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αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

Posted in Atherogenic Processes & Pathology, Coagulation Therapy and Internal Bleeding, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, tagged absciXimab, aIIb33 selectivity, Angioplasty, Antiplatelet drug, arginine-aspartic acid sequence, atherectomy, Centocor, clinitrials, Eric Topol, FDA approval, fibronectin, Glanzmann's thrombasthenia, GPIIb?IIIa receptor, integrin, merck, monoclonal antibody, platelet activation, platelets, RCD sequence, Robert Califf, Technology Transfer, vWF on October 12, 2013| Leave a Comment »

Larry H Bernstein, MD, FCAP, Reporter and curator

αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

http://phrmaceuticalintelligence.com/2013-10-12/larryhbern_BS-Coller/αllbβ3 Antagonists As An Example of Translational Medicine Therapeutics

by Barry S. Coller, MD
Rockefeller University

Introduction

This article is a segment in several articles about platelets, platelet function, and advances in applying the surge of knowledge to therapy. In acute coronary syndromes, plaque rupture leads to thrombotic occlusion. We have also seen that the development of a plaque occurs in 3 stages, only the last of which involves plaque rupture. Platelets interact with the vascular endothelium, and platelet-endothelial as well as platelet-platelet interactions are known to be important in atherogenesis. We learned that platelets are derived from megakaryocytes that break up and these elements are released into the blood stream. It has recently been discovered that platelets can replicate in the circulation. The turnover of platelets is rapid, and platelets sre stored at room temperature with shaking, and are viable for perhaps only 3-4 days once they are received in the blood bank for use. In cardiology, the identification, isolation, and characterization of GPIIb/IIIa from the platelet was a huge advance in the potential for coronary intervention, and that potential became of paramount importance with the introduction of GPIIb/IIIa inhibitors as a standard in coronary vascular therapeutic procedures. The following manuscript by Barry Coller, at Rockefeller University, is a presentation of the GPIIb/IIIa story as an excellent example of Translational Medicine.

Search for GPIIb/IIIa inhibitor of the (anti-αIIb133 (GPIIb/IIIa) receptor)

The deliberate search for drugs to inhibit the αIIb133 (GPIIb/IIIa) receptor ushered in the era of rationally designed antiplatelet therapy and thus represents an important milestone in the evolution of antiplatelet drug development. The selection of the αIIb133 receptor as a therapeutic target rested on a broad base of basic and clinical research conducted by many investigators in the 1960s and 1970s working in the fields of platelet physiology, the rare bleeding disorder Glanzmann thrombasthenia, platelet membrane glycoproteins, integrin receptors, coronary artery pathology, and experimental thrombosis. Thus, αIIb133 was found to mediate platelet aggregation by virtually all of the physiology agonists (e.g., ADP, epinephrine, and thrombin) through a mechanism in which platelet activation by these agents results in a change in the conformation of the receptor. This is followed by increased affinity of the receptor for the multivalent ligands fibrinogen and von Willebrand factor, both of which are capable of binding to receptors on two platelets simultaneously, producing platelet crosslinking and aggregation. At about the same time, experimental studies demonstrated platelet thrombus formation at sites of vascular injury, and biochemical studies in humans demonstrated evidence of platelet activation during acute ischemic cardiovascular events.

Our own studies initially focused on platelet-fibrinogen interactions using an assay in which normal platelets agglutinated fibrinogen-coated beads. The agglutination was enhanced with platelet activators. Platelets from patients with Glanzmann thrombasthenia, who lack the αIIb133 receptor, did not agglutinate the beads. We adapted this assay to a microtiter plate system to identify monoclonal antibodies that inhibited platelet-fibrinogen interactions and then demonstrated that these antibodies bound to αIIb133. They were also more potent inhibitors of platelet aggregation than any known antiplatelet agent and produced a pattern of aggregation that was virtually identical to that found using platelets from patients with Glanzmann thrombasthenia.

I recognized the theoretical potential of using an antibody to inhibit platelets in vivo but also recognized the challenges and limitations. Since experimental models of thrombosis had been developed in the dog, and since the antibody we initially worked with did not react with dog platelets, we had to go back to our original samples to identify an antibody (7E3) that reacted with dog platelets in addition to human platelets. Since coating platelets with immunoglobulins results in their rapid elimination of the platelets from the circulation, and since the clearance is mediated by the immunoglobulin Fc region, we prepared F(ab’)2 fragments of 7E3 for our in vivo studies. Additional challenges included preparing large quantities of antibody on a very limited budget and purifying the antibodies so they contained only minimal amounts of endotoxin. With the small amount of 7E3-F(ab’)2 we initially prepared, we were able to show dose response inhibition of platelet aggregation in three dogs, achieving greater inhibition than with aspirin or ticlopidine, the only antiplatelet agents approved for human use at that time. We also devised an assay using radiolabeled 7E3 to quantify the percentage of platelet αIIbβ3 receptors that were blocked when a specific dose of 7E3-F(ab’)2 was administered in vivo. This allowed us to directly measure the effect of the agent on its target receptor on its target cell.

I considered two criteria most important in selecting the initial animal models in which to test the efficacy and safety of administering 7E3-F(ab’)2:

1) the model had to convincingly simulate a human vascular disease, and
2) aspirin had to have failed to produce complete protection from thrombosis.

The latter criterion was particularly important because I planned to stop this line of research if the 7E3-F(ab’)2 was not more efficacious than aspirin.

Ultimately, we collaborated with Dr. John Folts of the University of Wisconsin, who had developed a dog model of unstable angina by attaching a short cylindrical ring to partially occlude a coronary artery and using a hemostat to induce vascular injury. Pretreatment of the animal with 7E3-F(ab’)2 was more effective than aspirin or any other compound Dr. Folts had previously tested in preventing platelet thrombus formation, as judged by its effects on the characteristic repetitive cycles of platelet deposition and embolization. Electron microscopy of the vessels confirmed the reduction in platelet thrombi by 7E3-F(ab’)2, with only a monolayer of platelets typically deposited.

Dr. Chip Gold and his colleagues at Massachusetts General Hospital had developed a dog model to assess the effects of tissue plasminogen activator (t-PA) on experimental thrombi induced in the dog coronary artery. Although t-PA was effective in lysing the thrombi, the blood vessels rapidly reoccluded with new thrombi that were rich in platelets. Aspirin could not prevent reocclusion, whereas 7E3-F(ab’)2 not only prevented reocclusion, but also increased the speed of reperfusion by t-PA.

The next steps in drug development could not be performed in my laboratory because they required resources far in excess of those in my grant from the National Heart, Lung, and Blood Institute to study basic platelet physiology. As a result, in 1986 the Research Foundation of the State University of New York licensed the 7E3 antibody to Centocor, Inc., a new biotechnology company specializing in the diagnostic and therapeutic application of monoclonal antibodies.

Subsequent Development of 7E3

The subsequent development of 7E3 as a therapeutic agent required extensive collaboration among myself, a large number of outstanding scientists at Centocor, and many leading academic cardiologists. Many decisions and hurdles remained for us, including the decision to develop a mouse/human chimeric 7E3 Fab (c7E3 Fab); the design and execution of the toxicology studies; the assessment of the potential toxicity of 7E3 crossreactivity with αVβ3; the development of sensitive and specific assays to assess immune responses to c7E3 Fab; the design, execution, and analysis of the Phase I, II, and III studies; and the preparation, submission, and presentation of the Product Licensing Application to the Food and Drug Administration, and comparable documents to European and Scandinavian agencies.

Based on the results of the 2,099 patient EPIC trial, in which conjunctive treatment with a bolus plus infusion of c7E3 Fab significantly reduced the risk of developing an ischemic complication (death, myocardial infarction, or need for urgent intervention) after coronary artery angioplasty or atherectomy in patients at high risk of such complications, the Food and Drug Administration approved the conjunctive use of c7E3 Fab (generic name, abciximab) in high-risk angioplasty and atherectomy on December 22, 1994. Since then it has been administered to more than 2.5 million patients in the U.S., Europe, Scandinavia, and Asia. Its optimal role in treating cardiovascular disease continues to evolve in response to the introduction of new anticoagulants, antiplatelet agents, stents, and procedures.

Extended Investigations

We have also been able to apply the monoclonal antibodies we prepared to αIIb33 to the prenatal detection of Glanzmann thrombasthenia, and have used the antibodies as probes for characterizing both the biogenesis of the receptor and the conformational changes that the receptor undergoes with activation. We have been able to precisely map the 7E3 epitope on 33, providing additional insights into the mechanism by which it prevents ligand binding. We have also exploited the ability of another antibody to αIIb33 to stabilize the receptor complex in order to facilitate production of crystals of the αIIb33 headpiece; the x-ray diffraction properties of these crystals were studied in collaboration with Dr. Timothy Springer’s group at Harvard and provide the first structural information on the receptor.

In landmark studies in the 1980s, Pierschbacher and Ruoslahti demonstrated the importance of the arginine-aspartic acid (RGD) sequence in the interaction of the integrin α531 with fibronectin, and they went on to show that peptides with the RGD sequence could inhibit this interaction. Subsequent studies by many groups demonstrated that these peptides could also inhibit the interaction of platelet αIIb33 with fibrinogen and von Willebrand factor. Dr. David Phillip and Dr. Robert Scarbrough led the team at Cor Therapeutics that made a cyclic pentapeptide with high selectivity for αIIb33 over αV33 by patterning their compound on the KGD sequence in the snake venom barbourin. The resulting antiplatelet agent, eptifibatide, received FDA approval in May 1998. At Merck, Dr. Robert Gould led the team that developed the nonpeptide RGD-mimetic tirofiban, which also is selective for αIIb33 compared to αV33. It also received FDA approval in May 1998. Our recent x-ray crystallographic studies in collaboration with Dr. Springer’s group provided structural information on the mechanisms and sites of binding of these drugs with αIIb33.

Translation of Basic Science into Therapy

Many important elements and an enormous amount of good fortune were needed for the translation of the basic science information about platelet aggregation into the drug abciximab, including, but not limited to:

1) the support of basic studies of platelet physiology by the National Institutes of Health in my laboratory and many other laboratories,
2) the creation and ongoing funding of a core facility available to all faculty members to prepare monoclonal antibodies at the State University of New York at Stony Brook under the direction of Dr. Arnold Levine,
3) the 1988 Bayh-Dole Act and its subsequent amendments, and the expertise of the Technology Transfer Office at Stony Brook in licensing 7E3 to Centocor, which then provided the capital and additional expertise required for its development, and
4) the expert and enthusiastic collaboration by two large and disciplined cooperative groups of interventional cardiologists (TAMI, EPIC) under the dynamic leadership of Drs. Eric Topol and Rob Califf,

tirofiban, that were eager to test the safety and efficacy of the 7E3 derivatives. Although the translation of each new scientific discovery into improved health via novel preventive, diagnostic, or therapeutic strategies requires the blazing of a unique path, optimizing these elements and similar ones may allow the path to be shorter and/or to be traversed more easily, at a lower cost, or in a shorter period of time.

Related articles in Pharmaceutical Intelligence:

Platelets in Translational Research – 1 Larry H. Bernstein, MD, FCAP
http://pharmaceuticalintelligence.com/10-6-2013/larryhbern/Platelets_in_Translational_Research-1
Platelets in Translational Research – 2 Larry H. Bernstein, MD, FCAP
http://phramaceuticalintelligence.com/2013-10-7/larryhbern/Platelets-in-Translational-Research-2/

Do Novel Anticoagulants Affect the PT/INR? The Cases of XARELTO (rivaroxaban) and PRADAXA (dabigatran)
Vivek Lal, MBBS, MD, FCIR, Justin D Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
http://pharmaceuticalintelligence.com/2013/09/23/do-novel-anticoagulants-affect-the-ptinr-the-cases-of-xarelto-rivaroxaban-and-pradaxa-dabigatran/

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What is the Future for Genomics in Clinical Medicine?

Posted in Bio Instrumentation in Experimental Life Sciences Research, Biological Networks, Gene Regulation and Evolution, Biomarkers & Medical Diagnostics, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Genome Biology, Genomic Testing: Methodology for Diagnosis, Infectious Disease & New Antibiotic Targets, International Global Work in Pharmaceutical, Interviews with Scientific Leaders, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Personalized and Precision Medicine & Genomic Research, Pharmaceutical Industry Competitive Intelligence, Population Health Management, Genetics & Pharmaceutical, Scientist: Career considerations, Stem Cells for Regenerative Medicine, Systemic Inflammatory Response Related Disorders, Technology Transfer: Biotech and Pharmaceutical, tagged biopharmaceutical research, Cell potency, Cellular differentiation, Code of Human Life, designer drugs, DNA, drug targets, Embryonic stem cell, gene expression, Genome Research, genomics to bedside, Human Genome Project, Human subject research, Max Planck Institute for Molecular Biomedicine, Medical research, microbiome, National Human Genome Research Institute, oligomers, oligonucleotide, Personalized medicine, pioneer transcription factros, polynucleotides, post translational modifications, reproductive research, Research Methodology, Scripps Research Institute, Scripps Translational Science Institute, SNPs, Stem cell, Stress, synthetic nucleotides, Technology Transfer, telomerase, telomere, transcription factor identification, transcription factors, Transcriptomics, Translational Medicine, University of Cambridge on February 17, 2013| 5 Comments »

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.

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 stem cell and possible uses on the circulatory, nervous, and immune systems. (Photo credit: Wikipedia)

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

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:

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

Retrovirus in the human genome is active in pluripotent stem cells (sciencedaily.com)
Learning from the linker: New study sheds light on cellular reprogramming (phys.org)
A Quantitative System for Discriminating Induced Pluripotent Stem Cells, Embryonic Stem Cells and Somatic Cells (plosone.org)
Stem Cell Therapies May Improve Following Finding That Retrovirus In The Human Genome Is Active In Pluripotent Stem Cells (medicalnewstoday.com)
Leprosy spreads by reprogramming nerve cells into migratory stem cells (guardian.co.uk)
Harvard Researchers: Embryonic Cells Can Cause Cancer (salem-news.com)

BEYOND THE “MALE MODEL”: AN ALTERNATIVE FEMALE MODEL OF SCIENCE, TECHNOLOGY AND INNOVATION

Posted in International Global Work in Pharmaceutical, Pharmaceutical Industry Competitive Intelligence, Scientist: Career considerations, Statistical Methods for Research Evaluation, Technology Transfer: Biotech and Pharmaceutical, tagged Careers in Life Sciences, Doctor of Philosophy, European Union, National Science Foundation, research, Technology Transfer, Thomas Hunt Morgan on August 1, 2012| 3 Comments »

Reporter: Aviva Lev-Ari, PhD, RN

This post addresses related issues On the Career of the Life Sciences Scientist and compliments the following two posts on this Scientific Web Site:

August 1, 2012 — Introducing Career Streams into Academic Research

http://pharmaceuticalintelligence.com/2012/08/01/introducing-career-streams-into-academic-research/

June 27, 2012 — Picturing US-Trained PhDs’ Paths and Pharmaceutical Industry’s Crisis of Productivity: Partnerships between Industry and Academia

http://pharmaceuticalintelligence.com/2012/06/27/picturing-us-trained-phds-paths-pharmaceutical-industrys-crisis-of-productivity-partnerships-between-industry-and-academia/

BEYOND THE “MALE MODEL”: AN ALTERNATIVE FEMALE MODEL OF SCIENCE, TECHNOLOGY AND INNOVATION

THE TRIPLE HELIX ASSOCIATION NEWSLETTER, VOLUME 1 ISSUE 3 JULY 2012

Hélice www.triplehelixassociation.org Triple Helix X, 2012, Bandung, Indonesia . . . www.th2012.org

by Professor Henry Etzkowitz, President of the Triple Helix Association, Senior Researcher, H-STAR Institute, Stanford University, Visiting Professor, Birkbeck, London University and Edinburgh University Business School

henry.etzkowitz@stanford.edu

Professor Henry Etzkowitz paper is based on his Keynote Address to the FemTalent Conference, Barcelona, Spain 2011

I am often asked: why is a man studying women in science? The answer to that question is: my mother. She graduated with high honors in Geology from Hunter College, a public women’s college in New York City during the 1930’s depression. I had long thought that the reason why she didn’t pursue a career in geology was

because of the depression, that there were simply no jobs. However, on a research trip to the University of Texas at Austin, I visited the Engineering School which had a “wall of recognition” at the main entrance, including the names of many distinguished professors and practitioners, all of them men, who had graduated during the 1930’s depression and pursued careers in Geology. Perhaps the reason why a woman did not pursue a career in geological science at the time, might be found in the gender dynamics of science and technology. The broader question is how the best results may be attained from societal investment in human capital formation.

Firstly, we will consider the implications of findings from a study done in the 1990s, in the United States, sponsored by the National Science Foundation, of women’s experience in academic science (Etzkowitz, Kemelgor and Uzzi, 2000) including over 400 in-depth qualitative interviews conducted in a dozen leading research universities in five disciplines: biology, physics, chemistry and computer science:

1. One of the lessons from this study is that in Europe and other countries, there is a move to introduce the American system of higher education, including tenure procedures, which put a very strong emphasis on early achievement which, as we shall see, has deleterious consequences for women. Higher education policy makers in Europe and other parts of the world may want to look more closely at its effects before introducing this system, which is now taken as the gold standard in higher education. The introduction of the tenure system is driven by

international ranking procedures which drive movement from a system of relatively equal universities. Before abandoning values of equality, it should be seriously asked if introducing extreme inequalities will overall advance or inhibit quality academic research, teaching, and innovation.

2. Secondly, I discuss the Vanish Box model, derived from a four country study, sponsored by the European Union, DG Research, on Women and Technology Transfer (Ranga, et. al. 2008). During interviews with women in US academic science on Athena Unbound study, some of them would talk about colleagues who were no longer in the department, they were now in jobs elsewhere. I interviewed some of these women leaving academic science, and found that they were taking up careers in science-related professions such as science journalism, technology transfer, museology. etc. They were using their scientific training in translating science into use and spreading the results of science to a broader public. Rather than being “lost to science” as presumed by the “Leaky pipeline” thesis of science career loss; they were pursuing work-life balance in their new careers. This finding inspired the study sponsored by the European Union on Women and Technology Transfer.

3. Third, I outline a four phase model of women’s experience in science, technology, and innovation, “the Vanish Box”: after the magic trick of the “disappearance of re-appearance of a woman”. “The vanish box” model shows the dynamics of the historical experience of women in science, and questions the taken for granted “male model” of science that does not work for women or men who seek work-life balance.

4. Finally, we address the question of whether the Gender Revolution in science and technology is stalled or moving forward.

Gender Inequalities in Academic Careers

The historical relationship between status and gender provides a clue to understanding the underlying dynamics of women and men’s careers in science. Typically, there is strong participation of women in the early stages of development of a new discipline, but as the new area becomes prestigious and rewards increase, women disappear. As fields attain recognition and fruition, and the Nobel and other prizes are awarded, it is men who are there to receive them. There were a significant number of women working in “the fly room,” the drosophila genetics lab headed by Thomas Hunt Morgan at Colombia University, but as the field became prestigious, women virtually disappeared from classical genetics (Kohler, 1994).

Does this historical relationship between gender and science still hold today or has it changed? The most important finding from all the specific instances that we came across was that the most important thing holding back women’s advance in academic science was “inflexibility” of rules and procedures. It didn’t matter what the specific procedure was. For example, in the US it’s expected that that you should pursue your PhD at a different University than your undergraduate degree. This is the highest route to achievement. If a woman has a relationship, and the man moves will she leave the relationship to seek her competitive advantage?

On the other hand, if the man in the relationship moves, and the woman goes along, she may then have to move to a school that is not as good as the one which she otherwise might have gotten into with a broader range of selection. Thus, this informal rule of exogamy, mandating leaving the previous school or worksite at each point of progression, from undergraduate degree to PhD to entry level position, works against women’s advancement.

On the other hand, in Sweden the rule is the opposite. Instead of saying that you should move from one university to another, the rule is that you should stay at your own University; that if you are a highly successful junior scholar you will be kept within that University. A Swedish professor said, “why would I send my best

graduate student away? He is going to replace me when I retire.” So the rule in Sweden is endogamy that you stay within one university. Again, if a woman’s partner moves, and she moves with him, it will hurt her career, because she has left her university of origin. So which ever way the rule is, it is an inflexible rule, it has more negative consequences for women than for men. The gender policy implication is to increase flexibility in the system.

A female model of science, balancing work and family life, has been invented but it is a subsidiary and undervalued format that needs to be brought to the forefront and institutionalized. However, this would necessitate re-thinking aspects of the academic system, especially the US model, that unintentionally yet systematically works against women’s inclusion in the higher level of academic science. The US model of academic hierarchy, front loading in the academic career with a strong emphasis on youth and achievement

in the early years, is partly based on a mistaken idea that youth are more productive in science than people who are of an older age. That finding was documented in a study done by Merton and Zuckerman of “Aging and Age Structure” (1973). They found that “productivity was as high or even higher at the later stages of a scientific career”, and that makes sense. When you are more advanced in your career you have more access to resources, more

graduate students, more research associates, more people working with you. Co-authorship arises from having members of your research group being highly productive. Nevertheless, there is a strong belief that youth makes disproportionate scientific advances, and this has been the basis of a system in which there is a strong emphasis on early achievement during the first years of your career. There is a race to accumulate publications and research grants in order to be given a permanent position in a high status American University.

In Europe, the tradition has been once you are hired there is a probationary period and then you continue to be promoted or not. But in the United States there is a very sharp dividing line: an “up or out” system. The implications for women’s advancement in science, includes the contradiction between the “tenure clock”, typically of seven years, and the “biological clock”, the time when is possible to have children, and these coincide. Thus, women have to make the choice to postpone having children to after tenure, which then becomes their middle or late 30s. There were some women who didn’t want to postpone, and some of them rethought

their commitment to academic science and left for that reason. Occasionally, in some universities there has been some reform of these procedures to try to accommodate women by extending the clock. i.e. you can apply for a year extension to reduce your time in the workplace and/or take a break in order to have more time for one’s young children. Even this attempt to ameliorate the male model of science and make it more amenable to women’s

participation contains a contradiction: women are concerned that if they apply for this privilege that it will be held against them in the final review. The academic system requires a demonstration of full commitment to racing the clock, otherwise you will be viewed as insufficiently competitive. Moderating the conditions will be held against you, or at least that is the fear. The attempted reform has its dangers since many women feel that they may be given points off for taking advantage of the attempt to change the rules. The contradiction between the tenure clock and the biological clock encourages some women in academic science to seek an alternative career path.

An American professor talked about a leading female student who stayed in the same city and took a job at a local teaching college. But an exception was made for her because she was such an outstanding scholar that she was then brought back to the leading University in the same city where she had received her PhD and allowed to pursue a career at that university. The rule was counterproductive to the best use of talent, but this case was an

unusual exception. However, it is one that can be more regularly made if we are thinking of how to revise the system that works against women by following an implicit male model of science. Another negative factor is the “two out of three” time bind. In interviews in the US and Mexico, it was found that if you are trying to do three things, most women usually find it was too much, they could do advanced research in a highly productive way, and manage their family relationship, but that didn’t leave time for spending time in local politicking and talking with people, which is the way towards advancing within the academic system. So they could advance in their research career, but not in the career that would lead to becoming a chair person or administrator within academia. So this is the two out of three time bind.

Traditionally there has been a gendered division of labor where men worked with men and women with women, in gendered occupations. Some think that this occurred as a basis of naturally occurring gender divisions. But historically we can see that these gendered occupations change over time. I did my masters thesis on the male nurse, titled “the Precarious Identity of the Male Nurse” (Etzkowitz, 1971). The nursing profession in the

nineteenth century was entirely male, and began to change over to a female profession by the end of the nineteenth century, and by the middle of the twentieth century it was virtually entirely female. Thus, gendered professions can change over time and are subject to revision.

From “Leaky Pipeline” to the “Vanish Box”

The pipeline model has been based upon the movement from schooling into higher education and into careers; the premise that there should be an unimpeded flow. Over a period of twenty years, at maximum, women should be at the highest level of any occupation. Recruitment has taken place: young women now make up equal numbers in bachelor’s degrees, and the numbers are moving up in the PhDs. But they have not moved up at the same rate to the associate and full professor levels. The pipeline has not worked by filling it at one end, and expecting a changed result at the other. We need to make changes in the system to make the pipeline work. A gender neutral occupation would be one with flexibility in the role, and with balance between on-site and off-site work, and the possibility of equal participation of both genders in the occupation. What happens to women who, for one reason or another, don’t continue in an academic career in science? This was the question that we posed in: “Women in Science and Technology”(WIST) sponsored by the European Union. We identified that women who had left academic science, were reappearing in science related professions, using their scientific, networking and social skills in these new professions. In the UK, when opportunities opened up in the mid 1990s, female PhDs entered this career, following the States where women had risen to the top of their profession as heads of offices at major universities. From this study of women disappearing and reappearing from academia to science related professions, we developed a concept that we called the “vanish box” model (Etzkowitz and Ranga, 2011), that takes place in four stages:

1. The first is the disappearance of women: the disappearance that we found in the Athena Unbound study, the exclusionary practices, or the taken for granted male model of science which did not take account of women’s needs, of women’s life chances and lifestyles. They weren’t found at the highest levels of academic science to the extent that would be expected if the pipeline was working as it was supposed to, with women flowing in and being promoted up over a period of time. So disappearance.

2. The disappeared women are in the reserve army: at home or in part time positions unemployed or underemployed. The reserve army is called back when there is an emergency or a shortage. For example, during World War ll women PhD’s who had been unemployed or working as volunteers in their husband’s laboratory were called into full-time positions in the Manhattan Project and other Labs. After the War, some began to get academic positions and rose to the highest level after having been in the reserve army for many years.

3. The third phase of the model is the creation of new opportunities; either by emergency situations, or by the creation of new professions that require people with scientific training. An example of this was the Technology Transfer profession that we studied: a new profession that required people with scientific training and background and business training, and typically people with a scientific background would learn the business skills, take courses or even a master’s degrees in business. This provided opportunities, but there are still limitations: the new profession wasn’t as prestigious as the old one, and it had both advantages and disadvantages: working in a technology transfer office gives more of an opportunity for a work life balance, but the prestige of the profession isn’t that high and the opportunities for advancement are limited.

4. On the other hand, as the knowledge society advances, professions that translate knowledge into use become more prestigious and the profession also rises in status and prestige over time. That is what has been happening with technology transfer. The question that arises is, will it follow the same model of classical genetics, or will it lead to a new model of a gender neutral profession, with men and women working at the highest levels in a situation that allows for work-life balance? There is some evidence that this may be happening in small biotech firms. A recent study found that women recruited into these firms were taken seriously in their work, they were being promoted, and so the start-up biotechnology firm has offered evidence that there may be a changing relationship between gender and career advancement and new possibilities available in this area.

Beyond the “Male Model”: An Alternative Female Model

The American sociologist Cecilia Ridgeway has set forth the thesis of a stalled revolution, in the 1970’s large numbers of women entered professions in law and medicine but more recently advancement of women has halted. Pay differentials continue to exist. Women have not risen to positions in board of directors of firms to the same extent that might be expected. On the other hand, women now make up a majority of bachelor’s degrees recipients – over fifty percent in some places and as high as sixty percent in others. Forty years ago the percentage of women at MIT could be counted on the fingers of one hand. Today half of the undergraduate students at MIT are female. Once you get to the level of twenty percent social relations within organizations start to change; but they really transform at the fifty percent level. Typically in women’s entrepreneurship the service occupations make up the majority; but in Catalonia, there is a major change going on as the majority of the women in a program to support

entrepreneurship were in science and technology related occupations. The woman running this program said that the key issue is working with these entrepreneurs is how to grow their firms and still retain a work life balance. So the revolution is moving here. Academia is still resistant to change, but business has been moving faster, and Academia has to learn from industry. That is the next stage in making the gender revolution in science and technology.

To this end, the relationship between career structure and life cycle needs to be rethought (Etzkowitz and Stein, 1978). The current taken-for-granted career path is based on implicit male assumptions that do not take into account women’s greater responsibilities for family maintenance and societal reproduction that persist, even given good faith efforts on the part of men to play a greater role in child care (Kayyem, 2012). In the male model,

imposed on women as well, significant early achievement, typically involving a high time commitment, is the prerequisite for subsequent high-level positions. It is hypothesized that women’s difficulties in conforming to this model explains at least part of the variance in the paucity of women in high-level positions even as their participation rates increase.

An alternative female model, with a higher time commitment after child-rearing years, may be discerned. A Rockefeller University Professor, who started on her PhD at a later than usual age, and US Secretary of State Hilary Clinton, exemplify this alternative model that needs to be legitimized as an alternative path to high achievement. The current offering of a relaxed early career path in law firms is stigmatized as a “mommy track”

and reified into a permanent blockage to later high flying. When an alternative “female model” is available for women and men, gender democracy in science, technology and innovation, as well as in the larger society, will be a reality.

REFERENCES

Etzkowitz, H (1971), The Male Nurse: Sexual Separation of Labor in Society, Journal of Marriage and the Family.

Etzkowitz, H, and P. Stein. (1978) The Life Spiral: Human Needs and Adult Roles Journal of Economic and Family Issues. 1:4: 434-446

Etzkowitz, H, Kemelgor, C and Uzzi, B (2000), Athena Unbound: The Advancement of Women in Science and Technology Cambridge University Press.

Etzkowitz, H and Ranga, M (2011), Gender Dynamics in Science and Technology: From the “Leaky Pipeline” to the Vanish Box, Brussels Economic Review, 54:2/3.

Kayyem, J. (2012) The working moms debate International Herald Tribune June 27 Wednesday p.8.

Kohler, R. (1994) Lords of the Fly: Drosophila Genetics and the Experimental Life. Chicago: University of Chicago Press

Ranga, M et al (2008), Gender Patterns in Technology Transfer: Social innovation in the making?, Research Global, 4-5.

Ridgeway, C (2011), Framed by Gender: How Gender Inequality Persists in the Modern World, Oxford: Oxford University Press.

Zuckerman, H and Merton R (1972), Age, Ageing and Age Structure in Science. In Ageing and Society, Riley, M, Johnson, M and Foner, A, eds, Vol 3. New York: Russell Sage Foundation.