine for proving, partly by accident, that bacteria and not stress was the main cause of painful ulcers of the stomach and intestine. Yes, a general practioner proved that ulcers are not caused by stress, spicy foods, or even cigarettes and alcohol, but rather by a bacterium. It was one of the most richly deserved Nobel Prizes in Medicine ever awarded.

Prior to the discovery, the number one surgical operation (in terms of revenue produced for the surgeons and hospitals) was the vagotomy and antrectomy. Prior to the discovery, the number one drugs were H2 receptor blockers like Tagamet and the then up and coming proton pump inhibitors like omeprazole. Single handedly, he got rid of both the number one surgical operation and the largest part of the market for the number one drugs, dealing a huge blow to both big surgery and big pharma, and making the lives of tens of millions much more pain free and enjoyable.

How many loved-ones had died of a bleeding ulcer? Today, they would have been cured permanently by a two week course of antibiotics. Oh! How having that big mug of thick black coffee without having to chase it with a half bottle of Maalox!

And the Aussie did it without any research grants and over the dead body opposition of the entire world of medicine, gastroenterology, surgery, the NIH, and the pharmaceutical industry. And, for gosh sakes, he used himself as the definitive guinea pig!

Dr. Julie Parsonnet, Professor of Medicine and of Health Research and Policy at Stanford University School of Medicine, wrote a Perspective of Marshall and Warren's achievement in the December 8, 2005 issue of the New England Journal of Medicine:

The insights that have already been gleaned are certain to be important for similar diseases in other organ systems. Indeed, the discovery of H. pylori has inspired researchers to examine previous convictions with new skepticism and to pursue more aggressively the possibility of infectious causes of myriad chronic diseases, including cancers, rheumatologic diseases, and atherosclerosis.

The clinical and scientific importance of H. pylori has exceeded everyone's wildest expectations. Yet the most important legacy of Marshall and Warren's award may have less to do with science than with the inspiration it should provide to medical practitioners worldwide. This year's (2005) Nobel Prize was a prize for clinicians. At the time of their discovery, Warren and Marshall were physicians doing their daily jobs.

They were not in the laboratory chasing after the Nobel Prize. They had no intention of being in the limelight. They had no research grants for studying ulcer disease. Rather, they happened upon something interesting, and driven by curiosity, they investigated and reported it. They proved again that we, as physicians, can make groundbreaking discoveries in the course of our clinical practices if we attend to our work with open eyes, a sense of curiosity, a desire to understand, and a willingness to pursue ideas to their completion.

In other words, these two doctors from Western Australia won the Nobel Prize by being exemplars of the great clinician described millennia ago by Hippocrates: "Many and elegant discoveries have been made . . . and others will yet be found out, if a person possessed of the proper ability, and knowing those discoveries which have been made, should proceed from them to prosecute his investigations." May Marshall and Warren inspire others to do likewise.
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Old 11-27-2012, 08:59 AM
gdpawel gdpawel is offline
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Default

In 1982, Dana Farber's Emil Frey won the Karnofsky Award. In his acceptance speech at ASCO, he decried the demise of the "discoverer" (risk taker, not so well organized, high failure rate, but big payoff when successful) because of the ascendancy of the "investigator" culture (no risk taking, well organized, exhaustive analysis of trivial hypotheses, huge payoff when successful).

The mindset of rewarding academic achievement and publication over all else. We would all like to think that organizations, government agencies, scientists, researchers, and even practitioners work together, sharing information "for the benefit of patients." However, each group has its own priorities and its own agenda. Moreover, the image of cooperation between these very different groups only gives the illusion that reform isn't needed. The present system exists to serve academic achievement and publication, but not to serve the best interests of people.

This is a perfect example of thirty-five years of the trial-and-error mind-set (empiricism) that has occupied cancer research. The cancer "investigator" culture that prizes itself on the exhaustive examination of trivial hypotheses, while eschewing support of cancer "discoverer" type research, attempting to create entirely new paradigms of cancer treatment. A dysfunctional culture that pushes tens of thousands of physicians and scientists toward the goal of finding the tiniest improvements in treatment rather than genuine breakthroughs, that rewards academic achievment and publication even though their proven "activity" has little to do with curing cancer.

If you ever get a chance to watch a 1940 movie, starring Edward G Robinson, called "Dr. Ehrlich's Magic Bullet," watch it. Ehrlich is one of the very first pioneers of chemotherapy. Ehrlich was the discoverer of the first effective treatment for syphillis. The movie is a very accurate foreshadowing of chemotherapy of cancer, one century later. Ehrlich's treatment was very toxic, but it worked, sometimes miraculously. This work inspired Alexander Fleming, who later discovered penicillin, which was a true magic bullet (Ehrlich's bullet was a wondrous bullet, but it wasn't magic).

The belief that research grants and the NIH will be our salvation can be so off base. Here and there around the world, there are decisions still being made by folks who aren't in the pockets of American Big Business. Individual intelligence, integrity and curiosity. Maybe there is a glimmer of hope?
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Last edited by gdpawel : 02-23-2014 at 07:52 PM. Reason: spelling error
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Old 04-06-2013, 05:10 PM
gdpawel gdpawel is offline
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Default Molecular genomics provides a veneer of information

Robert Nagourney, M.D.

Molecular genomics provides a veneer of information that has seduced our best scientists into the false belief that they know more than they really do.

We jumped off the bio-chemistry track and dove headlong into the gene. Today, we realize what an error this may have been. Over the past several years, genomics publications are slowly being replaced by metabolomics publications.

Metabolomics is the study of human metabolism at the level of enzyme and bio-chemical reaction. Scientists are slowly becoming scientists again.

Recognizing the shortcomings of information for information's sake, the molecular biologists, who thought they could leave their chemistry books behind and never open them again, are scrambling to relearn the basics.

It turns out the cancer isn't - in the truest sense of the word - a genetic disease. It is, instead a metabolic disorder. Cancer cells, like all members of multicellular organisms, function under the controlling influence of the organism as a whole.

The social contract of cellular biology dictates that each individual cell in a multicellular organism relinquishes its autonomy in return for the provision of nutrients, water, oxygen, protection and mobility. But cancer cells break the contract and escape the regulatory control so essential for orderly multicellular existence.

Veiwing cancer in this light, where we once saw cells as proteins and lipids driven by the information contained in their DNA, and defined them by their capacity for self-replication, we must now view these microscopic entities as complex biosystems that retrieve information from their DNA they way computers draw upon their disk drives.

It was our ill-conceived love affair with genomics that led us to the failed war on cancer, and it will be our renewed appreciation of biochemistry that digs us out of this mess. Scientists today are realizing that they must return to the fundamental principles of physiology, chemistry, and biology to unravel the mysteries of cancer.

Reference: Outliving Cancer, Robert A. Nagourney, Basic Health Publications, Inc. 2013

Systems Biology Is The Future Of Medical Research

[url]http://cancerfocus.org/forum/showthread.php?t=3473
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Old 10-11-2013, 10:12 AM
gdpawel gdpawel is offline
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Default Cancer is not a genomic disorder, but a metabolic disorder

Robert A. Nagourney, M.D.

A study conducted by Canadian investigators and reported in the September 1, 2013 issue of the Journal of Clinical Oncology examined the impact of Metformin use on mortality in men with diabetes and prostate cancer (Margel D. Urbach DR., Lipscombe LL, Metformin Use and All-Cause and Prostate Cancer-Specific Mortality Among Men with Diabetes, Journal of Clinical Oncology, volume 31, #25, pgs 3069-3075, 2013). The investigators examined 3837 patient with a median age of 75 years. They conducted a retrospective analysis examining the Ontario Province heath care records. The intent was to examine duration of exposure to Metformin as a diabetes management in patients with prostate cancer to assess the impact on all-cause and prostate cancer-specific mortality.

The results are impressive and instructive. There was a significant decrease in the risk of prostate cancer-specific and all-cause mortality, which related to the dose and duration of exposure to Metformin. The adjusted hazard ratio for the study of 0.76 indicates that there is a 24% reduction in mortality for prostate cancer-specific events with the use of Metformin. This study was not perfect, as it was retrospective, there was no randomization and it was impossible to control for all other variables such as exercise, smoking history and clinical parameters of prostate cancer. Nonetheless, there is a clear and important trend toward reduced prostate cancer and even overall mortality. This is but one of a series of clinical studies that have examined the impact of Metformin upon not only prostate cancer but also breast cancer. Much of this work was originally pioneered by Dr. Michael Pollack from McGill University in Montreal.

Metformin and the closely related Phenformin are members of the class of drugs known as biguanides. While the exact mode of action of the biguanides is not fully understood, they are known to disrupt mitochondrial respiration at complex I. This upregulates an enzyme known as adenosine monophosphate kinase (AMPK) thereby altering energy metabolism within the cell and down regulating mTOR. In diabetics, this drives down blood glucose to control the disease. However, in cancer patients, a profound effect is observed that suppresses synthetic pathways necessary for energy metabolism, cellular survival and cellular proliferation. These effects appear responsible for the impact upon prostate cancer. Interestingly, these drugs are more effective in controlling already transformed cells and less effective in the prevention of cancer. This is consistent with the observation that malignantly transformed cells change their state of metabolism.

This article is interesting on many levels. The first and most obvious is that this relatively inexpensive and well-tolerated drug can have an impact on prostate cancer.

Secondly, these effects appear to cross the lines of different cancer types, such that breast cancer and other forms of cancer might also be successfully treated.

The third note of interest shows that even patients without diabetes can tolerate Metformin, suggesting this as an adjunct to many different treatments. Finally and most importantly this represents the new and important recognition that cancer is not a genomic disorder, but a metabolic disorder. Cancer may utilize normal genetic elements to its own advantage. AMP kinase, LKB1 and mTOR are not unique to cancer, but instead, are found in every cell. These normal proteins are simply altered in their function in malignantly transformed cells. Metformin is one of what will soon be a large number of metabolomic agents entering the clinical arena as cancer research moves from the nucleus to the mitochondrion.

[url]http://jco.ascopubs.org/content/31/25/3069.abstract
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Old 10-17-2013, 12:46 AM
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Default Cell Biology

Robert A. Nagourney, M.D.
Medical Director
Rational Therapeutics
Long Beach, CA

On October 7, 2013, in Stockholm, Sweden, the Nobel Committee announced the winners of the Nobel Prize for Medicine and Physiology – two Americans and one German, all now located at institutions in the US. The discovery for which these three investigators share the prize involves their work over three decades studying the transport, packaging and trafficking of cellular proteins.

All cells must communicate and maintain their identity. To do so cells have developed intricate systems whereby neurotransmitters, proteins, hormones, and other species are encapsulated in small vesicles. These vesicles may be utilized to extrude materials into the extracellular domain or may store materials within the cell for later use. Working in model systems including yeast cells, these investigators showed the intricacy of cellular physiology associated with micro-vesicular function.

What makes these investigators’ work so interesting is that it is principally the study of cellular physiology, or what we call cell biology. While many breakthroughs and observations today reflect discoveries at the level of DNA, RNA and the genome, these investigators have pioneered protein kinetics and physiology. What is so exciting about this Nobel Prize is that it returns attention to the intricacies of cellular function at the level of phenotype. Protein biology represents the final common pathway from blueprint (DNA) to function. While genes that are detected within the nucleus (the purview of genomic analyses and many recent Nobel prizes) may or may not ultimately be expressed, depending upon splice variants, DNA methylation, histone acetylation, small interfering RNAs and non-coding DNAs among other phenomena, functional proteins are the active end-product and do very much exist.

We now recognize that cellular signaling, misfolded protein response, autophagy and apoptotic responses are tightly bound together. Among the most toxic phenomena for a cell is the misfolded protein signal, a signal that occurs far from the gene. This represents the target of the newest classes of drugs known as proteasome inhibitors and heat shock protein inhibitors, which function within the cytoplasm, not the nucleus.

It is exciting to imagine a day when physiologist, biochemist, enzymologist, physical chemist, and protein chemist regain their position as leaders in cancer research.

N.B: It should not go unmentioned that the assay offered by Rational Therapeutics is based on cellular function.
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Old 12-05-2013, 05:16 PM
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Default Methylation Signaling Controls Cancer Growth

A study led by researchers at Boston University School of Medicine (BUSM) demonstrates a new mechanism involving a signaling protein and its receptor that may block the formation of new blood vessels and cancer growth. The findings are published in the December issue of Science Signaling.

Angiogenesis creates new blood vessels in a process that can lead to the onset and progression of several diseases such as cancer and age-related macular degeneration.

Vascular endothelial growth factor (VEGF) is a signaling protein produced by damaged cells, which binds to one of its receptors VEGFR-2, located on the surface of blood vessel cells. Once VEGF is bound to its receptor, it is activated and sends a biochemical signal to the inside of the blood vessel cell to initiate angiogenesis. There are currently multiple Federal Drug Administration-approved medications that target this process. However these medications are limited by insufficient efficacy and the development of resistance.

The researchers demonstrated that a biochemical process called methylation, which can regulate gene expression, also affects VEGFR-2, and this can lead to angiogenesis. Using multiple methods, the researchers were able to interfere with the methylation process of VEGFR-2 and subsequently block angiogenesis and tumor growth.

“The study points to the methylation of VEGFR-2 as an exciting, yet unexplored drug target for cancer and ocular angiogenesis, ushering in a new paradigm in anti-angiogenesis therapy,” said Nader Rahimi, PhD, associate professor of pathology, BUSM, who served as the study’s senior investigator.

Source: Boston University Medical Center (2013, November 28). Methylation signaling controls cancer growth. ScienceDaily. December 2, 2013
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Old 12-05-2013, 05:18 PM
gdpawel gdpawel is offline
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Default Angiogenesis and cancer growth controlled by methylation signaling?

VEGF Inhibition Associated with Multiple Signaling Processes

There is much research devoted to measuring gene expression. A key challenge is differentiating changes in gene expression caused by changes in primary DNA sequence, versus those caused principally by modifications to histones and methylation of DNA.

Understanding the structural and functional relationships of cells and tissues is critical to advancements in key research disciplines, including molecular biology, genetics, reproductive function, immunology, cancer and neurobiology.

Now that the human genome has more or less been sequenced and the technologies developed to analyze numerous genes and gene products simultaneously (microarray technologies), the focus of scientific query will switch from simply identifying the gene/protein to investigating the function(s) and inter-relationships between specific gene products and specific cellular activities (drug selection).

There are small molecule tyrosine kinase inhibitors with broad specificity that target all VEGF receptors (VEGFR), the platelet-derived growth factor receptor, and c-KIT. They are multi-VEGFR inhibitors designed to block angiogenesis and lymphangiogenesis by binding the intracellular kinase domain of all three VEGFRs, VEGFR-1, (Fit-1), VEGFR-2 (KDR/Flk-1), and VEGFR-3 (Fit-4).

You need to target the activity of all known receptors that bind VEGF, inhibiting the formation of new blood vessels (angiogenesis). In some cases, these drugs kill tumor cells without killing microvascular cells in the same time frame. In other cases they kill microvascular cells without killing tumor cells. In yet other cases they kill both types of cells or neither type of cells. The ability of these agents to kill tumor and/or microvascular cells in the same tumor specimen is highly variable among the different agents.

The ability to monitor cell "function" provides scientists with a vital method to characterize and compare activity of cells. Programmed cell death, or apoptosis, is critical in embryonic development, cancer formation, and lowering inflammatory response and is often used to determine if cells are functioning properly.
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