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Posts Tagged ‘Transcriptomics’


Larry H Bernstein, MD, FCAP, Reporter and Curator

http://pharmaceyticalinnovation.com/7/10/2014/A new relationship identified in preterm stress and development of autism or schizophrenia/

 

This is a fascinating study.  It is of considerable interest because it deals with several items that need to be addressed with respect to neurodevelopmental disruptive disorders.  It leaves open some aspects that are known, but not subject to investigation in the experiments.  Then there is also no reporting of some associations that are known at the time of deveopment of these disorders – autism spectrum, and schizophrenia.  Of course, I don’t know how it would be possible to also look at prediction of a possible relationship to later development of mood disorders.

  1. The placenta functions as an endocrine organ in the conversion of androsteinedione to testosterone during pregnancy, which is delivered to the fetus.
  2. The conversion is by a known enzymatic pathway – and there is a sex difference in the depression of testosterone in males, females not affected.
  3. There is a greater susceptibility of males to autism and schizophrenia than of females, which I as reader, had not known, but if this is true, it would lend some credence to a biological advantage to protect the females of animal species, and might raise some interest into what relationship it has to protecting multitasking for females.
  4. It is well known that the twin studies that have been carried out determined that in identical twins, there is discordance as a rule.  Those studies are old, and they did not examine whether the other identical twin might be anywhere on the autism spectrum disorder (not then termed “spectrum”.
  5. However, there is a clear effect of stress on “gene expression”, and in this case we are looking at enzymation suppression at the placental level affecting trascriptional activity in the male fetus.  The same genetic signature exists in the male genetic profile, so we are not looking at a clear somatic mutation in this study.
  6. There is also much less specific an association with the MTHFR gene mutation at either one or two loci. This would have to be looked at as a possible separate post translational somatic mutation.
  7. Whether there is another component expressed later in the function of the zinc metalloproteinase under stress in the affected subject is worth considering, but can’t be commented on with respect to the study.

Penn Team Links Placental Marker of Prenatal Stress to Neurodevelopmental Problems 

By Ilene Schneider          July 8, 2014

When a woman experiences a stressful event early in pregnancy, the risk that her child will develop autism spectrum disorders or schizophrenia increases. The way in which maternal stress is transmitted to the brain of the developing fetus, leading to these problems in neurodevelopment, is poorly understood.

New findings by University of Pennsylvania School of Veterinary Medicine scientists suggest that an enzyme found in the placenta is likely playing an important role. This enzyme, O-linked-N-acetylglucosamine transferase, or OGT, translates maternal stress into a reprogramming signal for the brain before birth. The study was supported by the National Institute of Mental Health.

“By manipulating this one gene, we were able to recapitulate many aspects of early prenatal stress,” said Tracy L. Bale, senior author on the paper and a professor in the Department of Animal Biology at Penn Vet. “OGT seems to be serving a role as the ‘canary in the coal mine,’ offering a readout of mom’s stress to change the baby’s developing brain. Bale, who also holds an appointment in the Department of Psychiatry, co-authored tha paper with postdoctoral researcher Christopher L. Howerton, for PNAS.

OGT is known to play a role in gene expression through chromatin remodeling, a process that makes some genes more or less available to be converted into proteins. In a study published last year in PNAS, Bale’s lab found that placentas from male mice pups had lower levels of OGT than those from female pups, and placentas from mothers that had been exposed to stress early in gestation had lower overall levels of OGT than placentas from the mothers’ unstressed counterparts.

“People think that the placenta only serves to promote blood flow between a mom and her baby, but that’s really not all it’s doing,” Bale said. “It’s a very dynamic endocrine tissue and it’s sex-specific, and we’ve shown that tampering with it can dramatically affect a baby’s developing brain.”

To elucidate how reduced levels of OGT might be transmitting signals through the placenta to a fetus, Bale and Howerton bred mice that partially or fully lacked OGT in the placenta. They then compared these transgenic mice to animals that had been subjected to mild stressors during early gestation, such as predator odor, unfamiliar objects or unusual noises, during the first week of their pregnancies.

The researchers performed a genome-wide search for genes that were affected by the altered levels of OGT and were also affected by exposure to early prenatal stress using a specific activational histone mark and found a broad swath of common gene expression patterns.

They chose to focus on one particular differentially regulated gene called Hsd17b3, which encodes an enzyme that converts androstenedione, a steroid hormone, to testosterone. The researchers found this gene to be particularly interesting in part because neurodevelopmental disorders such as autism and schizophrenia have strong gender biases, where they either predominantly affect males or present earlier in males.

Placentas associated with male mice pups born to stressed mothers had reduced levels of the enzyme Hsd17b3, and, as a result, had higher levels of androstenedione and lower levels of testosterone than normal mice.

“This could mean that, with early prenatal stress, males have less masculinization,” Bale said. “This is important because autism tends to be thought of as the brain in a hypermasculinized state, and schizophrenia is thought of as a hypomasculinized state. It makes sense that there is something about this process of testosterone synthesis that is being disrupted.”

Furthermore, the mice born to mothers with disrupted OGT looked like the offspring of stressed mothers in other ways. Although they were born at a normal weight, their growth slowed at weaning. Their body weight as adults was 10 to 20 percent lower than control mice.

Because of the key role that that the hypothalamus plays in controlling growth and many other critical survival functions, the Penn Vet researchers then screened the mouse genome for genes with differential expression in the hypothalamus, comparing normal mice, mice with reduced OGT and mice born to stressed mothers.

They identified several gene sets related to the structure and function of mitochrondria, the powerhouses of cells that are responsible for producing energy. And indeed, when compared by an enzymatic assay that examines mitochondria biogenesis, both the mice born to stressed mothers and mice born to mothers with reduced OGT had dramatically reduced mitochondrial function in their hypothalamus compared to normal mice. These studies were done in collaboration with Narayan Avadhani’s lab at Penn Vet. Such reduced function could explain why the growth patterns of mice appeared similar until weaning, at which point energy demands go up.

“If you have a really bad furnace you might be okay if temperatures are mild,” Bale said. “But, if it’s very cold, it can’t meet demand. It could be the same for these mice. If you’re in a litter close to your siblings and mom, you don’t need to produce a lot of heat, but once you wean you have an extra demand for producing heat. They’re just not keeping up.”

Bale points out that mitochondrial dysfunction in the brain has been reported in both schizophrenia and autism patients. In future work, Bale hopes to identify a suite of maternal plasma stress biomarkers that could signal an increased risk of neurodevelopmental disease for the baby.

“With that kind of a signature, we’d have a way to detect at-risk pregnancies and think about ways to intervene much earlier than waiting to look at the term placenta,” she said.

 

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Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/7/7/2014/Bzzz! Are fruitflies like us?

We are following closely the developments in genomics that have had a progression since the Double-Helix dogma served the Nobel Prize to Watson and Crick, and that achievement led to the completion of a provisional Human Genome at the birth of the 21st century.  Since then there has been exploration of cellular regulation, signaling pathways, and protein-protein as well as protein membrane interactions in eukaryotes.  But we can go further back prior to the double-helix and remind ourselves of the huge contributions that led up to the double helix.  This was a time of great research that set the tone for what is now called molecular biology.  We associate the work with the genetic studies of Thomas Hunt Morgan on the fruit fly.  There may yet be a new chapter that is stradling the gap between DNA, RNA and transcription turning toward a deeper understanding of gene expression and organ specificities.  Is it a new beginning?  There is certainly going to be a deeper understanding of the several roles of RNA as well as proteins.

 

The Gateway Opens

THOMAS HUNT MORGAN AT COLUMBIA UNIVERSITY
Genes, Chromosomes, and the Origins of Modern Biology
Eric R. Kandel, MD
2000 recipent of Nobel Prize in Medicine

University Professor & Kavli Professor of Brain Science,
Co-director, Mind Brain Behavior Initiative
Director, The Kavli Institute for Brain Science
Senior Investigator, Howard Hughes Medical Institute
Columnia University

When future historians turn to examine the major intellectual accomplishments of the twentieth century, they will undoubtedly give a special place to the extraordinary achievements in biology, achievements that have revolutionized our understanding of life’s processes and of disease. Important intimations of what was to happen in biology were already apparent in the second half of the nineteenth century. Darwin had delineated the evolution of animal species, Mendel had discovered some basic rules about inheritance, and Weissman, Roux, Driesch, de Vries, and other embryologists were beginning to decipher how an organism develops from a single cell. What was lacking at the end of the nineteenth century, however, was an overarching sense of how these bold advances were related to one another.

The insight that unified these three fields- heredity, evolution, and development- and set biology on the course toward its current success came only at the beginning of the twentieth century. It derived from the discovery that the gene, localized to specific positions on the chromosome, was at once the unit of Mendelian heredity, the driving force for Darwinian evolution, and the control switch for development. This remarkable discovery can be traced directly to one person and to one institution: Thomas Hunt Morgan and Columbia University. Much as Darwin’s insights into the evolution of animal species first gave coherence to nineteenth-century biology as a descriptive science, Morgan’s findings about genes and their location on chromosomes helped transform biology into an experimental science.

aware that abstract thinking, remote from, and even antagonistic to the study of nature, leads easily into dogma, taboos and fettering of free thinking because it does not carry its own corrective, the recourse to factual evidence. The scientist, therefore, with all respect for the many facets of the human mind, is more impressed by the revolutions in thinking brought about by great factual discoveries, which by their very nature lead to generalizations which change at once the outlook of many, if not all, lines of thought.”

. . . . the rise and development of genetics to mature age is another instance of an all-comprising and all-affecting generalization based upon an overwhelming body of integrated facts, . . . [and] will rank in the history of science with such other great events ..”

Richard B. Goldschmidt, The Impact of Genetics Upon Science (1950)

Even more important, Morgan’s discoveries made it possible to address a series of questions regarding the function and structure of genes. What is their chemical nature? How do genes duplicate themselves? What goes wrong when genes mutate? How do genes provide the basis for understanding genetic disease? How do genes determine the properties of cells, the development of organisms, and the course of evolution?

Thomas Hunt Morgan

Thomas Hunt Morgan

 

Morgan

Morgan

 

Eric Kandel

Eric Kandel

 

New Study of Fruit Fly Genome Reveals Complexity of RNA and Provides a Model for Studying Mechanisms for Hereditary Diseases in Humans

July 7, 2014

This investigation of the fruit fly’s transcriptome—the complete collection of the genome’s RNA—unearthed thousands of new genes, transcripts, and proteins

Scientists have teased another level of information out of the genome. This time, the new insights were developed from studies of the fruit fly’s transcriptome. This knowledge will give pathologists another channel of information that may be useful in developing assays to support more precise diagnosis and therapeutic decisions.

The findings were published in a recent issue of Nature. The study focused on the transcriptome—a complete collection of the genome’s RNA—of the common fruit fly−Drosophila melangogaster.

Why Studies of the Fruit Fly Are Useful

The fruit fly has been used as a genetics model to study human genetics for more than a 100 years. Not only are they easy to care for and work with, but they share 75% of the same genes as humans. Today, the fruit fly genome has emerged as a critical tool tor understanding human biology and disease, by providing an understanding of genes and life processes that are conserved over extensive evolutionary changes.

The research consortium included 41 researchers from 11 universities and institutes that are members of the National Human Genome Research Institute’s Model Organism Encyclopedia of DNA Elements, called modENCODE for short. This project used state-of-the-art gene sequencing to study all of the expressed RNAs produced by a genome in greater detail than ever before accomplished.

RNA Sequenced in Diverse Tissues at Different Stages of Development

RNA was sequenced at different stages of development, in diverse tissues, in cells growing in culture, and in flies stressed by environmental contaminants, stated a IU press release issued by Indiana University Bloomington (IUB).

The modENCODE study revealed that the fruit fly genome is far more complex than previously suspected. These new findings suggest that this also may be true for the genomes of higher animals. Specifically, the researchers found that:

  • a small set of genes in the nervous system is responsible for much of the complexity;
  • long regulatory and antisense RNA (asRNA), a single-stranded RNA complementary to a messenger RNA transcribed within a cell, are prominent during gonadal development;
  • splicing factors, proteins involved in controlling maturation of RNAs, are themselves spliced in complex ways; and,
  • the fruit fly transcriptome undergoes major changes in response toenvironmental stressors.

How Study of Fruit Fly RNA Benefits Human Genome Research

“The modENCODE work is intended to provide a new baseline for research using Drosophila,” declared Peter Cherbas, Ph.D., an IUB Professor Emeritus of Biology and one of 10 IUB researchers who served as co-authors of the study. “The goal is to provide researchers working on particular processes with much of the detailed background information they would otherwise need to collect for themselves.

Click here for photo
Peter Cherbas, Ph.D. (pictured), Professor Emeritus of Biology at Indiana University Bloomington, says that the modENCORE study of the fruit fly’s complete RNA answered a lot of questions about the genome of organisms, but raised even more questions that science will want to answer. (Photo copyright Indiana University Bloomington.)

“As usual in science, we’ve answered a number of questions and raised even more,” observed Cherbas. “For example, we identified 1,468 new genes, of which 536 were found to reside in previously uncharacterized gene-free zones.”

“We think these results could influence gene regulation research in all animals,” added Thom Kaufman, Ph.D., IUB Distinguished Professor of Biology who also co-authored the study. “This exhaustive study also identified a number of phenomena previously reported only in mammals, and that alone is really telling about the versatility of Drosophila melanogaster as a model organism. The new work provides a number of new potential uses for this powerful model system,” he stressed.

Click here for photo
Thom Kaufman, Ph.D. (pictured), Indiana University Bloomington Distinguished Professor of Biology, says that the modENCORE study provides a powerful model for studying gene regulation in all organisms. (Photo copyright Indiana University Bloomington.)

Impact of Environmental Stressors on Gene Expression

Both Kaufman and Cherbas cited perturbation experiments that identified genes and transcripts. The new genes were identified after subjecting adult fruit flies to heat and cold shock, then exposing them to heavy metals, caffeine and the herbicide paraquat. Fruit fly larvae were treated with heavy metals, caffeine, ethanol, or the insecticide rotenone.

These environmental stressors generated small changes in the expression level of thousands of genes. One treatment experiment resulted in four newly modeled genes being expressed altogether differently, noted the researchers. Perturbation experiments, in fact, revealed a total of 5,249 transcript models for 811 genes.

In fact, the findings from these perturbation experiments mirror similar findings made following the 2010 British Petroleum Deepwater Horizon oil spill in the Gulf of Mexico. Researchers studying the impact on marsh fishes found that, similar to the fruit flies, these fish responded to chronic hydrocarbon exposure with a number of expressions beyond the heat shock pathway. These expressions included the down regulation of genes encoding eggshell and yolk proteins.

The response overlap between species indicates that the modENCODE consortium may have identified a conserved metazoan [animal] stress response that enhances metabolism and suppresses genes involved in reproduction.

What This Means for Pathologists and Laboratory Professionals

This study is significant for pathologists and medical laboratory professionals because it peels away another layer of information encoded in DNA and RNA. The findings of this study also show how genomic knowledge is moving to the next level in the quest to understand the origins of disease.
—by Patricia Kirk

 

Study of complete RNA collection of fruit fly uncovers unprecedented complexity

IU plays key role in consortium; 1,468 new genes discovered March 17, 2014  

BLOOMINGTON, Ind. — Scientists from Indiana University are part of a consortium that has described the transcriptome of the fruit fly Drosophila melanogaster in unprecedented detail, identifying thousands of new genes, transcripts and proteins.

In the new work, published Sunday in the journal Nature, scientists studied the transcriptome — the complete collection of RNAs produced by a genome — at different stages of development, in diverse tissues, in cells growing in culture, and in flies stressed by environmental contaminants. To do so, they used contemporary sequencing technology to sequence all of the expressed RNAs in greater detail than ever before possible.

The paper shows that the Drosophila genome is far more complex than previously suspected and suggests that the same will be true of the genomes of other higher organisms. The paper also reports a number of novel, particular results: that a small set of genes used in the nervous system are responsible for a disproportionate level of complexity; that long regulatory and so-called “antisense” RNAs are especially prominent during gonadal development; that “splicing factors” (proteins that control the maturation of RNAs by splicing) are themselves spliced in complex ways; and that the Drosophila transcriptome undergoes large and interesting changes in response to environmental stresses.

The importance of Drosophila melanogaster as a model system cannot be overstated. Using it, the mechanisms of heredity were worked out about 100 years ago. Today, as biologists have developed increasing appreciation of how well genes and critical life processes are conserved over long evolutionary distances, flies have emerged as critical tools for understanding human biology and disease. Drosophila research is an area that has long had associations with IU, beginning with Nobel Laureate Herman J. Muller.

IU has 10 co-authors on the paper from the IU Bloomington College of Arts and Sciences’ Department of Biology and the university’s Center for Genomics and Bioinformatics. They are included among the 41 co-authors from 11 universities and institutes that are members of the National Human Genome Research Institute’s Model Organism Encyclopedia of DNA Elements project, or modENCODE. Among the IU co-authors are Professor Emeritus of Biology Peter Cherbas, who helped manage the expansive project, and Distinguished Professor of Biology Thom Kaufman, who helped oversee design of the project and the production of biological samples.

“The modENCODE work is intended to provide a new baseline for research using Drosophila,” Cherbas said. “The goal is to provide researchers working on particular processes with much of the detailed background information they would otherwise need to collect for themselves.

“As usual in science, we’ve answered a number of questions and raised even more. For example, we identified 1,468 new genes, of which 536 were found to reside in previously uncharacterized gene-free zones.”

“We think these results could influence gene regulation research in all animals,” Kaufman said. “This exhaustive study also identified a number of phenomena previously reported only in mammals, and that alone is really telling about the versatility of Drosophila melanogaster as a model organism. The new work provides a number of new potential uses for this powerful model system.”

An example they pointed to was the perturbation experiments that identified new genes and transcripts. New genes were identified in experiments where adults were challenged with heat shock, cold shock, exposure to heavy metals, the drug caffeine and the herbicide paraquat, while larvae were treated with heavy metals, caffeine, ethanol or the insecticide rotenone.

Those environmental stresses resulted in small changes in expression level at thousands of genes; and in one treatment, four newly modeled genes were expressed altogether differently. In total, 5,249 transcript models for 811 genes were revealed only under perturbed conditions.

As did the flies in this new research, scientists who studied the Deepwater Horizon incident in the Gulf of Mexico found that marsh fishes responding to chronic hydrocarbon exposure had a number of expressional responses beyond the heat shock pathway, including the down regulation of genes encoding eggshell and yolk proteins as did the flies. To see this response overlap across phyla means the consortium may have identified a conserved metazoan stress response involving enhanced metabolism and the suppression of genes involved in reproduction.

Indiana University co-authors with Cherbas and Kaufman were co-first author Robert Eisman, Justen Andrews, Lucy Cherbas, Brian D. Eads, David Miller, Keithanne Mockaitis, Johnny Roberts and Dayu Zhang. All were associated with the Department of Biology and/or the Center for Genomics and Bioinformatics.

“Diversity and dynamics of the Drosophila transcriptome,” published March 16 in the journal Nature, also included 31 other co-authors whose affiliations were with the University of California, Berkeley; Lawrence Berkeley National Laboratory; University of Connecticut Health Center; Cold Spring Harbor Laboratory; Sloan-Kettering Institute; National Institute of Diabetes and Digestive and Kidney Diseases; RIKEN Yokohama Institute (Japan); Harvard Medical School; and Howard Hughes Medical Institute.

Antisense RNA

From Wikipedia, the free encyclopedia

Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Some authors have used the term micRNA (mRNA-interfering complementary RNA) to refer to these RNAs but it is not widely used.[1]

Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery.[2] [3] This effect is therefore stoichiometric. An example of naturally occurring mRNA antisense mechanism is the hok/sok system of the E. coli R1 plasmid. Antisense RNA has long been thought of as a promising technique for disease therapy; the only such case to have reached the market is the drug fomivirsen. One commentator has characterized antisense RNA as one of “dozens of technologies that are gorgeous in concept, but exasperating in [commercialization]”.[4] Generally, antisense RNA still lack effective design, biological activity, and efficient route of administration.[5]

Historically, the effects of antisense RNA have often been confused with the effects of RNA interference (RNAi), a related process in which double-stranded RNA fragments called small interfering RNAs trigger catalytically mediated gene silencing, most typically by targeting the RNA-induced silencing complex (RISC) to bind to and degrade the mRNA. Attempts to genetically engineer transgenic plants to express antisense RNA instead activate the RNAi pathway, although the processes result in differing magnitudes of the same downstream effect; gene silencing. Well-known examples include the Flavr Savr tomato and two cultivars of ringspot-resistant papaya.[6][7]

Transcription of longer cis-antisense transcripts is a common phenomenon in the mammalian transcriptome.[8] Although the function of some cases have been described, such as the Zeb2/Sip1 antisense RNA, no general function has been elucidated. In the case of Zeb2/Sip1,[9] the antisense noncoding RNA is opposite the 5′ splice site of an intron in the 5’UTR of the Zeb2 mRNA. Expression of the antisense ncRNA prevents splicing of an intron that contains a ribosome entry site necessary for efficient expression of the Zeb2 protein. Transcription of long antisense ncRNAs is often concordant with the associated protein-coding gene,[10] but more detailed studies have revealed that the relative expression patterns of the mRNA and antisense ncRNA are complex.[11][12]

 

 

Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase

Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase

 

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Structure of the N-terminal domain of the yeast Hsp90 chaperone

 

 

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?


Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

The evolution of progress we have achieved in cancer research, diagnosis, and therapeutics has  originated from an emergence of scientific disciplines and the focus on cancer has been recent. We can imagine this from a historical perspective with respect to two observations. The first is that the oldest concepts of medicine lie with the anatomic dissection of animals and the repeated recurrence of war, pestilence, and plague throughout the middle ages, and including the renaissance.  In the awakening, architecture, arts, music, math, architecture and science that accompanied the invention of printing blossomed, a unique collaboration of individuals working in disparate disciplines occurred, and those who were privileged received an education, which led to exploration, and with it, colonialism.  This also led to the need to increasingly, if not without reprisal, questioning long-held church doctrines.

It was in Vienna that Rokitansky developed the discipline of pathology, and his student Semelweis identified an association between then unknown infection and childbirth fever. The extraordinary accomplishments of John Hunter in anatomy and surgery came during the twelve years war, and his student, Edward Jenner, observed the association between cowpox and smallpox resistance. The development of a nursing profession is associated with the work of Florence Nightengale during the Crimean War (at the same time as Leo Tolstoy). These events preceded the work of Pasteur, Metchnikoff, and Koch in developing a germ theory, although Semelweis had committed suicide by infecting himself with syphilis. The first decade of the Nobel Prize was dominated by discoveries in infectious disease and public health (Ronald Ross, Walter Reed) and we know that the Civil War in America saw an epidemic of Yellow Fever, and the Armed Services Medical Museum was endowed with a large repository of osteomyelitis specimens. We also recall that the Russian physician and playwriter, Anton Checkov, wrote about the conditions in prison camps.

But the pharmacopeia was about to open with the discoveries of insulin, antibiotics, vitamins, thyroid action (Mayo brothers pioneered thyroid surgery in the thyroid iodine-deficient midwest), and pitutitary and sex hormones (isolatation, crystal structure, and synthesis years later), and Karl Landsteiner’s discovery of red cell antigenic groups (but he also pioneered in discoveries in meningitis and poliomyelitis, and conceived of the term hapten) with the introduction of transfusion therapy that would lead to transplantation medicine.  The next phase would be heralded by the discovery of cancer, which was highlighted by the identification of leukemia by Rudolph Virchow, who cautioned about the limitations of microscopy. This period is highlighted by the classic work – “Microbe Hunters”.

John Hunter

John Hunter

Walter Reed

Walter Reed

Robert Koch

Robert Koch

goldberger 1916 Pellagra

goldberger 1916 Pellagra

Louis Pasteur

Louis Pasteur

A multidisciplinary approach has led us to a unique multidisciplinary or systems view of cancer, with different fields of study offering their unique expertise, contributions, and viewpoints on the etiology of cancer.  Diverse fields in immunology, biology, biochemistry, toxicology, molecular biology, virology, mathematics, social activism and policy, and engineering have made such important contributions to our understanding of cancer, that without cooperation among these diverse fields our knowledge of cancer would never had evolved as it has. In a series of posts “Heroes in Medical Research:” the work of researchers are highlighted as examples of how disparate scientific disciplines converged to produce seminal discoveries which propelled the cancer field, although, at the time, they seemed like serendipitous findings.  In the post Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin (Translating Basic Research to the Clinic) discusses the seminal yet serendipitous discoveries by bacteriologist Dr. Barnett Rosenberg, which eventually led to the development of cisplatin, a staple of many chemotherapeutic regimens. Molecular biologist Dr. Robert Ting, working with soon-to-be Nobel Laureate virologist Dr. James Gallo on AIDS research and the associated Karposi’s sarcoma identified one of the first retroviral oncogenes, revolutionizing previous held misconceptions of the origins of cancer (described in Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer).   Located here will be a MONTAGE of PHOTOS of PEOPLE who made seminal discoveries and contributions in every field to cancer   Each of these paths of discovery in cancer research have led to the unique strategies of cancer therapeutics and detection for the purpose of reducing the burden of human cancer.  However, we must recall that this work has come at great cost, while it is indeed cause for celebration. The current failure rate of clinical trials at over 70 percent, has been a cause for disappointment, and has led to serious reconsideration of how we can proceed with greater success. The result of the evolution of the cancer field is evident in the many parts and chapters of this ebook.  Volume 4 contains chapters that are in a predetermined order:

  1. The concepts of neoplasm, malignancy, carcinogenesis,  and metastatic potential, which encompass:

(a)     How cancer cells bathed in an oxygen rich environment rely on anaerobic glycolysis for energy, and the secondary consequences of cachexia and sarcopenia associated with progression

invasion

invasion

ARTS protein and cancer

ARTS protein and cancer

Glycolysis

Glycolysis

Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue

akip1-expression-modulates-mitochondrial-function

akip1-expression-modulates-mitochondrial-function

(b)     How advances in genetic analysis, molecular and cellular biology, metabolomics have expanded our basic knowledge of the mechanisms which are involved in cellular transformation to the cancerous state.

nucleotides

nucleotides

Methylation of adenine

Methylation of adenine

ampk-and-ampk-related-kinase-ark-family-

ampk-and-ampk-related-kinase-ark-family-

ubiquitylation

ubiquitylation

(c)  How molecular techniques continue to advance our understanding  of how genetics, epigenetics, and alterations in cellular metabolism contribute to cancer and afford new pathways for therapeutic intervention.

 genomic effects

genomic effects

LKB1AMPK pathway

LKB1AMPK pathway

mutation-frequencies-across-12-cancer-types

mutation-frequencies-across-12-cancer-types

AMPK-activating drugs metformin or phenformin might provide protection against cancer

AMPK-activating drugs metformin or phenformin might provide protection against cancer

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

2. The distinct features of cancers of specific tissue sites of origin

3.  The diagnosis of cancer by

(a)     Clinical presentation

(b)     Age of onset and stage of life

(c)     Biomarker features

hairy cell leukemia

hairy cell leukemia

lymphoma leukemia

lymphoma leukemia

(d)     Radiological and ultrasound imaging

  1. Treatments
  2. Prognostic differences within and between cancer types

We have introduced the emergence of a disease of great complexity that has been clouded in more questions than answers until the emergence of molecular biology in the mid 20th century, and then had to await further discoveries going into the 21st century.  What gave the research impetus was the revelation of

1     the mechanism of transcription of the DNA into amino acid sequences

Proteins in Disease

Proteins in Disease

2     the identification of stresses imposed on cellular function

NO beneficial effects

NO beneficial effects

3     the elucidation of the substructure of the cell – cell membrane, mitochondria, ribosomes, lysosomes – and their functions, respectively

pone.0080815.g006  AKIP1 Expression Modulates Mitochondrial Function

AKIP1 Expression Modulates Mitochondrial Function

4     the elucidation of oligonucleotide sequences

nucleotides

nucleotides

dna-replication-unwinding

dna-replication-unwinding

dna-replication-ligation

dna-replication-ligation

dna-replication-primer-removal

dna-replication-primer-removal

dna-replication-leading-strand

dna-replication-leading-strand

dna-replication-lagging-strand

dna-replication-lagging-strand

dna-replication-primer-synthesis

dna-replication-primer-synthesis

dna-replication-termination

dna-replication-termination

5     the further elucidation of functionally relevant noncoding lncDNA

lncRNA-s   A summary of the various functions described for lncRNA

6     the technology to synthesis mRNA and siRNA sequences

RNAi_Q4 Primary research objectives

Figure. RNAi and gene silencing

7     the repeated discovery of isoforms of critical enzymes and their pleiotropic properties

8.     the regulatory pathways involved in signaling

signaling pathjways map

Figure. Signaling Pathways Map

This is a brief outline of the modern progression of advances in our understanding of cancer.  Let us go back to the beginning and check out a sequence of  Nobel Prizes awarded and related discoveries that have a historical relationship to what we know.  The first discovery was the finding by Louis Pasteur that fungi that grew in an oxygen poor environment did not put down filaments.  They did not utilize oxygen and they produced used energy by fermentation.  This was the basis for Otto Warburg sixty years later to make the comparison to cancer cells that grew in the presence of oxygen, but relied on anaerobic glycolysis. He used a manometer to measure respiration in tissue one cell layer thick to measure CO2 production in an adiabatic system.

video width=”1280″ height=”720″ caption=”1741-7007-11-65-s1 Macromolecular juggling by ubiquitylation enzymes.” mp4=”https://pharmaceuticalintelligence.files.wordpress.com/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4“][/video]

An Introduction to the Warburg Apparatus

http://www.youtube.com/watch?v=M-HYbZwN43o

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later:   Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166.

otto_heinrich_warburg

otto_heinrich_warburg

Warburg apparatus

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices.

The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his “discovery of the nature and mode of action of the respiratory enzyme” [1].

The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of ‘titrations’ can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C.

The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C.

Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations.

“The Nobel Prize in Physiology or Medicine 1931”. Nobelprize.org. 27 Dec 2011 www.nobelprize.org/nobel_prizes/medicine/laureates/1931/

  1. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.
  2. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  3. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. – “At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells.”

Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signalingcellular differentiationcell death, as well as the control of the cell cycle and cell growth.[3]   The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,[9   Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900.  Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[13] In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.[13]    

The Clark Oxygen Sensor

Dr. Leland Clark – inventor of the “Clark Oxygen Sensor” (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications .   ‘The convenience and simplicity of the polarographic ‘oxygen electrode’ technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the investigator’s ingenuity and specific requirements of the system under investigation.’Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.   “one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.” Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.

Great Figures in Cancer

Dr. Elizabeth Blackburn,

Dr. Elizabeth Blackburn,

j_michael_bishop onogene

j_michael_bishop onogene

Harold Varmus

Harold Varmus

Potts and Habener (PTH mRNA, Harvard MIT)  JCI

Potts and Habener (PTH mRNA, Harvard MIT) JCI

JCI Fuller Albright and hPTH AA sequence

JCI Fuller Albright and hPTH AA sequence

Dr. E. Donnall Thomas  Bone Marrow Transplants

Dr. E. Donnall Thomas Bone Marrow Transplants

Dr Haraldzur Hausen  EBV HPV

Dr Haraldzur Hausen EBV HPV

Dr. Craig Mello

Dr. Craig Mello

Dorothy Hodgkin  protein crystallography

Lee Hartwell - Hutchinson Cancer Res Center

Lee Hartwell – Hutchinson Cancer Res Center

Judah Folkman, MD

Judah Folkman, MD

Gertrude B. Elien (1918-1999)

Gertrude B. Elien (1918-1999)

The Nobel Prize in Physiology or Medicine 1922   

Archibald V. Hill, Otto Meyerhof

AV Hill –

“the production of heat in the muscle” Hill started his research work in 1909. It was due to J.N. Langley, Head of the Department of Physiology at that time that Hill took up the study on the nature of muscular contraction. Langley drew his attention to the important (later to become classic) work carried out by Fletcher and Hopkins on the problem of lactic acid in muscle, particularly in relation to the effect of oxygen upon its removal in recovery. In 1919 he took up again his study of the physiology of muscle, and came into close contact with Meyerhof of Kiel who, approaching the problem differently, arrived at results closely analogous to his study. In 1919 Hill’s friend W. Hartree, mathematician and engineer, joined in the myothermic investigations – a cooperation which had rewarding results.

Otto Meyerhof

otto-fritz-meyerhof

otto-fritz-meyerhof

lactic acid production in muscle contraction Under the influence of Otto Warburg, then at Heidelberg, Meyerhof became more and more interested in cell physiology.  In 1923 he was offered a Professorship of Biochemistry in the United States, but Germany was unwilling to lose him.  In 1929 he was he was placed in charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.  From 1938 to 1940 he was Director of Research at the Institut de Biologie physico-chimique at Paris, but in 1940 he moved to the United States, where the post of Research Professor of Physiological Chemistry had been created for him by the University of Pennsylvania and the Rockefeller Foundation.  Meyerhof’s own account states that he was occupied chiefly with oxidation mechanisms in cells and with extending methods of gas analysis through the calorimetric measurement of heat production, and especially the respiratory processes of nitrifying bacteria. The physico-chemical analogy between oxygen respiration and alcoholic fermentation caused him to study both these processes in the same subject, namely, yeast extract. By this work he discovered a co-enzyme of respiration, which could be found in all the cells and tissues up till then investigated. At the same time he also found a co-enzyme of alcoholic fermentation. He also discovered the capacity of the SH-group to transfer oxygen; after Hopkins had isolated from cells the SH bodies concerned, Meyerhof showed that the unsaturated fatty acids in the cell are oxidized with the help of the sulfhydryl group. After studying closer the respiration of muscle, Meyerhof investigated the energy changes in muscle. Considerable progress had been achieved by the English scientists Fletcher and Hopkins by their recognition of the fact that lactic acid formation in the muscle is closely connected with the contraction process. These investigations were the first to throw light upon the highly paradoxical fact, already established by the physiologist Hermann, that the muscle can perform a considerable part of its external function in the complete absence of oxygen.

But it was indisputable that in the last resort the energy for muscle activity comes from oxidation, so the connection between activity and combustion must be an indirect one, and observed that in the absence of oxygen in the muscle, lactic acid appears, slowly in the relaxed state and rapidly in the active state, disappearing in the presence of oxygen. Obviously, then, oxygen is involved when muscle is in the relaxed state. http://upload.wikimedia.org/wikipedia/commons/e/e1/Glycolysis.jpg

The Nobel Prize committee had been receiving nominations intermittently for the previous 14 years (for Eijkman, Funk, Goldberger, Grijns, Hopkins and Suzuki but, strangely, not for McCollum in this period). Tthe Committee for the 1929 awards apparently agreed that it was high time to honor the discoverer(s) of vitamins; but who were they? There was a clear case for Grijns, but he had not been re-nominated for that particular year, and it could be said that he was just taking the relatively obvious next steps along the new trail that had been laid down by Eijkman, who was also now an old man in poor health, but there was no doubt that he had taken the first steps in the use of an animal model to investigate the nutritional basis of a clinical disorder affecting millions. Goldberger had been another important contributor, but his recent death put him out of consideration. The clearest evidence for lack of an unknown “something” in a mammalian diet was presented by Gowland Hopkins in 1912. This Cambridge biochemist was already well known for having isolated the amino acid tryptophan from a protein and demonstrated its essential nature. He fed young rats on an experimental diet, half of them receiving a daily milk supplement, and only those receiving milk grew well, Hopkins suggested that this was analogous to human diseases related to diet, as he had suggested already in a lecture published in 1906. Hopkins, the leader of the “dynamic biochemistry” school in Britain and an influential advocate for the importance of vitamins, was awarded the prize jointly with Eijkman. A door was opened. Recognition of work on the fat-soluble vitamins begun by McCollum. The next award related to vitamins was given in 1934 to George WhippleGeorge Minot and William Murphy “for their discoveries concerning liver therapy in cases of [then incurable pernicious] anemia,” The essential liver factor (cobalamin, or vitamin B12) was isolated in 1948, and Vitamin B12  was absent from plant foods. But William Castle in 1928 had demonstrated that the stomachs of pernicious anemia patients were abnormal in failing to secrete an “intrinsic factor”.

1937   Albert von Szent-Györgyi Nagyrápolt

” the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

http://www.biocheminfo.org/klotho/html/fumarate.html

structure of fumarate

Szent-Györgyi was a Hungarian biochemist who had worked with Otto Warburg and had a special interest in oxidation-reduction mechanisms. He was invited to Cambridge in England in 1927 after detecting an antioxidant compound in the adrenal cortex, and there, he isolated a compound that he named hexuronic acid. Charles Glen King of the University of Pittsburgh reported success In isolating the anti-scorbutic factor in 1932, and added that his crystals had all the properties reported by Szent-Györgyi for hexuronic acid. But his work on oxidation reactions was also important. Fumarate is an intermediate in the citric acid cycle used by cells to produce energy in the form of adenosine triphosphate (ATP) from food. It is formed by the oxidation of succinate by the enzyme succinate dehydrogenase. Fumarate is then converted by the enzyme fumarase to malate. An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group.

In the same year, Norman Haworth from the University of Birmingham in England received a Nobel prize from the Chemistry Committee for having advanced carbohydrate chemistry and, specifically, for having worked out the structure of Szent-Györgyi’s crystals, and then been able to synthesize the vitamin. This was a considerable achievement. The Nobel Prize in Chemistry was shared with the Swiss organic chemist Paul Karrer, cited for his work on the structures of riboflavin and vitamins A and E as well as other biologically interesting compounds. This was followed in 1938 by a further Chemistry award to the German biochemist Richard Kuhn, who had also worked on carotenoids and B-vitamins, including riboflavin and pyridoxine. But Karrer was not permitted to leave Germany at that time by the Nazi regime. However, the American work with radioisotopes at Lawrence Livermore Laboratory, UC Berkeley, was already ushering in a new era of biochemistry that would enrich our studies of metabolic pathways. The importance of work involving vitamins was acknowledged in at least ten awards in the 20th century.

1.   Carpenter, K.J., Beriberi, White Rice and Vitamin B, University of California Press, Berkeley (2000).

2.  Weatherall, M.W. and Kamminga, H., The making of a biochemist: the construction of Frederick Gowland Hopkins’ reputation. Medical History vol.40, pp. 415-436 (1996).

3.  Becker, S.L., Will milk make them grow? An episode in the discovery of the vitamins. In Chemistry and Modern Society (J. Parascandela, editor) pp. 61-83, American Chemical Society,

Washington, D.C. (1983).

4.  Carpenter, K.J., The History of Scurvy and Vitamin C, Cambridge University Press, New York (1986).

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle.

D R FinderC D Hardin

Molecular and Cellular Biochemistry (Impact Factor: 2.33). 05/1999; 195(1-2):113-21.  http://dx.doi.org/10.1023/A:1006976432578

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

C D HardinL RaeymaekersR J Paul

The Journal of General Physiology (Impact Factor: 4.73). 12/1991; 99(1):21-40.   http://dx.doi.org:/10.1085/jgp.99.1.21

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

C HohlR OestreichP RösenR WiesnerM Grieshaber

Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 01/1988; 259(2):527-35. http://dx.doi.org:/10.1016/0003-9861(87)90519-4   It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive.

We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized.

In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

1953 Hans Adolf Krebs –

 ” discovery of the citric acid cycle” and In the course of the 1920’s and 1930’s great progress was made in the study of the intermediary reactions by which sugar is anaerobically fermented to lactic acid or to ethanol and carbon dioxide. The success was mainly due to the joint efforts of the schools of Meyerhof, Embden, Parnas, von Euler, Warburg and the Coris, who built on the pioneer work of Harden and of Neuberg. This work brought to light the main intermediary steps of anaerobic fermentations.

In contrast, very little was known in the earlier 1930’s about the intermediary stages through which sugar is oxidized in living cells. When, in 1930, I left the laboratory of Otto Warburg (under whose guidance I had worked since 1926 and from whom I have learnt more than from any other single teacher), I was confronted with the question of selecting a major field of study and I felt greatly attracted by the problem of the intermediary pathway of oxidations.

These reactions represent the main energy source in higher organisms, and in view of the importance of energy production to living organisms (whose activities all depend on a continuous supply of energy) the problem seemed well worthwhile studying.   http://www.johnkyrk.com/krebs.html

Interactive Krebs cycle

There are different points where metabolites enter the Krebs’ cycle. Most of the products of protein, carbohydrates and fat metabolism are reduced to the molecule acetyl coenzyme A that enters the Krebs’ cycle. Glucose, the primary fuel in the body, is first metabolized into pyruvic acid and then into acetyl coenzyme A. The breakdown of the glucose molecule forms two molecules of ATP for energy in the Embden Meyerhof pathway process of glycolysis.

On the other hand, amino acids and some chained fatty acids can be metabolized into Krebs intermediates and enter the cycle at several points. When oxygen is unavailable or the Krebs’ cycle is inhibited, the body shifts its energy production from the Krebs’ cycle to the Embden Meyerhof pathway of glycolysis, a very inefficient way of making energy.  

Fritz Albert Lipmann –

 “discovery of co-enzyme A and its importance for intermediary metabolism”.

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation.

For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.   In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann.

In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[13]The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[13]

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The phosphate effect was removed by washing with a phosphate free acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

A coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. Addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate.   The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. Acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis to combine with another acetic acid molecule and begin the Krebs’ Cycle again.

Hugo Theorell

the nature and effects of oxidation enzymes”

From 1933 until 1935 Theorell held a Rockefeller Fellowship and worked with Otto Warburg at Berlin-Dahlem, and here he became interested in oxidation enzymes. At Berlin-Dahlem he produced, for the first time, the oxidation enzyme called «the yellow ferment» and he succeeded in splitting it reversibly into a coenzyme part, which was found to be flavin mononucleotide, and a colourless protein part. On return to Sweden, he was appointed Head of the newly established Biochemical Department of the Nobel Medical Institute, which was opened in 1937.

Succinate is oxidized by a molecule of FAD (Flavin Adenine Dinucleotide). The FAD removes two hydrogen atoms from the succinate and forms a double bond between the two carbon atoms to create fumarate.

1953

double-stranded-dna

double-stranded-dna

crick-watson-with-their-dna-model.

crick-watson-with-their-dna-model.

Watson & Crick double helix model 

A landmark in this journey

They followed the path that became clear from intense collaborative work in California by George Beadle, by Avery and McCarthy, Max Delbruck, TH Morgan, Max Delbruck and by Chargaff that indicated that genetics would be important.

1965

François Jacob, André Lwoff and Jacques Monod  –

” genetic control of enzyme and virus synthesis”.

In 1958 the remarkable analogy revealed by genetic analysis of lysogeny and that of the induced biosynthesis of ß-galactosidase led François Jacob, with Jacques Monod, to study the mechanisms responsible for the transfer of genetic information as well as the regulatory pathways which, in the bacterial cell, adjust the activity and synthesis of macromolecules. Following this analysis, Jacob and Monod proposed a series of new concepts, those of messenger RNA, regulator genes, operons and allosteric proteins.

Francois Jacob

Having determined the constants of growth in the presence of different carbohydrates, it occurred to me that it would be interesting to determine the same constants in paired mixtures of carbohydrates. From the first experiment on, I noticed that, whereas the growth was kinetically normal in the presence of certain mixtures (that is, it exhibited a single exponential phase), two complete growth cycles could be observed in other carbohydrate mixtures, these cycles consisting of two exponential phases separated by a-complete cessation of growth.

Lwoff, after considering this strange result for a moment, said to me, “That could have something to do with enzyme adaptation.”

“Enzyme adaptation? Never heard of it!” I said.

Lwoff’s only reply was to give me a copy of the then recent work of Marjorie Stephenson, in which a chapter summarized with great insight the still few studies concerning this phenomenon, which had been discovered by Duclaux at the end of the last century.  Studied by Dienert and by Went as early as 1901 and then by Euler and Josephson, it was more or less rediscovered by Karström, who should be credited with giving it a name and attracting attention to its existence.

Lwoff’s intuition was correct. The phenomenon of “diauxy” that I had discovered was indeed closely related to enzyme adaptation, as my experiments, included in the second part of my doctoral dissertation, soon convinced me. It was actually a case of the “glucose effect” discovered by Dienert as early as 1900.   That agents that uncouple oxidative phosphorylation, such as 2,4-dinitrophenol, completely inhibit adaptation to lactose or other carbohydrates suggested that “adaptation” implied an expenditure of chemical potential and therefore probably involved the true synthesis of an enzyme.

With Alice Audureau, I sought to discover the still quite obscure relations between this phenomenon and the one Massini, Lewis, and others had discovered: the appearance and selection of “spontaneous” mutants.   We showed that an apparently spontaneous mutation was allowing these originally “lactose-negative” bacteria to become “lactose-positive”. However, we proved that the original strain (Lac-) and the mutant strain (Lac+) did not differ from each other by the presence of a specific enzyme system, but rather by the ability to produce this system in the presence of lactose.  This mutation involved the selective control of an enzyme by a gene, and the conditions necessary for its expression seemed directly linked to the chemical activity of the system.

1974

Albert Claude, Christian de Duve and George E. Palade –

” the structural and functional organization of the cell”.

I returned to Louvain in March 1947 after a period of working with Theorell in Sweden, the Cori’s, and E Southerland in St. Louis, fortunate in the choice of my mentors, all sticklers for technical excellence and intellectual rigor, those prerequisites of good scientific work. Insulin, together with glucagon which I had helped rediscover, was still my main focus of interest, and our first investigations were accordingly directed on certain enzymatic aspects of carbohydrate metabolism in liver, which were expected to throw light on the broader problem of insulin action. But I became distracted by an accidental finding related to acid phosphatase, drawing most of my collaborators along with me. The studies led to the discovery of the lysosome, and later of the peroxisome.

In 1962, I was appointed a professor at the Rockefeller Institute in New York, now the Rockefeller University, the institution where Albert Claude had made his pioneering studies between 1929 and 1949, and where George Palade had been working since 1946.  In New York, I was able to develop a second flourishing group, which follows the same general lines of research as the Belgian group, but with a program of its own.

1968

Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg –

“interpretation of the genetic code and its function in protein synthesis”.

1969

Max Delbrück, Alfred D. Hershey and Salvador E. Luria –

” the replication mechanism and the genetic structure of viruses”.

1975 David Baltimore, Renato Dulbecco and Howard Martin Temin –

” the interaction between tumor viruses and the genetic material of the cell”.

1976

Baruch S. Blumberg and D. Carleton Gajdusek –

” new mechanisms for the origin and dissemination of infectious diseases” The editors of the Nobelprize.org website of the Nobel Foundation have asked me to provide a supplement to the autobiography that I wrote in 1976 and to recount the events that happened after the award. Much of what I will have to say relates to the scientific developments since the last essay. These are described in greater detail in a scientific memoir first published in 2002 (Blumberg, B. S., Hepatitis B. The Hunt for a Killer Virus, Princeton University Press, 2002, 2004).

1980

Baruj Benacerraf, Jean Dausset and George D. Snell 

” genetically determined structures on the cell surface that regulate immunological reactions”.

1992

Edmond H. Fischer and Edwin G. Krebs 

“for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”

1994

Alfred G. Gilman and Martin Rodbell –

“G-proteins and the role of these proteins in signal transduction in cells”

2011

Bruce A. Beutler and Jules A. Hoffmann –

the activation of innate immunity and the other half to Ralph M. Steinman – “the dendritic cell and its role in adaptive immunity”.

Renato L. Baserga, M.D.

Kimmel Cancer Center and Keck School of Medicine

Dr. Baserga’s research focuses on the multiple roles of the type 1 insulin-like growth factor receptor (IGF-IR) in the proliferation of mammalian cells. The IGF-IR activated by its ligands is mitogenic, is required for the establishment and the maintenance of the transformed phenotype, and protects tumor cells from apoptosis. It, therefore, serves as an excellent target for therapeutic interventions aimed at inhibiting abnormal growth. In basic investigations, this group is presently studying the effects that the number of IGF-IRs and specific mutations in the receptor itself have on its ability to protect cells from apoptosis.

This investigation is strictly correlated with IGF-IR signaling, and part of this work tries to elucidate the pathways originating from the IGF-IR that are important for its functional effects. Baserga’s group has recently discovered a new signaling pathway used by the IGF-IR to protect cells from apoptosis, a unique pathway that is not used by other growth factor receptors. This pathway depends on the integrity of serines 1280-1283 in the C-terminus of the receptor, which bind 14.3.3 and cause the mitochondrial translocation of Raf-1.

Another recent discovery of this group has been the identification of a mechanism by which the IGF-IR can actually induce differentiation in certain types of cells. When cells have IRS-1 (a major substrate of the IGF-IR), the IGF-IR sends a proliferative signal; in the absence of IRS-1, the receptor induces cell differentiation. The extinction of IRS-1 expression is usually achieved by DNA methylation.

Janardan Reddy, MD

Northwestern University

The central effort of our research has been on a detailed analysis at the cellular and molecular levels of the pleiotropic responses in liver induced by structurally diverse classes of chemicals that include fibrate class of hypolipidemic drugs, and phthalate ester plasticizers, which we designated hepatic peroxisome proliferators. Our work has been central to the establishment of several principles, namely that hepatic peroxisome proliferation is associated with increases in fatty acid oxidation systems in liver, and that peroxisome proliferators, as a class, are novel nongenotoxic hepatocarcinogens.

We introduced the concept that sustained generation of reactive oxygen species leads to oxidative stress and serves as the basis for peroxisome proliferator-induced liver cancer development. Furthermore, based on the tissue/cell specificity of pleiotropic responses and the coordinated transcriptional regulation of fatty acid oxidation system genes, we postulated that peroxisome proliferators exert their action by a receptor-mediated mechanism. This receptor concept laid the foundation for the discovery of

  • a three member peroxisome proliferator-activated receptor (PPARalpha-, ß-, and gamma) subfamily of nuclear receptors.
  •  PPARalpha is responsible for peroxisome proliferator-induced pleiotropic responses, including
    • hepatocarcinogenesis and energy combustion as it serves as a fatty acid sensor and regulates fatty acid oxidation.

Our current work focuses on the molecular mechanisms responsible for PPAR action and generation of fatty acid oxidation deficient mouse knockout models. Transcription of specific genes by nuclear receptors is a complex process involving the participation of multiprotein complexes composed of transcription coactivators.  

Jose Delgado de Salles Roselino, Ph.D.

Leloir Institute, Brazil

Warburg effect, in reality “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end metabolic products, ethanol and carbon dioxide that was observed when yeast cells were transferred from anaerobic environmental condition to an aerobic one. In Pasteur´s works, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis condition and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took very long time to create a rather selective anaerobic condition. This selective culture media was led by the carbon dioxide higher levels produced by fast growing yeast cells and by a great alcohol content in the yeast culture media. This finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits.

In much resumed form, these observations indicates the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells. Biology inside classical thermo dynamics poses some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to decrease in V (volume) all this in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters. In a reversible system, a decrease in V, under same condition, will led to an increase in P.

In biochemistry, reversible usually indicates a reaction that easily goes from A to B or B to A. This observation confirms the important contribution of E Schrodinger in his What´s Life: “This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject-matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

Hans Krebs describes the cyclic nature of the citrate metabolism. Two major research lines search to understand the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. A major leader of this research line was B. Chance who tried to account for two carbon atoms of acetyl released as carbon dioxide in the series of Krebs cycle reactions. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. Alternatively, under the possible influence of the excellent results of Hodgkin and Huxley a second line of research appears.

The work of Hodgkin & Huxley indicated the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of P Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for energetic transfer mechanism required for ATP synthesis. Paul Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility). Nonetheless, a victorious Peter Mitchell obtained the correct result in the conceptual dispute, over the B. Chance point of view, after he used E. Coli mutants to show H gradients in membrane and its use as energy source.

However, this should not detract from the important work of Chance. B. Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that bring us back to Pasteur. This second result seems to have been neglected in searching for a single molecular mechanism required for the understanding of the buildup of chemical reserve in our body. In respiring mitochondria the rate of electron transport, and thus the rate of ATP production, is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has very low respiratory rate.

I have had an ongoing discussion with Jose Eduardo de Salles Roselino, inBrazil. He has made important points that need to be noted.

  1. The constancy of composition which animals maintain in the environment surrounding their cells is one of the dominant features of their physiology. Although this phenomenon, homeostasis, has held the interest of biologists over a long period of time, the elucidation of the molecular basis for complex processes such as temperature control and the maintenance of various substances at constant levels in the blood has not yet been achieved. By comparison, metabolic regulation in microorganisms is much better understood, in part because the microbial physiologist has focused his attention on enzyme-catalyzed reactions and their control, as these are among the few activities of microorganisms amenable to quantitative study. Furthermore, bacteria are characterized by their ability to make rapid and efficient adjustments to extensive variations in most parameters of their environment; therefore, they exhibit a surprising lack of rigid requirements for their environment, and appears to influence it only as an incidental result of their metabolism. Animal cells on the other hand have only a limited capacity for adjustment and therefore require a constant milieu. Maintenance of such constancy appears to be a major goal in their physiology (Regulation of Biosynthetic Pathways H.S. Moyed and H EUmbarger Phys Rev,42 444 (1962)).
  2. A living cell consists in a large part of a concentrated mixture of hundreds of different enzymes, each a highly effective catalyst for one or more chemical reactions involving other components of the cell. The paradox of intense and highly diverse chemical activity on the one hand and strongly poised chemical stability (biological homeostasis) on the other is one of the most challenging problems of biology (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). Almost nothing is known concerning the actual molecular basis for modulation of an enzyme`s kinetic behavior by interaction with a small molecule. (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). In the same article, since the core of Atkinson´s thinking seems to be strongly linked with Adenylates as regulatory effectors, the previous phrases seems to indicate a first step towards the conversion of homeostasis to an intracellular phenomenon and therefore, one that contrary to Umbarger´s consideration could be also studied in microorganisms.
  3.  Most biochemical studies using bacteria, were made before the end of the third upper part of log growth phase. Therefore, they could be considered as time-independent as S Luria presented biochemistry in Life an Unfinished Experiment. The sole ingredient on the missing side of the events that led us into the molecular biology construction was to consider that proteins, a macromolecule, would never be affected by small molecules translational kinetic energy. This, despite the fact that in a catalytic environment and its biological implications S Grisolia incorporated A K Balls observation indicating that the word proteins could be related to Proteus an old sea god that changed its form whenever he was subjected to inquiry (Phys Rev v 4,657 (1964).
  1. In D.E. Atkinson´s work (Science vol 150 p 851, 1965), changes in protein synthesis acting together with factors that interfere with enzyme activity will lead to “fine-tuned” regulation better than enzymatic activity regulation alone. Comparison of glycemic regulation in granivorous and carnivorous birds indicate that when no important nutritional source of glucose is available, glycemic levels can be kept constant in fasted and fed birds. The same was found in rats and cats fed on high protein diets. Gluconeogenesis is controlled by pyruvate kinase inhibition. Therefore, the fact that it can discriminate between fasting alone and fasting plus exercise (carbachol) requirement of gluconeogenic activity (correspondent level of pyruvate kinase inhibition) the control of enzyme activity can be made fast and efficient without need for changes in genetic expression (20 minute after stimulus) ( Migliorini,R.H. et al Am J. Physiol.257 (Endocrinol. Met. 20): E486, 1989). Regrettably, this was not discussed in the quoted work. So, when the control is not affected by the absorption of nutritional glucose it can be very fast, less energy intensive and very sensitive mechanism of control despite its action being made in the extracellular medium (homeostasis).

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What is the Future for Genomics in Clinical Medicine?


What is the Future for Genomics in Clinical Medicine?

Author and Curator: Larry H Bernstein, MD, FCAP

 

Introduction

This is the last in a series of articles looking at the past and future of the genome revolution.  It is a revolution indeed that has had a beginning with the first phase discovery leading to the Watson-Crick model, the second phase leading to the completion of the Human Genome Project, a third phase in elaboration of ENCODE.  But we are entering a fourth phase, not so designated, except that it leads to designing a path to the patient clinical experience.
What is most remarkable on this journey, which has little to show in treatment results at this time, is that the boundary between metabolism and genomics is breaking down.  The reality is that we are a magnificent “magical” experience in evolutionary time, functioning in a bioenvironment, put rogether like a truly complex machine, and with interacting parts.  What are those parts – organelles, a genetic message that may be constrained and it may be modified based on chemical structure, feedback, crosstalk, and signaling pathways.  This brings in diet as a source of essential nutrients, exercise as a method for delay of structural loss (not in excess), stress oxidation, repair mechanisms, and an entirely unexpected impact of this knowledge on pharmacotherapy.  I illustrate this with some very new observations.

Gutenberg Redone

The first is a recent talk on how genomic medicine has constructed a novel version of the “printing press”, that led us out of the dark ages.

Topol_splash_image

In our series The Creative Destruction of Medicine, I’m trying to get into critical aspects of how we can Schumpeter or reboot the future of healthcare by leveraging the big innovations that are occurring in the digital world, including digital medicine.

We have this big thing about evidence-based medicine and, of course, the sanctimonious randomized, placebo-controlled clinical trial. Well, that’s great if one can do that, but often we’re talking about needing thousands, if not tens of thousands, of patients for these types of clinical trials. And things are changing so fast with respect to medicine and, for example, genomically guided interventions that it’s going to become increasingly difficult to justify these very large clinical trials.

For example, there was a drug trial for melanoma and the mutation of BRAF, which is the gene that is found in about 60% of people with malignant melanoma. When that trial was done, there was a placebo control, and there was a big ethical charge asking whether it is justifiable to have a body count. This was a matched drug for the biology underpinning metastatic melanoma, which is essentially a fatal condition within 1 year, and researchers were giving some individuals a placebo.

The next observation is a progression of what he have already learned. The genome has a role is cellular regulation that we could not have dreamed of 25 years ago, or less. The role is far more than just the translation of a message from DNA to RNA to construction of proteins, lipoproteins, cellular and organelle structures, and more than a regulation of glycosidic and glycolytic pathways, and under the influence of endocrine and apocrine interactions. Despite what we have learned, the strength of inter-molecular interactions, strong and weak chemical bonds, essential for 3-D folding, we know little about the importance of trace metals that have key roles in catalysis and because of their orbital structures, are essential for organic-inorganic interplay. This will not be coming soon because we know almost nothing about the intracellular, interstitial, and intrvesicular distributions and how they affect the metabolic – truly metabolic events.

I shall however, use some new information that gives real cause for joy.

Reprogramming Alters Cells’ Fate

Kathy Liszewski
Gordon Conference  Report: June 21, 2012;32(11)
New and emerging strategies were showcased at Gordon Conference’s recent “Reprogramming Cell Fate” meeting. For example, cutting-edge studies described how only a handful of key transcription factors were needed to entirely reprogram cells.
M. Azim Surani, Ph.D., Marshall-Walton professor at the Gurdon Institute, University of Cambridge, U.K., is examining cellular reprogramming in a mouse model. Epiblast stem cells are derived from the early-stage embryonic stage after implantation of blastocysts, about six days into development, and retain the potential to undergo reversion to embryonic stem cells (ESCs) or to PGCs.”  They report two critical steps both of which are needed for exploring epigenetic reprogramming.  “Although there are two X chromosomes in females, the inactivation of one is necessary for cell differentiation. Only after epigenetic reprogramming of the X chromosome can pluripotency be acquired. Pluripotent stem cells can generate any fetal or adult cell type but are not capable of developing into a complete organism.”
The second read-out is the activation of Oct4, a key transcription factor involved in ESC development. The expression of Oct4 in epiSCs requires its proximal enhancer.  Dr. Surani said that their cell-based system demonstrates how a systematic analysis can be performed to analyze how other key genes contribute to the many-faceted events involved in reprogramming the germline.
Reprogramming Expressway
A number of other recent studies have shown the importance of Oct4 for self-renewal of undifferentiated ESCs. It is sufficient to induce pluripotency in neural tissues and somatic cells, among others. The expression of Oct4 must be tightly regulated to control cellular differentiation. But, Oct4 is much more than a simple regulator of pluripotency, according to Hans R. Schöler, Ph.D., professor in the department of cell and developmental biology at the Max Planck Institute for Molecular Biomedicine.
Oct4 has a critical role in committing pluripotent cells into the somatic cellular pathway. When embryonic stem cells overexpress Oct4, they undergo rapid differentiation and then lose their ability for pluripotency. Other studies have shown that Oct4 expression in somatic cells reprograms them for transformation into a particular germ cell layer and also gives rise to induced pluripotent stem cells (iPSCs) under specific culture conditions.
Oct4 is the gatekeeper into and out of the reprogramming expressway. By modifying experimental conditions, Oct4 plus additional factors can induce formation of iPSCs, epiblast stem cells, neural cells, or cardiac cells. Dr. Schöler suggests that Oct4 a potentially key factor not only for inducing iPSCs but also for transdifferention.  “Therapeutic applications might eventually focus less on pluripotency and more on multipotency, especially if one can dedifferentiate cells within the same lineage. Although fibroblasts are from a different germ layer, we recently showed that adding a cocktail of transcription factors induces mouse fibroblasts to directly acquire a neural stem cell identity.
Stem cell diagram illustrates a human fetus st...

Stem cell diagram illustrates a human fetus stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

English: Embryonic Stem Cells. (A) shows hESCs...

English: Embryonic Stem Cells. (A) shows hESCs. (B) shows neurons derived from hESCs. (Photo credit: Wikipedia)

Transforming growth factor beta (TGF-β) is a s...

Transforming growth factor beta (TGF-β) is a secreted protein that controls proliferation, cellular differentiation, and other functions in most cells. http://en.wikipedia.org/wiki/TGFbeta (Photo credit: Wikipedia)

Pioneer Transcription Factors

Pioneer transcription factors take the lead in facilitating cellular reprogramming and responses to environmental cues. Multicellular organisms consist of functionally distinct cellular types produced by differential activation of gene expression. They seek out and bind specific regulatory sequences in DNA. Even though DNA is coated with and condensed into a thick fiber of chromatin. The pioneer factor, discovered by Prof. KS Zaret at UPenn SOM in 1996, he says, endows the competence for gene activity, being among the first transcription factors to engage and pry open the target sites in chromatin.
FoxA factors, expressed in the foregut endoderm of the mouse,are necessary for induction of the liver program. They found that nearly one-third of the DNA sites bound by FoxA in the adult liver occur near silent genes

A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication

ME Hubbi, K Shitiz, DM Gilkes, S Rey,….GL Semenza. Johns Hopkins University SOM
Sci. Signal 2013; 6(262) 10pgs. [DOI: 10.1126/scisignal.2003417]   http:dx.doi.org/10.1126/scisignal.2003417

http://SciSignal.com/A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication/

Many of the cellular responses to reduced O2 availability are mediated through the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). We report a role for the isolated HIF-1α subunit as an inhibitor of DNA replication, and this role was independent of HIF-1β and transcriptional regulation. In response to hypoxia, HIF-1α bound to Cdc6, a protein that is essential for loading of the mini-chromosome maintenance (MCM) complex (which has DNA helicase activity) onto DNA, and promoted the interaction between Cdc6 and the MCM complex. The binding of HIF-1α to the complex decreased phosphorylation and activation of the MCM complex by the kinase Cdc7. As a result, HIF-1α inhibited firing of replication origins, decreased DNA replication, and induced cell cycle arrest in various cell types. To whom correspondence should be addressed. E-mail: gsemenza@jhmi.edu
Citation: M. E. Hubbi, Kshitiz, D. M. Gilkes, S. Rey, C. C. Wong, W. Luo, D.-H. Kim, C. V. Dang, A. Levchenko, G. L. Semenza, A Nontranscriptional Role for HIF-1α as a Direct Inhibitor of DNA Replication. Sci. Signal. 6, ra10 (2013).

Identification of a Candidate Therapeutic Autophagy-inducing Peptide

Nature 2013;494(7436).    http://nature.com/Identification_of_a_candidate_therapeutic_autophagy-inducing_peptide/   http://www.ncbi.nlm.nih.gov/pubmed/23364696
http://www.readcube.com/articles/10.1038/nature11866

Beth Levine and colleagues have constructed a cell-permeable peptide derived from part of an autophagy protein called beclin 1. This peptide is a potent inducer of autophagy in mammalian cells and in vivo in mice and was effective in the clearance of several viruses including chikungunya virus, West Nile virus and HIV-1.

Could this small autophagy-inducing peptide may be effective in the prevention and treatment of human diseases?

PR-Set7 Is a Nucleosome-Specific Methyltransferase that Modifies Lysine 20 of

Histone H4 and Is Associated with Silent Chromatin

K Nishioka, JC Rice, K Sarma, H Erdjument-Bromage, …, D Reinberg.   Molecular Cell, Vol. 9, 1201–1213, June, 2002, Copyright 2002 by Cell Press   http://www.cell.com/molecular-cell/abstract/S1097-2765(02)00548-8

http://www.sciencedirect.com/science/article/pii/S1097276502005488           http://www.ncbi.nlm.nih.gov/pubmed/12086618
http://www.cienciavida.cl/publications/b46e8d324fa4aefa771c4d6ece4d2e27_PR-Set7_Is_a_Nucleosome-Specific.pdf

We have purified a human histone H4 lysine 20methyl-transferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second in-star larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions polytene chromosomes, including chromocenter euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

Next-Generation Sequencing vs. Microarrays

Shawn C. Baker, Ph.D., CSO of BlueSEQ
GEN Feb 2013
With recent advancements and a radical decline in sequencing costs, the popularity of next generation sequencing (NGS) has skyrocketed. As costs become less prohibitive and methods become simpler and more widespread, researchers are choosing NGS over microarrays for more of their genomic applications. The immense number of journal articles citing NGS technologies it looks like NGS is no longer just for the early adopters. Once thought of as cost prohibitive and technically out of reach, NGS has become a mainstream option for many laboratories, allowing researchers to generate more complete and scientifically accurate data than previously possible with microarrays.

Gene Expression

Researchers have been eager to use NGS for gene expression experiments for a detailed look at the transcriptome. Arrays suffer from fundamental ‘design bias’ —they only return results from those regions for which probes have been designed. The various RNA-Seq methods cover all aspects of the transcriptome without any a priori knowledge of it, allowing for the analysis of such things as novel transcripts, splice junctions and noncoding RNAs. Despite NGS advancements, expression arrays are still cheaper and easier when processing large numbers of samples (e.g., hundreds to thousands).
Methylation
While NGS unquestionably provides a more complete picture of the methylome, whole genome methods are still quite expensive. To reduce costs and increase throughput, some researchers are using targeted methods, which only look at a portion of the methylome. Because details of exactly how methylation impacts the genome and transcriptome are still being investigated, many researchers find a combination of NGS for discovery and microarrays for rapid profiling.

Diagnostics

They are interested in ease of use, consistent results, and regulatory approval, which microarrays offer. With NGS, there’s always the possibility of revealing something new and unexpected. Clinicians aren’t prepared for the extra information NGS offers. But the power and potential cost savings of NGS-based diagnostics is alluring, leading to their cautious adoption for certain tests such as non-invasive prenatal testing.
Cytogenetics
Perhaps the application that has made the least progress in transitioning to NGS is cytogenetics. Researchers and clinicians, who are used to using older technologies such as karyotyping, are just now starting to embrace microarrays. NGS has the potential to offer even higher resolution and a more comprehensive view of the genome, but it currently comes at a substantially higher price due to the greater sequencing depth. While dropping prices and maturing technology are causing NGS to make headway in becoming the technology of choice for a wide range of applications, the transition away from microarrays is a long and varied one. Different applications have different requirements, so researchers need to carefully weigh their options when making the choice to switch to a new technology or platform. Regardless of which technology they choose, genomic researchers have never had more options.

Sequencing Hones In on Targets

Greg Crowther, Ph.D.

GEN Feb 2013

Cliff Han, PhD, team leader at the Joint Genome Institute in the Los Alamo National Lab, was one of a number of scientists who made presentations regarding target enrichment at the “Sequencing, Finishing, and Analysis in the Future” (SFAF) conference in Santa Fe, which was co-sponsored by the Los Alamos National Laboratory and DOE Joint Genome Institute. One of the main challenges is that of target enrichment: the selective sequencing of genomic or transcriptomic regions. The polymerase chain reaction (PCR) can be considered the original target-enrichment technique and continues to be useful in contexts such as genome finishing. “One target set is the unique gaps—the gaps in the unique sequence regions. Another is to enrich the repetitive sequences…ribosomal RNA regions, which together are about 5 kb or 6 kb.” The unique-sequence gaps targeted for PCR with 40-nucleotide primers complementary to sequences adjacent to the gaps, did not yield the several-hundred-fold enrichment expected based on previously published work. “We got a maximum of 70-fold enrichment and generally in the dozens of fold of enrichment,” noted Dr. Han.

“We enrich the genome, put the enriched fragments onto the Pacific Biosciences sequencer, and sequence the repeats,” continued Dr. Han. “In many parts of the sequence there will be a unique sequence anchored at one or both ends of it, and that will help us to link these scaffolds together.” This work, while promising, will remain unpublished for now, as the Joint Genome Institute has shifted its resources to other projects.
At the SFAF conference Dr. Jones focused on going beyond basic target enrichment and described new tools for more efficient NGS research. “Hybridization methods are flexible and have multiple stop-start sites, and you can capture very large sizes, but they require library prep,” said Jennifer Carter Jones, Ph.D., a genomics field applications scientist at Agilent. “With PCR-based methods, you have to design PCR primers and you’re doing multiplexed PCR, so it’s limited in the size that you can target. But the workflow is quick because there’s no library preparation; you’re just doing PCR.” She discussed Agilent’s recently acquired HaloPlex technology, a hybrid system that includes both a hybridization step and a PCR step. Because no library preparation is required, sequencing results can be obtained in about six hours, making it suitable for clinical uses. However, the hybridization step allows capture of targets of up to 5 megabases—longer than purely PCR-based methods can deliver. The Agilent talk also provided details on the applications of SureSelect, the company’s hybridization technology, to Methyl-Seq and RNA-Seq research. With this technology, 120-mer baits hybridize to targets, then are pulled down with streptavidin-coated magnetic beads.
These are selections from the SFAF conference, which is expected to be a boost to work on the microbiome, and lead to infectious disease therapeutic approaches.

Summary

We have finished a breathtaking ride through the genomic universe in several sessions.  This has been a thorough review of genomic structure and function in cellular regulation.  The items that have been discussed and can be studied in detail include:

  1.  the classical model of the DNA structure
  2. the role of ubiquitinylation in managing cellular function and in autophagy, mitophagy, macrophagy, and protein degradation
  3. the nature of the tight folding of the chromatin in the nucleus
  4. intramolecular bonds and short distance hydrophobic and hydrophilic interactions
  5. trace metals in molecular structure
  6. nuclear to membrane interactions
  7. the importance of the Human Genome Project followed by Encode
  8. the Fractal nature of chromosome structure
  9. the oligomeric formation of short sequences and single nucletide polymorphisms (SNPs)and the potential to identify drug targets
  10. Enzymatic components of gene regulation (ligase, kinases, phosphatases)
  11. Methods of computational analysis in genomics
  12. Methods of sequencing that have become more accurate and are dropping in cost
  13. Chromatin remodeling
  14. Triplex and quadruplex models not possible to construct at the time of Watson-Crick
  15. sequencing errors
  16. propagation of errors
  17. oxidative stress and its expected and unintended effects
  18. origins of cardiovascular disease
  19. starvation and effect on protein loss
  20. ribosomal damage and repair
  21. mitochondrial damage and repair
  22. miscoding and mutational changes
  23. personalized medicine
  24. Genomics to the clinics
  25. Pharmacotherapy horizons
  26. driver mutations
  27. induced pluripotential embryonic stem cell (iPSCs)
  28. The association of key targets with disease
  29. The real possibility of moving genomic information to the bedside
  30. Requirements for the next generation of electronic health record to enable item 29

Other Related articles on this Open Access Online Scientific Journal, include the following:

https://pharmaceuticalintelligence.com/2013/01/14/oogonial-stem-cells-purified-a-view-towards-the-future-of-reproductive-biology/   SSaha

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ RSaxena

https://pharmaceuticalintelligence.com/2012/08/22/a-possible-light-by-stem-cell-therapy-in-painful-dark-of-osteoarthritis-kartogenin-a-small-molecule-differentiates-stem-cells-to-chondrocyte-healthy-cartilage-cells/   ASarkar and RSaxena

https://pharmaceuticalintelligence.com/2012/08/07/human-embryonic-pluripotent-stem-cells-and-healing-post-myocardial-infarction/    LHB

https://pharmaceuticalintelligence.com/2013/02/03/genome-wide-detection-of-single-nucleotide-and-copy-number-variation-of-a-single-human-cell/  SJWilliams

https://pharmaceuticalintelligence.com/2013/01/09/gene-therapy-into-healthy-heart-muscle-reprogramming-scar-tissue-in-damaged-hearts/ ALev-Ari

https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/  SJWilliams

https://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/  TBarliya

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Directions for Genomics in Personalized Medicine

Author: Larry H. Bernstein, MD, FCAP

 

J. Craig Venter

J. Craig Venter (Photo credit: Wikipedia)

Otto Heinrich Warburg

Otto Heinrich Warburg (Photo credit: Wikipedia)

Purpose

This discussion will identify the huge expansion of genomic technology in the search for  biopharmacotherapeutic targets that continue to be explored involving different levels and interacting signaling pathways.   There are several methods of analyzing gene expression that will be discussed. Great primary emphasis required investigation of combinations of mutations expressed in different cancer types.  James Watson has proposed a major hypothesis that expresses the need to focus on “central” “driver mutations” that correspond with the regulation of gene expression, cell proliferation, and cell metabolism eith a critical rejection of antioxiant benefits.  What hasn’t been know is why drug resistance develops and whether the cellular migration and aerobic glycolysis can be redirected after cell metastasis occurs.  I attempt to bring out the complexities of current efforts.

.Introduction

  • This discussion is a continuation of a previous discussion on the role of genomics if discovery of therapeutic targets for cancer, each somewhat different, but all related to:
  • The reversal of carcinoma by targeting a key driver of multiple signaling pathways that activate cell proliferation
  • Pinpointing a stage in a multistage process at which tumor progression links to changes in morphology from basal cells to invasive carcinoma with changes in polarity and loss of glandular architecture
  • Reversal of the carcinoma through using a small molecule that either is covalently bound to a nanoparticle delivery system that blocks or reverses tumor development
  • Synthesis of a small molecule that interacts with the translation of the genome either by substitution of a key driver molecule or by blocking at the mRNA stage of translation
  • Blocking more than one signaling pathway that are links to carcinogenesis and cellular proliferation and invasion

Difficulty of the problem

A problem expressed by James Watson is that the investigations that are ongoing

  • are following a pathway that is not driven by attacking the “primary” driver of carcinogenesis.

He uses the Myc gene as an example, as noted in the previous discussion. The problem may be more complicated than he envisions.

  • The most consistent problem in chemotherapy, irrespective of the design and the target has been cancer remission for a short time followed by recurrence, and then
  • switching to another drug, or combination chemotherapy.

It is common to “clean” the field at the time of resection using radiotherapy before chemotherapy.

  • But the goal is understood to be “palliation”, not cure.

This raises a serious issue in the hypothesis posed by Watson. The issue is

  • whether there is a core locus of genetic regulation that is common to carcinogenesis irrespective of tissue metabolic expression.
  • This is supported by the observation that tissue specific express is lost in cancer cells by de-differentiation.

Historical Perspective

AEROBIC GLYCOLYSIS

In 1967 Otto Warburg published his view in a paper “The prime cause and prevention of cancer”.
There are primary and secondary causes of all diseases

  • plague – primary: plague bacillus
  • plague – secondary: filth, rats, and fleas

cancer, above all diseases,

  • has countless seconday causes
  • primary – replacement of respiration of oxygen in normal body tissue by fermentation of glucose with conversion from obligate aerobic to anaerobic, as in bacterial cells

The cornerstone to understanding cancer is in study of the energetics of life

This thinking came out of decades of work in the Dahlem Institute Kaiser Wilhelm pre WWII and Max Planck Institute after WWII, supported by the Rockefeller Foundation.

  • The oxygen- and hydrogen-transferring enzymes were discovered and isolated.
  • The methods were elegant for that time, using a manometer that improved on the method used by Haldane, that did not allow the leakage of O2 or CO2.
  • The interest was initiated by the increased growth of Sea Urchin eggs after fertization, which turned out to be not comparable to the rapid growth of cancer cells.
  • Warburg used both normal and cancer tissue and measured the utilization of O2. He found
    • that the normal tissue did not accumulate lactic acid.
    • Cancer tissue generated lactic acid
    • the rate of O2 consumption the same as normal tissue, but
    • the rate of lactate formation far exceeded any tissue, except the retina.
    • This was a discovery studied by “Pasteur” 60 years earlier (facultative aerobes), which he called the Pasteur effect.
    • Hematopoietic cells of bone marrow develop aerobic glycolysis when exposed to a low oxygen condition.

He then followed on an observation by Otto Meyerhoff (Embden-Myerhoff cycle) that in muscle

  • the consumption of one molecule of oxygen generates two molecules of lactate, but in aerobic glycolysis, the relationship disappears.
  • He expressed the effectiveness of respiration by the ‘Meyerhoff quotient’.
  • He found that cancer cells didn’t have a quotient of ‘2’

The role of the allosteric enzyme phosphofructokinase (PFK) not then known, would tie together the glycolytic and gluconeogenic pathways.
He used a heavy metal ion chelator ethylcarbylamine to

  • sever the link and turn on aerobic glycolysis.

The explanation for this was provided years later by the work fleshed out by Lynen, Bucher, Lowry, Racker, and Sols.

  • The rate-limiting enzyme, PFK is regulated by the concentrations of ATP, ADP, and inorganic phosphate. The ethylcarbylamide was an ‘uncoupler’ of oxidative phosphorylation.

Warburg understood that when normal cells switched to aerobic glycolysis

  • it is a re-orientation of normal cell expression.
  • this provides the basis for the inference that neoplastic cells become more like each other than their cell of origin.
  • embryonic cells can be transformed into cancer cells under hypoxic conditions
  • re-exposure to higher oxygen did not cause reversion back to normal cells.

Warburg publically expressed the rejected view in 1954 (at age 83) that restriction of chemical wastes, food additives, and air pollution would substantially reduce cancer rates.

His emphasis on the impairment of respiration was inadequate.

  • the prevailing view today is loss of controlled growth of normal cells in cancer cells.

Otto Warburg: Cell Physiologist, Biochemist, and Eccentric. Hans Krebs, in collaboration with Roswitha Schmid. Clarendon Press, Oxford. 1991.ISBN 0-19-858171-8.

The Human Genome Project

The Human Genome Project, driven by Francis Collins at NIH, and by Craig Venter at the Institute for Genome Research (TIGR) had parallel projects to map the human chromosome, completed in 2003. It originally aimed to map the nucleotides contained in a human haploid reference genome (more than three billion). TIGR was the first complete genomic sequencing of a free living organism, Haemophilus influenzae, in 1995. This used a shotgun sequencing technique pioneered earlier, but which had never been used for a whole bacterium.
Venter broke away from the HGP and started Celera in 1998 because of resistance to the shotgun sequency method, and his team completed the genome sequence in three years – seven years’ less time than the HGP timetable (using the gene of Dr. Venter). TIGR eventually sequenced and analyzed more than 50 microbial genomes. Its bioinformatics group developed

  • pioneering software algorithms that were used to analyze these genomes,
  • including the automatic gene finder GLIMMER and
  • the sequence alignment program MUMmer.

In 2002, Venter created and personally funded the J. Craig Venter Institute (JCVI) Joint Technology Center (JTC), which specialized in high throughput sequencing.  The JTC, in the top ranks of scientific institutions worldwide, sequenced nearly 100 million base pairs of DNA per day for its affiliated institutions (JCVI) .

He received his his Ph.D. degree in physiology and pharmacology from the University of California, San Diego in 1975 under biochemist Nathan O. Kaplan. A full professor at the State University of New York at Buffalo, he joined the National Institutes of Health in 1984. There he learned of a technique for rapidly identifying all of the mRNAs present in a cell and began to use it to identify human brain genes. The short cDNA sequence fragments discovered by this method are called expressed sequence tags (ESTs), a name coined by Anthony Kerlavage at TIGR.
Venter believed that shotgun sequencing was the fastest and most effective way to get useful human genome data. There was a belief that shotgun sequencing was less accurate than the clone-by-clone method chosen by the HGP, but the technique became widely accepted by the scientific community and is still the de facto standard used today.

References

Shreeve, James (2004). The Genome War: How Craig Venter Tried to Capture the Code of Life and Save the World. Knopf. ISBN 0375406298.
Sulston, John (2002). The Common Thread: A Story of Science, Politics, Ethics and the Human Genome. Joseph Henry Press. ISBN 0309084091.
“The Human Genome Project Race”. Center for Biomolecular Science & Engineering, UC Santa Cruz. Retrieved 20 March 2012.
Venter, J. Craig (2007). A Life Decoded: My Genome: My Life. Viking Adult. ISBN 0670063584.

Use of a Fluorophor Probe

An article has been discussed by Dr.  Tilda Barilya on use of a sensitive fluorescent probe in the near IR spectrum at > 700 nm to identify malignant ovarian cells in-vivo in abdominal exploration by tagging an overexpressed FR-α (folate-FITA)
The author makes the point that:

  • In ovarian cancer, the FR-α appears to constitute a good target because it is overexpressed in 90–95% of malignant tumors, especially serous carcinomas.
  • Targeting ligand, folate, is attractive as it is nontoxic, inexpensive and relatively easily conjugated to a fluorescent dye to create a tumor-specific fluorescent contrast agent.
  • The report is identified as “ the first in-human proof-of-principle of the use of intraoperative tumor-specific fluorescence imaging in staging and debulking surgery for ovarian cancer using the systemically administered targeted fluorescent agent folate-FITC.”

While this does invoke possibilities for prognosis, the decision to perform the surgery, whether laparoscopic or open, is late in the discovery process. However, it does suggest the possibility that the discovery and the treatment might be combined if the biomarker itself had the fluorescence to identify the overexpression, but it also is combined with a tag to block the overexpession. This hypothetical possibility is now expressed below.
https://pharmaceuticalintelligence.com/2013/01/19/ovarian-cancer-and-fluorescence-guided-surgery-a-report/

Gene Editing

Dr. Aviva Lev-Ari reports that a new technique developed at MIT Broad Institute and the Rockefeller University can edit DNA in precise locations taken from Science News titled Editing Genome With High Precision: New Method to Insert Multiple Genes in Specific Locations, Delete Defective Genes”.

Using this system, scientists can alter

  • several genome sites simultaneously and
  • can achieve much greater control over where new genes are inserted

According to Feng Zhang, this is an improvement beyond splicing the gene in specific locations and insertion of complexes difficult to assemble known as transcription activator-like effector nucleases (TALENs).

  • The researchers create DNA-editing complexes
  • using naturally occurring bacterial protein-RNA systems
  • that recognize and snip viral DNA, including
  • a nuclease called Cas9 bound to short RNA sequences.
  • they target specific locations in the genome, and
  • when they encounter a match, Cas9 cuts the DNA.

This approach can be used either to

  • disrupt the function of a gene or
  • to replace it with a new one.
  • To replace the gene, a DNA template for the new gene has to be copied into the genome after the DNA is cut. The method is also very precise —
  • if there is a single base-pair difference between the RNA targeting sequence and the genome sequence, Cas9 is not activated.

In its first iteration, it appears comparable in efficiency to what zinc finger nucleases and TALENs have to offer.
The research team has deposited the necessary genetic components with a nonprofit called Addgene, and they have also created a website with tips and tools for using this new technique.
The above story is reprinted from materials provided by Massachusetts Institute of Technology. The original article was written by Anne Trafton.
Le Cong, F. Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D. Hsu, Xuebing Wu, Wenyan Jiang, Luciano Marraffini, and Feng Zhang. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science, 3 January 2013 DOI: 10.1126/science.1231143. http://Science.com. Editing genome with high precision: New method to insert multiple genes in specific locations, delete defective genes. ScienceDaily. Retrieved January 20, 2013, from http://www.sciencedaily.com­ /releases/2013/01/130103143205.htm?goback=%2Egde_4346921_member_205356312.

Dr. Lev-Ari also reports on a study of early detection of breast cancer in “Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment“, by Dr. Rotem Karni and PhD student Vered Ben Hur at the Institute for Medical Research Israel-Canada of the Hebrew University. https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-detection-treatment/
These researchers have discovered a new mechanism by which breast cancer cells switch on their aggressive cancerous behavior. The discovery provides a valuable marker for the early diagnosis and follow-up treatment of malignant growths.
The method they use is

  • RNA splicing and insertion.
  • The information needed for the production of a mature protein is encoded in segments called exons .
  • In the splicing process, the non-coding segments of the RNA (introns) are spliced from the pre-mRNA and
  • the exons are joined together.

Alternative splicing is when a specific ”scene” (or exon) is either inserted or deleted from the movie (mRNA), thus changing its meaning.

  • Over 90 percent of the genes in our genome undergo alternative splicing of one or more of their exons, and
  • the resulting changes in the proteins encoded by these different mRNAs are required for normal function.
  • the normal process of alternative splicing is altered in cancer, and
  • ”bad” protein forms are generated that aid cancer cell proliferation and survival.

The researchers reported in online Cell Reports that breast cancer cells

  • change the alternative splicing of an important enzyme, called S6K1, which is
  • a protein involved in the transmission of information into the cell.
  • when this happens, breast cancer cells start to produce shorter versions of this enzyme and
  • these shorter versions transmit signals ordering the cells to grow, proliferate, survive and invade other tissues (otherwise proliferation is suppressed)

The application to biotherapeutics would be to ”reverse” the alternative splicing of S6K1 in cancer cells back to the normal situation as a novel anti-cancer therapy.

Additional Developments:

A*STAR Scientists Pinpoint Genetic Changes that Spell Cancer: Fruit flies light the way for scientists to uncover genetic changes.

With a new approach, researchers may rapidly distinguish the range of

  • genetic changes that are causally linked to cancer (i.e. “driver” mutations)
  • versus those with limited impact on cancer progression.

This study published in the prestigious journal Genes & Development could pave the way to design more targeted treatment against different cancer types, based on the specific cancer-linked mutations present in the patient, an advance in the development of personalized medicine.

Signaling pathways involved in tumour formation are conserved from fruit flies to humans. In fact, about 75 percent of known human disease genes have a recognizable match in the genome of fruit flies.
Leveraging on their genetic similarities, Dr Hector Herranz, a post-doctorate from the Dr. Stephen Cohen’s team developed an innovative strategy to genetically screen the whole fly genome for “cooperating” cancer genes.

  • These genes appear to have little or no impact on cancer.
  • However, they cooperate with other cancer genes, so that
  • the combination causes aggressive cancer, which
  • neither would cause alone.

In this study, the team was specifically looking for genes that

  • could cooperate with EGFR “driver” mutation,
  • a genetic change commonly associated with breast and lung cancers in humans.
  • SOCS5 (reported in this paper) is one of the several new “cooperating” cancer genes to be identified.

Already, there are indications that levels of SOCS5 expression are

  • reduced in breast cancer, and
  • patients with low levels of SOCS5 have poor prognosis.”

The IMCB team is preparing to explore the use of SOCS5 as a biomarker in diagnosis for cancer.
http://genes&development.com

Probing What Fuels Cancer

‘Altered cellular metabolism is a hallmark of cancer,’ says Dr Patrick Pollard, in the Nuffield Department of Clinical Medicine at Oxford. Most cancer cells get the energy they need predominantly through a high rate of glycolysis, allowing cancer cells deal with the low oxygen levels that tend to be present in a tumour.

But whether dysfunctional metabolism causes cancer, as Warburg believed, or is something that happens afterwards is a different question. In the meantime, gene studies rapidly progressed and indicated that genetic changes occur in cancer.

DNA mutations spring up all the time in the body’s cells, but

  • most are quickly repaired.
  • Alternatively the cell might shut down or be killed off (apoptosis) before any damage is caused. However, the repair machinery is not perfect.
  • If changes occur that bypass parts of the repair machinery or sabotage it,
  • the cell can escape the body’s normal controls on growth and
  • DNA changes can begin to accumulate as the cell becomes cancerous.

Patrick believes certain changes in cells can’t always be accounted for by ‘genetics.’
He is now collaborating with Professor Tomoyoshi Soga’s large lab at Keio University in Japan, which has been at the forefront of developing the technology for metabolomics research over the past couple of decades.

The Japanese lab’s ability to

  • screen samples for thousands of compounds and metabolites at once, and
  • the access to tumour material and cell and animal models of disease
  • enables them to probe the metabolic changes that occur in cancer.

There is reason to believe that

  • dysfunctional cell metabolism is important in cancer.
  • genes with metabolic functions are associated with some cancers
  • changes in the function of a metabolic enzyme have been implicated in the development of gliomas.

These results have led to the idea that some metabolic compounds, or metabolites, when they accumulate in cells, can cause changes to metabolic processes and set cells off on a path towards cancer.

Patrick Pollard and colleagues have now published a perspective article in the journal Frontiers in Molecular and Cellular Oncology that proposes fumarate as such an ‘oncometabolite’. Fumarate is a standard compound involved in cellular metabolism.

The researchers summarize evidence that shows how

  • accumulation of fumarate when an enzyme goes wrong affects various biological pathways in the cell.
  • It shifts the balance of metabolic processes and disrupts the cell in ways that could favour development of cancer.

Patrick and colleagues write in their latest article that the shift in focus of cancer research to include cancer cell metabolism ‘has highlighted how woefully ignorant we are about the complexities and interrelationships of cellular metabolic pathways’.

http://FrontiersMolecularCellularOncology.com

Extensive Promoter-Centered Chromatin Interactions Provide a Topological Basis for Transcription Regulation
(Li G, Ruan X, Auerbach RK, Sandhu KS, et al.) Cell 2012; 148(1-2): 84-98. http://cell.com

Using genome-wide Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET),
mapped long-range chromatin interactions associated with RNA polymerase II in human cells
uncovered widespread promoter-centered intragenic, extragenic, and intergenic interactions.

  • These interactions further aggregated into higher-order clusters
  • proximal and distal genes were engaged through promoter-promoter interactions.
  • most genes with promoter-promoter interactions were active and transcribed cooperatively
  • some interacting promoters could influence each other implying combinatorial complexity of transcriptional controls.

Comparative analyses of different cell lines showed that

  • cell-specific chromatin interactions could provide structural frameworks for cell-specific transcription,
  • and suggested significant enrichment of enhancer-promoter interactions for cell-specific functions.
  • genetically-identified disease-associated noncoding elements were spatially engaged with corresponding genes through long-range interactions.

Overall, our study provides insights into transcription regulation by

  • three-dimensional chromatin interactions for both housekeeping and
  • cell-specific genes in human cells.

New Nucleoporin: Regulator of Transcriptional Repression and Beyond.

(NJ Sarma and K Willis) Nucleus 2012; 3(6): 1–8; http://Nucleus.com © 2012 Landes Bioscience

Transcriptional regulation is a complex process that requires the integrated action of many multi-protein complexes.
The way in which a living cell coordinates the action of these complexes in time and space is still poorly understood.

  • nuclear pores, well known for their role in 3′ processing and export of transcripts, also participate in the control of transcriptional initiation.
  • nuclear pores interface with the well-described machinery that regulates initiation.

This work led to the discovery that

  • specific nucleoporins are required for binding of the repressor protein Mig1 to its site in target promoters.
  • Nuclear pores are involved in repressing, as well as activating, transcription.

Here we discuss in detail the main models explaining our result and consider what each implies about the roles that nuclear pores play in the regulation of gene expression.

Prediction of Breast Cancer Metastasis by Gene Expression Profiles: A Comparison of Metagenes and Single Genes.

(M Burton, M Thomassen, Q Tan, and TA Kruse.) Cancer Informatics 2012:11 193–217 doi: 10.4137/CIN.S10375

The popularity of a large number of microarray applications has in cancer research led to the development of predictive or prognostic gene expression profiles. However, the diversity of microarray platforms has made the full validation of such profiles and their related gene lists across studies difficult and, at the level of classification accuracies, rarely validated in multiple independent datasets. Frequently, while the individual genes between such lists may not match, genes with same function are included across such gene lists. Development of such lists does not take into account the fact that

  • genes can be grouped together as metagenes (MGs) based on common characteristics such as pathways, regulation, or genomic location.

In this study we compared the performance of either metagene- or single gene-based feature sets and classifiers using random forest and two support vector machines for classifier building. The performance

  • within the same dataset,
  • feature set validation perfor­mance, and
  • validation performance of entire classifiers in strictly independent datasets

were assessed by

  • 10 times repeated 10-fold cross validation,
  • leave-one-out cross validation, and
  • one-fold validation, respectively.

To test the significance of the performance difference between MG- and SG-features/classifiers, we used a repeated down-sampled binomial test approach.

MG- and SG-feature sets are transferable and perform well for training and testing prediction of metastasis outcome

  • in strictly independent data sets, both
  • between different and
  • within similar microarray platforms, while
  • classifiers had a poorer performance when validated in strictly independent datasets.

The study showed that MG- and SG-feature sets perform equally well in classifying indepen­dent data. Furthermore, SG-classifiers significantly outperformed MG-classifier

  • when validation is conducted between datasets using similar platforms, while
  • no significant performance difference was found when validation was performed between different platforms.

Prediction of metastasis outcome in lymph node–negative patients by MG- and SG-classifiers showed that SG-classifiers performed significantly better than MG-classifiers when validated in independent data based on the same microarray platform as used for developing the classifier. However, the MG- and SG-classifiers had similar performance when conducting classifier validation in independent data based on a different microarray platform. The latter was also true when only validating sets of MG- and SG-features in independent datasets, both between and within similar and different platforms.

Identification and Insilico Analysis of Retinoblastoma Serum microRNA Profile and Gene Targets Towards Prediction of Novel Serum Biomarkers.

M Beta, A Venkatesan, M Vasudevan, U Vetrivel, et al. Identification and Insilico Analysis of Retinoblastoma Serum microRNA Profile and Gene Targets Towards Prediction of Novel Serum Biomarkers.

http://Bioinformatics and Biology Insights 2013:7 21–34. doi: 10.4137/BBI.S10501

This study was undertaken

  • to identify the differentially expressed miRNAs in the serum of children with RB in comparison with the normal age matched serum,
  • to analyze its concurrence with the existing RB tumor miRNA profile,
  • to identify its novel gene targets specific to RB, and
  • to study the expression of a few of the identified oncogenic miRNAs in the advanced stage primary RB patient’s serum sample.

MiRNA profiling performed on 14 pooled serum from chil­dren with advanced RB and 14 normal age matched serum samples

  • 21 miRNAs found to be upregulated (fold change > 2.0, P < 0.05) and
  • 24 downregulated (fold change > 2.0, P < 0.05).

Intersection of 59 significantly deregulated miRNAs identified from RB tumor profiles with that of miRNAs detected in serum profile revealed that

  • 33 miRNAs had followed a similar deregulation pattern in RB serum.

Later we validated a few of the miRNAs (miRNA 17-92) identified by microarray in the RB patient serum samples (n = 20) by using qRT-PCR.

Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum samples

  • exploring the potential of serum miRNAs identification as noninvasive diagnosis.

Moreover, from miRNA gene target prediction, key regulatory genes of

  • cell proliferation,
  • apoptosis, and
  • positive and negative regulatory networks

involved in RB progression were identified in the gene expression profile of RB tumors.
Therefore, these identified miRNAs and their corresponding target genes could give insights on

  • potential biomarkers and key events involved in the RB pathway.

Computational Design of Targeted Inhibitors of Polo-Like Kinase 1 ( lk1).

(KS Jani and DS Dalafave) Bioinformatics and Biology Insights 2012:6 23–31.doi: 10.4137/BBI.S8971.

Computational design of small molecule putative inhibitors of Polo-like kinase 1 (Plk1) is presented. Plk1, which regulates the cell cycle, is often over expressed in cancers.

  • Down regulation of Plk1 has been shown to inhibit tumor progression.
  • Most kinase inhibitors interact with the ATP binding site on Plk1, which is highly conserved.
  • This makes the development of Plk1-specific inhibitors challenging, since different kinases have similar ATP sites.

However, Plk1 also contains a unique region called the polo-box domain (PBD), which is absent from other kinases.

  • the PBD site was used as a target for designed Plk1 putative inhibitors.
  • Common structural features of several experimentally known Plk1 ligands were first identified.
  • The findings were used to design small molecules that specifically bonded Plk1.
  • Drug likeness and possible toxicities of the molecules were investigated.
  • Molecules with no implied toxicities and optimal drug likeness values were used for docking studies.
  • Several molecules were identified that made stable complexes only with Plk1 and LYN kinases, but not with other kinases.
  • One molecule was found to bind exclusively the PBD site of Plk1.

Possible utilization of the designed molecules in drugs against cancers with over expressed Plk1 is discussed.

Conclusions

The previous discussions reviewed the status of an evolving personalized medicine multicentered and worldwide enterprise.  It is also clear from these reports that the search for targeted drugs matched to a cancer profile or signature has identified several approaches that show great promise.

  • We know considerably  more about metabolic pathways and linked changes in transcription that occur in neoplastic development.
  • There are several methods used to do highly accurate  insertions in gene sequences that are linked to specific metabolic changes, and
  • some may have significant implications for therapeutics, if
    • the link is a change that is associated with a driver mutation
    • the link can be identified by a fluorescent or other probe
    • the link is tied to a mRNA or peptide product that is a biomarker measured in the circulation
  • We have probes to genetic links to the control of many and interacting signaling pathways.
  • We know more about transcription through mRNA.
  • We are closer to the possibility that metabolic substrates, like ‘fumarate’ (a key intermediate in the TCA cycle), may provide a means to reverse regulate the neoplastic cells.
  • We may also find metabolic channels that drive the cells from proliferation to apoptosis or normal activity.

Summary

This discussion identified the huge expansion of genomic technology in the investigation of biopharmacotherapeutic targets that have been identified involving different levels and interacting signaling pathways.   There are several methods of analyzing gene expression, and a primary emphasis is given to combinations of mutations expressed in different cancer types.  There is a major hypothesis that expresses the need to focus on “central” “driver mutations” that correspond with the regulation of gene expression, cell proliferation, and cell metabolism.  What hasn’t been know is why drug resistance develops and whether the cellular migration and aerobic glycolysis can be redirected after cell metastasis occurs.

.

A slight mutation in the matched nucleotides c...

A slight mutation in the matched nucleotides can lead to chromosomal aberrations and unintentional genetic rearrangement. (Photo credit: Wikipedia)

Deutsch: Regulation der Phosphofructokinase

Deutsch: Regulation der Phosphofructokinase (Photo credit: Wikipedia)

Additional Related articles

Other posts related to this discussion were published on this Open Source  Online Scientific Journal from Leaders in Pharmaceutical Business  Intelligence:

Big Data in Genomic Medicine, LHB
https://pharmaceuticalintelligence.com/2012/12/17/big-data-in-genomic-medicine/

A New Therapy for Melanoma, LHB
https://pharmaceuticalintelligence.com/2012/09/15/a-new-therapy-for-melanoma/

BRCA1 a tumour suppressor in breast and ovarian cancer – functions in transcription, ubiquitination and DNA repair,  S Saha
https://pharmaceuticalintelligence.com/2012/12/04/brca1-a-tumour-suppressor-in-breast-and-ovarian-cancer-functions-in-transcription-ubiquitination-and-dna-repair/

Judging ‘Tumor response’-there is more food for thought,  R Saxena
https://pharmaceuticalintelligence.com/2012/12/04/judging-the-tumor-response-there-is-more-food-for-thought/

Computational Genomics Center: New Unification of Computational Technologies at Stanford, A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/12/03/computational-genomics-center-new-unification-of-computational-technologies-at-stanford/

Ovarian Cancer and fluorescence-guided surgery: A report, T.  Barliya
https://pharmaceuticalintelligence.com/2013/01/19/ovarian-cancer-and-fluorescence-guided-surgery-a-report/

Personalized medicine gearing up to tackle cancer ,  R. Saxena
https://pharmaceuticalintelligence.com/2013/01/07/personalized-medicine-gearing-up-to-tackle-cancer/

Exploring the role of vitamin C in Cancer therapy,   R. Saxena
https://pharmaceuticalintelligence.com/2013/01/15/exploring-the-role-of-vitamin-c-in-cancer-therapy/

Differentiation Therapy – Epigenetics Tackles Solid Tumors,    SJ Williams
https://pharmaceuticalintelligence.com/2013/01/03/differentiation-therapy-epigenetics-tackles-solid-tumors/

Mechanism involved in Breast Cancer Cell Growth: Function in Early Detection & Treatment,   A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/17/mechanism-involved-in-breast-cancer-cell-growth-function-in-early-detection-treatment/

Personalized Medicine: Cancer Cell Biology and Minimally Invasive Surgery (MIS),  A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/12/01/personalized-medicine-cancer-cell-biology-and-minimally-invasive-surgery-mis/

Role of Primary Cilia in Ovarian Cancer,  A. Awan
https://pharmaceuticalintelligence.com/2013/01/15/role-of-primary-cilia-in-ovarian-cancer-2/

The Molecular Pathology of Breast Cancer Progression,  T. Bailiya`
https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/

Stanniocalcin: A Cancer Biomarker,   A. Awan
https://pharmaceuticalintelligence.com/2012/12/25/stanniocalcin-a-cancer-biomarker/

Nanotechnology, personalized medicine and DNA sequencing,  T. Barliya
https://pharmaceuticalintelligence.com/2013/01/09/nanotechnology-personalized-medicine-and-dna-sequencing/

Gastric Cancer: Whole-genome reconstruction and mutational signatures,  A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/12/24/gastric-cancer-whole-genome-reconstruction-and-mutational-signatures-2/

Paradigm Shift in Human Genomics – Predictive Biomarkers and Personalized Medicine – Part 1, A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/13/paradigm-shift-in-human-genomics-predictive-biomarkers-and-personalized-medicine-part-1/

LEADERS in Genome Sequencing of Genetic Mutations for Therapeutic Drug Selection in Cancer Personalized Treatment: Part 2,  A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/13/leaders-in-genome-sequencing-of-genetic-mutations-for-therapeutic-drug-selection-in-cancer-personalized-treatment-part-2/

Personalized Medicine: An Institute Profile – Coriell Institute for Medical Research: Part 3, A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/13/personalized-medicine-an-institute-profile-coriell-institute-for-medical-research-part-3/

The Consumer Market for Personal DNA Sequencing: Part 4, A. Lev-Ari

https://pharmaceuticalintelligence.com/2013/01/13/consumer-market-for-personal-dna-sequencing-part-4/

Harnessing Personalized Medicine for Cancer Management, Prospects of Prevention and Cure: Opinions of Cancer Scientific Leaders @ http://pharmaceuticalintelligence.com   A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/13/7000/

GSK for Personalized Medicine using Cancer Drugs needs Alacris systems biology model to determine the in silico effect of the inhibitor in its “virtual clinical trial”  A Lev-Ari
https://pharmaceuticalintelligence.com/2012/11/14/gsk-for-personalized-medicine-using-cancer-drugs-needs-alacris-systems-biology-model-to-determine-the-in-silico-effect-of-the-inhibitor-in-its-virtual-clinical-trial/

Recurrent somatic mutations in chromatin-remodeling and ubiquitin ligase complex genes in serous endometrial tumors,  S. Saha
https://pharmaceuticalintelligence.com/2012/11/19/recurrent-somatic-mutations-in-chromatin-remodeling-and-ubiquitin-ligase-complex-genes-in-serous-endometrial-tumors/

Metabolic drivers in aggressive brain tumors,  pkandala
https://pharmaceuticalintelligence.com/2012/11/11/metabolic-drivers-in-aggressive-brain-tumors/

Personalized medicine-based cure for cancer might not be far away, R. Saxena
https://pharmaceuticalintelligence.com/2012/11/20/personalized-medicine-based-cure-for-cancer-might-not-be-far-away/

Response to Multiple Cancer Drugs through Regulation of TGF-β Receptor Signaling: a MED12 Control, A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/11/21/response-to-multiple-cancer-drugs-through-regulation-of-tgf-%CE%B2-receptor-signaling-a-med12-control/

Human Variome Project: encyclopedic catalog of sequence variants indexed to the human genome sequence,  A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/11/24/human-variome-project-encyclopedic-catalog-of-sequence-variants-indexed-to-the-human-genome-sequence/

Prostate Cancer Cells: Histone Deacetylase Inhibitors Induce Epithelial-to-Mesenchymal Transition,  SJ Williams
https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/

Tumor Imaging and Targeting: Predicting Tumor Response to Treatment: Where we stand?, R. Saxena
https://pharmaceuticalintelligence.com/2012/12/13/imaging-and-targeting-the-tumor-predicting-tumor-response-where-we-stand/

Nanotechnology: Detecting and Treating metastatic cancer in the lymph node, T. Barliya
https://pharmaceuticalintelligence.com/2012/12/19/nanotechnology-detecting-and-treating-metastatic-cancer-in-the-lymph-node/

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin, SJ Williams
https://pharmaceuticalintelligence.com/2013/01/12/heroes-in-medical-research-barnett-rosenberg-and-the-discovery-of-cisplatin/

Inspiration From Dr. Maureen Cronin’s Achievements in Applying Genomic Sequencing to Cancer Diagnostics,  A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/10/inspiration-from-dr-maureen-cronins-achievements-in-applying-genomic-sequencing-to-cancer-diagnostics/

The “Cancer establishments” examined by James Watson, co-discoverer of DNA w/Crick, 4/1953,      A. Lev-Ari
https://pharmaceuticalintelligence.com/2013/01/09/the-cancer-establishments-examined-by-james-watson-co-discover-of-dna-wcrick-41953/

Nanotech Therapy for Breast Cancer. T. Barlyia
https://pharmaceuticalintelligence.com/2012/12/09/naotech-therapy-for-breast-cancer/

Dasatinib in Combination With Other Drugs for Advanced, Recurrent Ovarian Cancer,  pkandala
https://pharmaceuticalintelligence.com/2012/12/08/dasatinib-in-combination-with-other-drugs-for-advanced-recurrent-ovarian-cancer/

Squeezing Ovarian Cancer Cells to Predict Metastatic Potential: Cell Stiffness as Possible Biomarker, pkandala
https://pharmaceuticalintelligence.com/2012/12/08/squeezing-ovarian-cancer-cells-to-predict-metastatic-potential-cell-stiffness-as-possible-biomarker/

Hypothesis – following on James Watson,  LHB
https://pharmaceuticalintelligence.com/2013/01/27/novel-cancer-h…ts-are-harmful/

Otto Warburg, A Giant of Modern Cellular Biology, LHB
https://pharmaceuticalintelligence.com/2012/11/02/otto-warburg-a-giant-of-modern-cellular-biology/

Is the Warburg Effect the cause or the effect of cancer: A 21st Century View?  LHB
https://pharmaceuticalintelligence.com/2012/10/17/is-the-warburg-effect-the-cause-or-the-effect-of-cancer-a-21st-century-view/

Remembering a Great Scientist among Mentors,  LHB
https://pharmaceuticalintelligence.com/2013/01/26/remembering-a-great-scientist-among-mentors/

Portrait of a great scientist and mentor: Nathan Oram Kaplan,   LHB
https://pharmaceuticalintelligence.com/2013/01/26/portrait-of-a-great-scientist-and-mentor-nathan-oram-kaplan/

Predicting Tumor Response, Progression, and Time to Recurrence, LHB
https://pharmaceuticalintelligence.com/2012/12/20/predicting-tumor-response-progression-and-time-to-recurrence/

Directions for genomics in personalized medicine,   LHB
https://pharmaceuticalintelligence.com/2013/01/27/directions-for-genomics-in-personalized-medicine/

How mobile elements in “Junk” DNA promote cancer. Part 1: Transposon-mediated tumorigenesis,  Sjwilliams
https://pharmaceuticalintelligence.com/2012/10/31/how-mobile-elements-in-junk-dna-prote-cacner-part1-transposon-mediated-tumorigenesis/

Novel Cancer Hypothesis Suggests Antioxidants Are Harmful, LHB
https://pharmaceuticalintelligence.com/2013/01/27/novel-cancer-hypothesis-suggests-antioxidants-are-harmful/

Mitochondria: Origin from oxygen free environment, role in aerobic glycolysis, metabolic adaptation,  LHB
https://pharmaceuticalintelligence.com/2012/09/26/mitochondria-origin-from-oxygen-free-environment-role-in-aerobic-glycolysis-metabolic-adaptation/

Advances in Separations Technology for the “OMICs” and Clarification of Therapeutic Targets, LHB
https://pharmaceuticalintelligence.com/2012/10/22/advances-in-separations-technology-for-the-omics-and-clarification-of-therapeutic-targets/

Cancer Innovations from across the Web, LHB
https://pharmaceuticalintelligence.com/2012/11/02/cancer-innovations-from-across-the-web/

Mitochondrial Damage and Repair under Oxidative Stress, LHB
https://pharmaceuticalintelligence.com/2012/10/28/mitochondrial-damage-and-repair-under-oxidative-stress/

Mitochondria: More than just the “powerhouse of the cell” R. Saxena
https://pharmaceuticalintelligence.com/2012/07/09/mitochondria-more-than-just-the-powerhouse-of-the-cell/

Mitochondria and Cancer: An overview of mechanisms, R. Saxena
https://pharmaceuticalintelligence.com/2012/09/01/mitochondria-and-cancer-an-overview/

Mitochondrial fission and fusion: potential therapeutic targets?  R. Saxena
https://pharmaceuticalintelligence.com/2012/10/31/mitochondrial-fission-and-fusion-potential-therapeutic-target/

Mitochondrial mutation analysis might be “1-step” away, R. Saxena
https://pharmaceuticalintelligence.com/2012/08/14/mitochondrial-mutation-analysis-might-be-1-step-away/

β Integrin emerges as an important player in mitochondrial dysfunction associated Gastric Cancer,       R. Saxena
https://pharmaceuticalintelligence.com/2012/09/10/%CE%B2-integrin-emerges-as-an-important-player-in-mitochondrial-dysfunction-associated-gastric-cancer/

mRNA interference with cancer expression, LHB
https://pharmaceuticalintelligence.com/2012/10/26/mrna-interference-with-cancer-expression/

What can we expect of tumor therapeutic response?  LHB
https://pharmaceuticalintelligence.com/2012/12/05/what-can-we-expect-of-tumor-therapeutic-response/

Expanding the Genetic Alphabet and linking the genome to the metabolome, LHB
https://pharmaceuticalintelligence.com/2012/09/24/expanding-the-genetic-alphabet-and-linking-the-genome-to-the-metabolome/

Breast Cancer, drug resistance, and biopharmaceutical targets, LHB
https://pharmaceuticalintelligence.com/2012/09/18/breast-cancer-drug-resistance-and-biopharmaceutical-targets/

Breast Cancer: Genomic Profiling to Predict Survival: Combination of Histopathology and Gene Expression Analysis, A. Lev-Ari
https://pharmaceuticalintelligence.com/2012/12/24/breast-cancer-genomic-profiling-to-predict-survival-combination-of-histopathology-and-gene-expression-analysis/

Ubiquinin-Proteosome pathway, autophagy, the mitochondrion, proteolysis and cell apoptosis,   LHB
https://pharmaceuticalintelligence.com/2012/10/30/ubiquinin-proteosome-pathway-autophagy-the-mitochondrion-proteolysis-and-cell-apoptosis/

Identification of Biomarkers that are Related to the Actin Cytoskeleton, LHB
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

Nitric Oxide has a ubiquitous role in the regulation of glycolysis -with a concomitant influence on mitochondrial function, LHB
https://pharmaceuticalintelligence.com/2012/09/16/nitric-oxide-has-a-ubiquitous-role-in-the-regulation-of-glycolysis-with-a-concomitant-influence-on-mitochondrial-function/

Genomic Analysis: FLUIDIGM Technology in the Life Science and Agricultural Biotechnology,  A. Lev-Ari https://pharmaceuticalintelligence.com/2012/08/22/genomic-analysis-fluidigm-technology-in-the-life-science-and-agricultural-biotechnology/

Nanotechnology: Detecting and Treating metastatic cancer in the lymph node, T. Barliya
https://pharmaceuticalintelligence.com/2012/12/19/nanotechnology-detecting-and-treating-metastatic-cancer-in-the-lymph-node/

 

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