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Live Notes, Real Time Conference Coverage AACR 2020: Tuesday June 23, 2020 3:00 PM-5:30 PM Educational Sessions

Reporter: Stephen J. Williams, PhD

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uesday, June 23

3:00 PM – 5:00 PM EDT

Virtual Educational Session
Tumor Biology, Bioinformatics and Systems Biology

The Clinical Proteomic Tumor Analysis Consortium: Resources and Data Dissemination

This session will provide information regarding methodologic and computational aspects of proteogenomic analysis of tumor samples, particularly in the context of clinical trials. Availability of comprehensive proteomic and matching genomic data for tumor samples characterized by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) and The Cancer Genome Atlas (TCGA) program will be described, including data access procedures and informatic tools under development. Recent advances on mass spectrometry-based targeted assays for inclusion in clinical trials will also be discussed.

Amanda G Paulovich, Shankha Satpathy, Meenakshi Anurag, Bing Zhang, Steven A Carr

Methods and tools for comprehensive proteogenomic characterization of bulk tumor to needle core biopsies

Shankha Satpathy
  • TCGA has 11,000 cancers with >20,000 somatic alterations but only 128 proteins as proteomics was still young field
  • CPTAC is NCI proteomic effort
  • Chemical labeling approach now method of choice for quantitative proteomics
  • Looked at ovarian and breast cancers: to measure PTM like phosphorylated the sample preparation is critical

 

Data access and informatics tools for proteogenomics analysis

Bing Zhang
  • Raw and processed data (raw MS data) with linked clinical data can be extracted in CPTAC
  • Python scripts are available for bioinformatic programming

 

Pathways to clinical translation of mass spectrometry-based assays

Meenakshi Anurag

·         Using kinase inhibitor pulldown (KIP) assay to identify unique kinome profiles

·         Found single strand break repair defects in endometrial luminal cases, especially with immune checkpoint prognostic tumors

·         Paper: JNCI 2019 analyzed 20,000 genes correlated with ET resistant in luminal B cases (selected for a list of 30 genes)

·         Validated in METABRIC dataset

·         KIP assay uses magnetic beads to pull out kinases to determine druggable kinases

·         Looked in xenografts and was able to pull out differential kinomes

·         Matched with PDX data so good clinical correlation

·         Were able to detect ESR1 fusion correlated with ER+ tumors

Tuesday, June 23

3:00 PM – 5:00 PM EDT

Virtual Educational Session
Survivorship

Artificial Intelligence and Machine Learning from Research to the Cancer Clinic

The adoption of omic technologies in the cancer clinic is giving rise to an increasing number of large-scale high-dimensional datasets recording multiple aspects of the disease. This creates the need for frameworks for translatable discovery and learning from such data. Like artificial intelligence (AI) and machine learning (ML) for the cancer lab, methods for the clinic need to (i) compare and integrate different data types; (ii) scale with data sizes; (iii) prove interpretable in terms of the known biology and batch effects underlying the data; and (iv) predict previously unknown experimentally verifiable mechanisms. Methods for the clinic, beyond the lab, also need to (v) produce accurate actionable recommendations; (vi) prove relevant to patient populations based upon small cohorts; and (vii) be validated in clinical trials. In this educational session we will present recent studies that demonstrate AI and ML translated to the cancer clinic, from prognosis and diagnosis to therapy.
NOTE: Dr. Fish’s talk is not eligible for CME credit to permit the free flow of information of the commercial interest employee participating.

Ron C. Anafi, Rick L. Stevens, Orly Alter, Guy Fish

Overview of AI approaches in cancer research and patient care

Rick L. Stevens
  • Deep learning is less likely to saturate as data increases
  • Deep learning attempts to learn multiple layers of information
  • The ultimate goal is prediction but this will be the greatest challenge for ML
  • ML models can integrate data validation and cross database validation
  • What limits the performance of cross validation is the internal noise of data (reproducibility)
  • Learning curves: not the more data but more reproducible data is important
  • Neural networks can outperform classical methods
  • Important to measure validation accuracy in training set. Class weighting can assist in development of data set for training set especially for unbalanced data sets

Discovering genome-scale predictors of survival and response to treatment with multi-tensor decompositions

Orly Alter
  • Finding patterns using SVD component analysis. Gene and SVD patterns match 1:1
  • Comparative spectral decompositions can be used for global datasets
  • Validation of CNV data using this strategy
  • Found Ras, Shh and Notch pathways with altered CNV in glioblastoma which correlated with prognosis
  • These predictors was significantly better than independent prognostic indicator like age of diagnosis

 

Identifying targets for cancer chronotherapy with unsupervised machine learning

Ron C. Anafi
  • Many clinicians have noticed that some patients do better when chemo is given at certain times of the day and felt there may be a circadian rhythm or chronotherapeutic effect with respect to side effects or with outcomes
  • ML used to determine if there is indeed this chronotherapy effect or can we use unstructured data to determine molecular rhythms?
  • Found a circadian transcription in human lung
  • Most dataset in cancer from one clinical trial so there might need to be more trials conducted to take into consideration circadian rhythms

Stratifying patients by live-cell biomarkers with random-forest decision trees

Stratifying patients by live-cell biomarkers with random-forest decision trees

Guy Fish CEO Cellanyx Diagnostics

 

Tuesday, June 23

3:00 PM – 5:00 PM EDT

Virtual Educational Session
Tumor Biology, Molecular and Cellular Biology/Genetics, Bioinformatics and Systems Biology, Prevention Research

The Wound Healing that Never Heals: The Tumor Microenvironment (TME) in Cancer Progression

This educational session focuses on the chronic wound healing, fibrosis, and cancer “triad.” It emphasizes the similarities and differences seen in these conditions and attempts to clarify why sustained fibrosis commonly supports tumorigenesis. Importance will be placed on cancer-associated fibroblasts (CAFs), vascularity, extracellular matrix (ECM), and chronic conditions like aging. Dr. Dvorak will provide an historical insight into the triad field focusing on the importance of vascular permeability. Dr. Stewart will explain how chronic inflammatory conditions, such as the aging tumor microenvironment (TME), drive cancer progression. The session will close with a review by Dr. Cukierman of the roles that CAFs and self-produced ECMs play in enabling the signaling reciprocity observed between fibrosis and cancer in solid epithelial cancers, such as pancreatic ductal adenocarcinoma.

Harold F Dvorak, Sheila A Stewart, Edna Cukierman

 

The importance of vascular permeability in tumor stroma generation and wound healing

Harold F Dvorak

Aging in the driver’s seat: Tumor progression and beyond

Sheila A Stewart

Why won’t CAFs stay normal?

Edna Cukierman

 

Tuesday, June 23

3:00 PM – 5:00 PM EDT

 

 

 

 

 

 

 

Other Articles on this Open Access  Online Journal on Cancer Conferences and Conference Coverage in Real Time Include

Press Coverage
Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Symposium: New Drugs on the Horizon Part 3 12:30-1:25 PM
Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on NCI Activities: COVID-19 and Cancer Research 5:20 PM
Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on Evaluating Cancer Genomics from Normal Tissues Through Metastatic Disease 3:50 PM
Live Notes, Real Time Conference Coverage 2020 AACR Virtual Meeting April 28, 2020 Session on Novel Targets and Therapies 2:35 PM

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Structure-guided Drug Discovery: (1) The Coronavirus 3CL hydrolase (Mpro) enzyme (main protease) essential for proteolytic maturation of the virus and (2) viral protease, the RNA polymerase, the viral spike protein, a viral RNA as promising two targets for discovery of cleavage inhibitors of the viral spike polyprotein preventing the Coronavirus Virion the spread of infection

 

Curators and Reporters: Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN

 

Therapeutical options to coronavirus (2019-nCoV) include consideration of the following:

(a) Monoclonal and polyclonal antibodies

(b)  Vaccines

(c)  Small molecule treatments (e.g., chloroquinolone and derivatives), including compounds already approved for other indications 

(d)  Immuno-therapies derived from human or other sources

 

 

Structure of the nCoV trimeric spike

The World Health Organization has declared the outbreak of a novel coronavirus (2019-nCoV) to be a public health emergency of international concern. The virus binds to host cells through its trimeric spike glycoprotein, making this protein a key target for potential therapies and diagnostics. Wrapp et al. determined a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy. Using biophysical assays, the authors show that this protein binds at least 10 times more tightly than the corresponding spike protein of severe acute respiratory syndrome (SARS)–CoV to their common host cell receptor. They also tested three antibodies known to bind to the SARS-CoV spike protein but did not detect binding to the 2019-nCoV spike protein. These studies provide valuable information to guide the development of medical counter-measures for 2019-nCoV. [Bold Face Added by ALA]

Science, this issue p. 1260

Abstract

The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo–electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.

SOURCE
Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation
  1. Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.

  2. 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
  1. Corresponding author. Email: jmclellan@austin.utexas.edu
  1. * These authors contributed equally to this work.

Science  13 Mar 2020:
Vol. 367, Issue 6483, pp. 1260-1263
DOI: 10.1126/science.abb2507

 

02/04/2020

New Coronavirus Protease Structure Available

PDB data provide a starting point for structure-guided drug discovery

A high-resolution crystal structure of COVID-19 (2019-nCoV) coronavirus 3CL hydrolase (Mpro) has been determined by Zihe Rao and Haitao Yang’s research team at ShanghaiTech University. Rapid public release of this structure of the main protease of the virus (PDB 6lu7) will enable research on this newly-recognized human pathogen.

Recent emergence of the COVID-19 coronavirus has resulted in a WHO-declared public health emergency of international concern. Research efforts around the world are working towards establishing a greater understanding of this particular virus and developing treatments and vaccines to prevent further spread.

While PDB entry 6lu7 is currently the only public-domain 3D structure from this specific coronavirus, the PDB contains structures of the corresponding enzyme from other coronaviruses. The 2003 outbreak of the closely-related Severe Acute Respiratory Syndrome-related coronavirus (SARS) led to the first 3D structures, and today there are more than 200 PDB structures of SARS proteins. Structural information from these related proteins could be vital in furthering our understanding of coronaviruses and in discovery and development of new treatments and vaccines to contain the current outbreak.

The coronavirus 3CL hydrolase (Mpro) enzyme, also known as the main protease, is essential for proteolytic maturation of the virus. It is thought to be a promising target for discovery of small-molecule drugs that would inhibit cleavage of the viral polyprotein and prevent spread of the infection.

Comparison of the protein sequence of the COVID-19 coronavirus 3CL hydrolase (Mpro) against the PDB archive identified 95 PDB proteins with at least 90% sequence identity. Furthermore, these related protein structures contain approximately 30 distinct small molecule inhibitors, which could guide discovery of new drugs. Of particular significance for drug discovery is the very high amino acid sequence identity (96%) between the COVID-19 coronavirus 3CL hydrolase (Mpro) and the SARS virus main protease (PDB 1q2w). Summary data about these closely-related PDB structures are available (CSV) to help researchers more easily find this information. In addition, the PDB houses 3D structure data for more than 20 unique SARS proteins represented in more than 200 PDB structures, including a second viral protease, the RNA polymerase, the viral spike protein, a viral RNA, and other proteins (CSV).

Public release of the COVID-19 coronavirus 3CL hydrolase (Mpro), at a time when this information can prove most vital and valuable, highlights the importance of open and timely availability of scientific data. The wwPDB strives to ensure that 3D biological structure data remain freely accessible for all, while maintaining as comprehensive and accurate an archive as possible. We hope that this new structure, and those from related viruses, will help researchers and clinicians address the COVID-19 coronavirus global public health emergency.

Update: Released COVID-19-related PDB structures include

  • PDB structure 6lu7 (X. Liu, B. Zhang, Z. Jin, H. Yang, Z. Rao Crystal structure of COVID-19 main protease in complex with an inhibitor N3 doi: 10.2210/pdb6lu7/pdb) Released 2020-02-05
  • PDB structure 6vsb (D. Wrapp, N. Wang, K.S. Corbett, J.A. Goldsmith, C.-L. Hsieh, O. Abiona, B.S. Graham, J.S. McLellan (2020) Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation Science doi: 10.1126/science.abb2507) Released 2020-02-26
  • PDB structure 6lxt (Y. Zhu, F. Sun Structure of post fusion core of 2019-nCoV S2 subunit doi: 10.2210/pdb6lxt/pdb) Released 2020-02-26
  • PDB structure 6lvn (Y. Zhu, F. Sun Structure of the 2019-nCoV HR2 Domain doi: 10.2210/pdb6lvn/pdb) Released 2020-02-26
  • PDB structure 6vw1
    J. Shang, G. Ye, K. Shi, Y.S. Wan, H. Aihara, F. Li Structural basis for receptor recognition by the novel coronavirus from Wuhan doi: 10.2210/pdb6vw1/pdb
    Released 2020-03-04
  • PDB structure 6vww
    Y. Kim, R. Jedrzejczak, N. Maltseva, M. Endres, A. Godzik, K. Michalska, A. Joachimiak, Center for Structural Genomics of Infectious Diseases Crystal Structure of NSP15 Endoribonuclease from SARS CoV-2 doi: 10.2210/pdb6vww/pdb
    Released 2020-03-04
  • PDB structure 6y2e
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure of the free enzyme of the SARS-CoV-2 (2019-nCoV) main protease doi: 10.2210/pdb6y2e/pdb
    Released 2020-03-04
  • PDB structure 6y2f
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure (monoclinic form) of the complex resulting from the reaction between SARS-CoV-2 (2019-nCoV) main protease and tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate (alpha-ketoamide 13b) doi: 10.2210/pdb6y2f/pdb
    Released 2020-03-04
  • PDB structure 6y2g
    L. Zhang, X. Sun, R. Hilgenfeld Crystal structure (orthorhombic form) of the complex resulting from the reaction between SARS-CoV-2 (2019-nCoV) main protease and tert-butyl (1-((S)-1-(((S)-4-(benzylamino)-3,4-dioxo-1-((S)-2-oxopyrrolidin-3-yl)butan-2-yl)amino)-3-cyclopropyl-1-oxopropan-2-yl)-2-oxo-1,2-dihydropyridin-3-yl)carbamate (alpha-ketoamide 13b) doi: 10.2210/pdb6y2g/pdb
    Released 2020-03-04
First page image

Abstract

Coronavirus disease 2019 (COVID-19) is a global pandemic impacting nearly 170 countries/regions and more than 285,000 patients worldwide. COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), which invades cells through the angiotensin converting enzyme 2 (ACE2) receptor. Among those with COVID-19, there is a higher prevalence of cardiovascular disease and more than 7% of patients suffer myocardial injury from the infection (22% of the critically ill). Despite ACE2 serving as the portal for infection, the role of ACE inhibitors or angiotensin receptor blockers requires further investigation. COVID-19 poses a challenge for heart transplantation, impacting donor selection, immunosuppression, and post-transplant management. Thankfully there are a number of promising therapies under active investigation to both treat and prevent COVID-19. Key Words: COVID-19; myocardial injury; pandemic; heart transplant

SOURCE

https://www.ahajournals.org/doi/pdf/10.1161/CIRCULATIONAHA.120.046941

ACE2

  • Towler P, Staker B, Prasad SG, Menon S, Tang J, Parsons T, Ryan D, Fisher M, Williams D, Dales NA, Patane MA, Pantoliano MW (Apr 2004). “ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis”The Journal of Biological Chemistry279 (17): 17996–8007. doi:10.1074/jbc.M311191200PMID 14754895.

 

  • Turner AJ, Tipnis SR, Guy JL, Rice G, Hooper NM (Apr 2002). “ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors”Canadian Journal of Physiology and Pharmacology80 (4): 346–53. doi:10.1139/y02-021PMID 12025971.

 

  •  Zhang, Haibo; Penninger, Josef M.; Li, Yimin; Zhong, Nanshan; Slutsky, Arthur S. (3 March 2020). “Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target”Intensive Care Medicine. Springer Science and Business Media LLC. doi:10.1007/s00134-020-05985-9ISSN 0342-4642PMID 32125455.

 

  • ^ Gurwitz, David (2020). “Angiotensin receptor blockers as tentative SARS‐CoV‐2 therapeutics”Drug Development Researchdoi:10.1002/ddr.21656PMID 32129518.

 

Angiotensin converting enzyme 2 (ACE2)

is an exopeptidase that catalyses the conversion of angiotensin I to the nonapeptide angiotensin[1-9][5] or the conversion of angiotensin II to angiotensin 1-7.[6][7] ACE2 has direct effects on cardiac functiona and is expressed predominantly in vascular endothelial cells of the heart and the kidneys.[8] ACE2 is not sensitive to the ACE inhibitor drugs used to treat hypertension.[9]

ACE2 receptors have been shown to be the entry point into human cells for some coronaviruses, including the SARS virus.[10] A number of studies have identified that the entry point is the same for SARS-CoV-2,[11] the virus that causes COVID-19.[12][13][14][15]

Some have suggested that a decrease in ACE2 could be protective against Covid-19 disease[16], but others have suggested the opposite, that Angiotensin II receptor blocker drugs could be protective against Covid-19 disease via increasing ACE2, and that these hypotheses need to be tested by datamining of clinical patient records.[17]

REFERENCES

https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme_2

 

FOLDING@HOME TAKES UP THE FIGHT AGAINST COVID-19 / 2019-NCOV

We need your help! Folding@home is joining researchers around the world working to better understand the 2019 Coronavirus (2019-nCoV) to accelerate the open science effort to develop new life-saving therapies. By downloading Folding@Home, you can donate your unused computational resources to the Folding@home Consortium, where researchers working to advance our understanding of the structures of potential drug targets for 2019-nCoV that could aid in the design of new therapies. The data you help us generate will be quickly and openly disseminated as part of an open science collaboration of multiple laboratories around the world, giving researchers new tools that may unlock new opportunities for developing lifesaving drugs.

2019-nCoV is a close cousin to SARS coronavirus (SARS-CoV), and acts in a similar way. For both coronaviruses, the first step of infection occurs in the lungs, when a protein on the surface  of the virus binds to a receptor protein on a lung cell. This viral protein is called the spike protein, depicted in red in the image below, and the receptor is known as ACE2. A therapeutic antibody is a type of protein that can block the viral protein from binding to its receptor, therefore preventing the virus from infecting the lung cell. A therapeutic antibody has already been developed for SARS-CoV, but to develop therapeutic antibodies or small molecules for 2019-nCoV, scientists need to better understand the structure of the viral spike protein and how it binds to the human ACE2 receptor required for viral entry into human cells.

Proteins are not stagnant—they wiggle and fold and unfold to take on numerous shapes.  We need to study not only one shape of the viral spike protein, but all the ways the protein wiggles and folds into alternative shapes in order to best understand how it interacts with the ACE2 receptor, so that an antibody can be designed. Low-resolution structures of the SARS-CoV spike protein exist and we know the mutations that differ between SARS-CoV and 2019-nCoV.  Given this information, we are uniquely positioned to help model the structure of the 2019-nCoV spike protein and identify sites that can be targeted by a therapeutic antibody. We can build computational models that accomplish this goal, but it takes a lot of computing power.

This is where you come in! With many computers working towards the same goal, we aim to help develop a therapeutic remedy as quickly as possible. By downloading Folding@home here [LINK] and selecting to contribute to “Any Disease”, you can help provide us with the computational power required to tackle this problem. One protein from 2019-nCoV, a protease encoded by the viral RNA, has already been crystallized. Although the 2019-nCoV spike protein of interest has not yet been resolved bound to ACE2, our objective is to use the homologous structure of the SARS-CoV spike protein to identify therapeutic antibody targets.

This illustration, created at the Centers for Disease Control and Prevention (CDC), reveals ultrastructural morphology exhibited by coronaviruses. Note the spikes that adorn the outer surface of the virus, which impart the look of a corona surrounding the virion, when viewed electron microscopically. A novel coronavirus virus was identified as the cause of an outbreak of respiratory illness first detected in Wuhan, China in 2019.

Image and Caption Credit: Alissa Eckert, MS; Dan Higgins, MAM available at https://phil.cdc.gov/Details.aspx?pid=23311

Structures of the closely related SARS-CoV spike protein bound by therapeutic antibodies may help rapidly design better therapies. The three monomers of the SARS-CoV spike protein are shown in different shades of red; the antibody is depicted in green. [PDB: 6NB7 https://www.rcsb.org/structure/6nb7]

(post authored by Ariana Brenner Clerkin)

References:

PDB 6lu7 structure summary ‹ Protein Data Bank in Europe (PDBe) ‹ EMBL-EBI https://www.ebi.ac.uk/pdbe/entry/pdb/6lu7 (accessed Feb 5, 2020).

Tian, X.; Li, C.; Huang, A.; Xia, S.; Lu, S.; Shi, Z.; Lu, L.; Jiang, S.; Yang, Z.; Wu, Y.; et al. Potent Binding of 2019 Novel Coronavirus Spike Protein by a SARS Coronavirus-Specific Human Monoclonal Antibody; preprint; Microbiology, 2020. https://doi.org/10.1101/2020.01.28.923011.

Walls, A. C.; Xiong, X.; Park, Y. J.; Tortorici, M. A.; Snijder, J.; Quispe, J.; Cameroni, E.; Gopal, R.; Dai, M.; Lanzavecchia, A.; et al. Unexpected Receptor Functional Mimicry Elucidates Activation of Coronavirus Fusion. Cell 2019176, 1026-1039.e15. https://doi.org/10.2210/pdb6nb7/pdb.

SOURCE

https://foldingathome.org/2020/02/27/foldinghome-takes-up-the-fight-against-covid-19-2019-ncov/

UPDATED 3/13/2020

I am reposting the following Science blog post from Derrick Lowe as is and ask people go browse through the comments on his Science blog In the Pipeline because, as Dr. Lowe states that in this current crisis it is important to disseminate good information as quickly as possible so wanted the readers here to have the ability to read his great posting on this matter of Covid-19.  Also i would like to direct readers to the journal Science opinion letter concerning how important it is to rebuild the trust in good science and the scientific process.  The full link for the following In the Pipeline post is: https://blogs.sciencemag.org/pipeline/archives/2020/03/06/covid-19-small-molecule-therapies-reviewed

A Summary of current potential repurposed therapeutics for COVID-19 Infection from In The Pipeline: A Science blog from Derick Lowe

Covid-19 Small Molecule Therapies Reviewed

Let’s take inventory on the therapies that are being developed for the coronavirus epidemic. Here is a very thorough list of at Biocentury, and I should note that (like Stat and several other organizations) they’re making all their Covid-19 content free to all readers during this crisis. I’d like to zoom in today on the potential small-molecule therapies, since some of these have the most immediate prospects for use in the real world.

The ones at the front of the line are repurposed drugs that are already approved for human use, for a lot of obvious reasons. The Biocentury list doesn’t cover these, but here’s an article at Nature Biotechnology that goes into detail. Clinical trials are a huge time sink – they sort of have to be, in most cases, if they’re going to be any good – and if you’ve already done all that stuff it’s a huge leg up, even if the drug itself is not exactly a perfect fit for the disease. So what do we have? The compound that is most advanced is probably remdesivir from Gilead, at right. This has been in development for a few years as an RNA virus therapy – it was originally developed for Ebola, and has been tried out against a whole list of single-strand RNA viruses. That includes the related coronaviruses SARS and MERS, so Covid-19 was an obvious fit.

The compound is a prodrug – that phosphoramide gets cleaved off completely, leaving the active 5-OH compound GS-44-1524. It mechanism of action is to get incorporated into viral RNA, since it’s taken up by RNA polymerase and it largely seems to evade proofreading. This causes RNA termination trouble later on, since that alpha-nitrile C-nucleoside is not exactly what the virus is expecting in its genome at that point, and thus viral replication is inhibited.

There are five clinical trials underway (here’s an overview at Biocentury). The NIH has an adaptive-design Phase II trial that has already started in Nebraska, with doses to be changed according to Bayesian readouts along the way. There are two Phase III trials underway at China-Japan Friendship Hospital in Hubei, double-blinded and placebo-controlled (since placebo is, as far as drug therapy goes, the current standard of care). And Gilead themselves are starting two open-label trials, one with no control arm and one with an (unblinded) standard-of-care comparison arm. Those might read out first, depending on when they get off the ground, but will be only rough readouts due to the fast-and-loose trial design. The two Hubei trials and the NIH one will add some rigor to the process, but I’m not sure when they’re going to report. My personal opinion is that I like the chances of this drug more than anything else on this list, but it’s still unlikely to be a game-changer.

There’s an RNA polymerase inhibitor (favipiravir) from Toyama, at right, that’s in a trial in China. It’s a thought – a broad-spectrum agent of this sort would be the sort of thing to try. But unfortunately, from what I can see, it has already turned up as ineffective in in vitro tests. The human trial that’s underway is honestly the sort of thing that would only happen under circumstances like the present: a developing epidemic with a new pathogen and no real standard of care. I hold out little hope for this one, but given that there’s nothing else at present, it probably should be tried. As you’ll see, this is far from the only situation like this.

One of the screens of known drugs in China that also flagged remdesivir noted that the old antimalarial drug chloroquine seemed to be effective in vitro. It had been reported some years back as a possible antiviral, working through more than one mechanism, probably both at viral entry and intracellularly thereafter. That part shouldn’t be surprising – chloroquine’s actual mode(s) of action against malaria parasites are still not completely worked out, either, and some of what people thought they knew about it has turned out to be wrong. There are several trials underway with it at Chinese facilities, some in combination with other agents like remdesivir. Chloroquine has of course been taken for many decades as an antimalarial, but it has a number of liabilities, including seizures, hearing damage, retinopathy and sudden effects on blood glucose. So it’s going to be important to establish just how effective it is and what doses will be needed. Just as with vaccine candidates, it’s possible to do more harm with a rushed treatment than the disease is doing itself

There are several other known antiviral drugs are being tried in China, but I don’t have too much hope for those, either. The neuraminidase inhibitors such as oseltamivir (better known as Tamiflu) were tried against SARS and were ineffective; there is no reason to expect anything versus Covid-19 although these drugs are a component of some drug cocktail trials. The HIV protease therapies such as darunavir and the combination therapy Kaletra are in trials, but that’s also a rather desperate long shot, since there’s no particular reason to think that they will have any such protease inhibition against what this new virus has to offer (and indeed, such agents weren’t much help against SARS in the end, either). The classic interferon/ribavirin combination seems to have had some activity against SARS and MERS, and is in two trials from what I can see. That’s not an awful idea by any means, but it’s not a great one, either: if your viral disease has interferon/ribavirin as a front line therapy, it generally means that there’s nothing really good available. No, unless we get really lucky none of these ideas are going to slow the disease down much.

There are a few other repurposed-protease-inhibitors ideas out there, such as this one. (Edit: I had seen this paper but couldn’t track it down, so thanks to those who sent it along). This paper suggests that the TMPRSS2 protease is important for viral entry on the human-cell-side of the process, a pathway that has been noted for other coronaviruses. And it points out that there is a an approved inhibitor (in Japan) for this enzyme (camostat), so that would definitely seem to be worth a trial, probably in combination with remdesivir.

That’s about it for the existing small molecules, from what I can see. What about new ones? Don’t hold your breath, is all I can say. A drug discovery program from scratch against a new pathogen is, as many readers here well know, not a trivial exercise. As this Bloomberg article details, many such efforts in the past (small molecules and vaccines alike) have come to grief because by the time they had anything to deliver the epidemic itself had passed. Indeed, Gilead’s remdesivir had already been dropped as a potential Ebola therapy.

You will either need to have a target in mind up front or go phenotypic. For the former, what you’d see are better characterizations of the viral protease and more extensive screens against it. Two other big target areas are viral entry (which involves the “spike” proteins on the virus surface and the ACE2 protein on human cells) and viral replication. To the former, it’s worth quickly noting that ACE2 is so much unlike the more familiar ACE protein that none of the cardiovascular ACE inhibitors do anything to it at all. And targeting the latter mechanisms is how remdesivir was developed as a possible Ebola agent, but as you can see, that took time, too. Phenotypic screens are perfectly reasonable against viral pathogens as well, but you’ll need to put time and effort into that assay up front, just as with any phenotypic effort, because as anyone who does that sort of work will tell you, a bad phenotypic screen is a complete waste of everyone’s time.

One of the key steps for either route is identifying an animal model. While animal models of infectious disease can be extremely well translated to human therapy, that doesn’t happen by accident: you need to choose the right animal. Viruses in general (and coronaviruses are no exception) vary widely in their effects in different species, and not just across the gaps of bird/reptile/human and the like. No, you’ll run into things where even the usual set of small mammals are acting differently from each other, with some of them not even getting sick at all. This current virus may well have gone through a couple of other mammalian species before landing on us, but you’ll note that dogs (to pick one) don’t seem to have any problem with it.

All this means that any new-target new-chemical-matter effort against Covid-19 (or any new pathogen) is going to take years, and there is just no way around that. Update: see here for just such an effort to start finding fragment hits for the viral protease. This puts small molecules in a very bimodal distribution: you have the existing drugs that might be repurposed, and are presumably available right now. Nothing else is! At the other end, for completely new therapies you have the usual prospects of drug discovery: years from now, lots of money, low success rate, good luck to all of us. The gap between these two could in theory be filled by vaccines and antibody therapies (if everything goes really, really well) but those are very much their own area and will be dealt with in a separate post.

Either way, the odds are that we (and I mean “we as a species” here) are going to be fighting this epidemic without any particularly amazing pharmacological weapons. Eventually we’ll have some, but I would advise people, pundits, and politicians not to get all excited about the prospects for some new therapies to come riding up over the hill to help us out. The odds of that happening in time to do anything about the current outbreak are very small. We will be going for months, years, with the therapeutic options we have right now. Look around you: what we have today is what we have to work with.

Other related articles published in this Open Access Online Scientific Journal include the following:

 

Group of Researchers @ University of California, Riverside, the University of Chicago, the U.S. Department of Energy’s Argonne National Laboratory, and Northwestern University solve COVID-19 Structure and Map Potential Therapeutics

Reporters: Stephen J Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2020/03/06/group-of-researchers-solve-covid-19-structure-and-map-potential-therapeutic/

Predicting the Protein Structure of Coronavirus: Inhibition of Nsp15 can slow viral replication and Cryo-EM – Spike protein structure (experimentally verified) vs AI-predicted protein structures (not experimentally verified) of DeepMind (Parent: Google) aka AlphaFold

Curators: Stephen J. Williams, PhD and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2020/03/08/predicting-the-protein-structure-of-coronavirus-inhibition-of-nsp15-can-slow-viral-replication-and-cryo-em-spike-protein-structure-experimentally-verified-vs-ai-predicted-protein-structures-not/

 

Coronavirus facility opens at Rambam Hospital using new Israeli tech

https://www.jpost.com/Israel-News/Coronavirus-facility-opens-at-Rambam-Hospital-using-new-Israeli-tech-619681

 

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5:00 – 5:45 PM Early Diagnosis Through Predictive Biomarkers, NonInvasive Testing

Reporter: Stephen J. Williams, Ph.D.

 

Diagnosing cancer early is often the difference between survival and death. Hear from experts regarding the new and emerging technologies that form the next generation of cancer diagnostics.

Moderator: Heather Rose, Director of Licensing, Thomas Jefferson University
Speakers:
Bonnie Anderson, Chairman and CEO, Veracyte @BonnieAndDx
Kevin Hrusovsky, Founder and Chairman, Powering Precision Health @KevinHrusovsky

Bonnie Anderson and Veracyte produces genomic tests for thyroid and other cancer diagnosis.  Kevin Hrusovksy and Precision Health uses peer reviewed evidence based medicine to affect precision medicine decision.

Bonnie: aim to get a truth of diagnosis.  Getting tumor tissue is paramount as well as properly preserved tissue.  They use deep RNA sequencing  and machine learning  in their clinically approved tests.

Kevin: Serial biospace entrepreneur.  Two diseases, cancer and neurologic, have been diseases which have been hardest to get reproducible and validated biomarkers of early disease.  He concentrates on protein biomarkers.

Heather:  FDA has recently approved drugs for early disease intervention.  However the use of biomarkers can go beyond patient stratification in clinical trials.

Kevin: 15 approved drugs for MS but the markers are scans looking for brain atrophy which is too late of an endpoint.  So we need biomarkers of early disease progression.  We can use those early biomarkers of disease progression so pharma can target those early biomarkers and or use those early biomarkers of disease progression  for endpoint

Bonnie: exciting time in the early diagnostics field. She prefers transcriptomics to DNA based methods such as WES or WGS (whole exome or whole genome sequencing).  It was critical to show data on the cost savings imparted by their transcriptomic based thryoid cancer diagnostic test for payers to consider this test eligible for reimbursement.

Kevin: There has been 20 million  CAT scans for  cancer but it is estimated 90% of these scans led to misdiagnosis. Biomarker  development  has revolutionized diagnostics in this disease area.  They have developed a breakthrough panel of ten protein biomarkers in serum which he estimates may replace 5 million mammograms.

All panelists agreed on the importance of regulatory compliance and the focus of new research should be on early detection.  In addition they believe that Dr. Gotlieb’s appointment to the FDA is a positive for the biomarker development field, as Dr. Gotlieb understands the potential and importance of early detection and prevention of disease.  Kevin also felt Dr. Gotlieb understands the importance of incorporating biomarkers as endpoints in clinical trials.  Over 750 phase 1,2, and 3 clinical trials use biomarker endpoints but the pharma companies still need to prove the biomarkers clinical relevance to the FDA.They also agreed it would be helpful to involve advocacy groups in putting more pressure on the healthcare providers and policy makers on this importance of diagnostics as a preventative measure.

In addition, the discovery and use of biomarkers as disease endpoints has led to a resurgence of Alzheimer’s disease drug development by companies which have previously given up on these type of neurodegenerative diseases.

Kevin feels proteomics offers great advantages over DNA-based diagnostics, especially in cancer such as ovarian cancer, where a high degree of specificity for a diagnostic test is required to ascertain if a woman should undergo prophylactic oophorectomy.  He suggests that a new blood-based protein biomarker panel is being developed for early detection of some forms of ovarian cancer.

Please follow on Twitter using the following #hash tags and @pharma_BI

#MCConverge

#cancertreatment

#healthIT

#innovation

#precisionmedicine

#healthcaremodels

#personalizedmedicine

#healthcaredata

And at the following handles:

@pharma_BI

@medcitynews

 

Please see related articles on Live Coverage of Previous Meetings on this Open Access Journal

LIVE – Real Time – 16th Annual Cancer Research Symposium, Koch Institute, Friday, June 16, 9AM – 5PM, Kresge Auditorium, MIT

Real Time Coverage and eProceedings of Presentations on 11/16 – 11/17, 2016, The 12th Annual Personalized Medicine Conference, HARVARD MEDICAL SCHOOL, Joseph B. Martin Conference Center, 77 Avenue Louis Pasteur, Boston

Tweets Impression Analytics, Re-Tweets, Tweets and Likes by @AVIVA1950 and @pharma_BI for 2018 BioIT, Boston, 5/15 – 5/17, 2018

BIO 2018! June 4-7, 2018 at Boston Convention & Exhibition Center

https://pharmaceuticalintelligence.com/press-coverage/

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Protein profiling in cancer and metabolic diseases

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Deep Protein Profiling Key

Company has encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment.

http://www.technologynetworks.com/Proteomics/news.aspx?ID=190145

Proteome Sciences plc has strongly encouraged by two recent reports that emphasise the importance of protein profiling to improve outcomes in cancer treatment. These highlight the growing need for more detailed, personal assessment of protein profiles to improve the management of cancer treatment.

In the first study two groups from University College London and Cancer Research UK demonstrated that genetic mutations in cancer can lead to changes in the proteins on the cell surface1. These are new sequences which are seen as foreign by the body’s immune system and, with appropriate immunotherapy, the level of response in lung cancer was greatly enhanced.

However many of the patients with these types of mutations unfortunately still did not respond which highlighted the need for deeper analysis of the protein expression in tumours in order to better appreciate the mechanisms that contribute to treatment failure.

The second study, led by Professor Nigel Bundred of Manchester University, reported that use of two drugs that act on the same breast cancer target, an over-expressing protein called Her-2, were able to eradicate detectable tumours in around 10% of those treated in just 11 days, with 87% of those treated having a proteomic change indicating cells had stopped growing and/or cell death had increased2.

Whilst these results appear very promising it is worth noting that the over-expressing Her-2 target is only present in about 20% of breast tumours meaning this combination therapy was successful in clearing tumours in just 2% of the total breast cancer population.

Dr. Ian Pike, Chief Operating Officer of Proteome Sciences commented, “Both these recent studies should rightly be recognised as important steps forward towards better cancer treatment. However, in order to overcome the limitations of current drug therapy programs, a much deeper and more comprehensive analysis of the complex protein networks that regulate tumour growth and survival is required and will be essential to achieve a major advance in the battle to treat cancer.

“Our SysQuant® workflows provide that solution. As an example, in pancreatic cancer3 we have successfully mapped the complex network of regulatory processes and demonstrate the ability to devise personalised treatment combinations on an individual basis for each patient. A retrospective study with SysQuant® to predict response to the targeted drug Sorafenib in liver cancer is in process and we are planning further prospective trials to guide personalised treatment selection in liver cancer.

“We are already delivering systems-wide biology solutions through SysQuant® and TMTcalibrator™ programs to our clients that are generating novel biological data and results using more sensitive profiling that are helping them to better understand their drug development programs and to provide new biomarkers for tracking patient response in clinical trials.

“We are strongly positioned to deliver more comprehensive analysis of proteins and cellular pathways across other areas of disease and in particular to extend the use of SysQuant® with other leading cancer research groups in liver and other cancers.”

Proteome Sciences has also expanded its offering in personalised medicine through the use of its TMTcalibrator™ technology to uniquely identify protein biomarkers that reveal active cancer and other disease processes in body fluid samples. The importance of these ‘mechanistic’ biomarkers is that they are essential to monitor that drugs are being effective and that they can be used as early biomarkers of disease recurrence.

Using SysQuant® and TMTcalibrator™, Proteome Sciences can deliver more comprehensive analysis and provide unparalleled levels of sensitivity and breadth of coverage of the proteome, enabling faster, more efficient drug development and more accurate disease diagnosis.

 

Discovering ‘Outlier’ Enzymes

Researchers at TSRI and Salk Institute have discovered ‘Outlier’ enzymes that could offer new targets to treat type 2 diabetes and inflammatory disorders.

A team led by scientists at The Scripps Research Institute (TSRI) and the Salk Institute for Biological Studies have discovered two enzymes that appear to play a role in metabolism and inflammation—and might someday be targeted with drugs to treat type 2 diabetes and inflammatory disorders. The discovery is unusual because the enzymes do not bear a resemblance—in their structures or amino-acid sequences—to any known class of enzymes.

The team of scientists nevertheless identified them as “outlier” members of the serine/threonine hydrolase class, using newer techniques that detect biochemical activity. “A huge fraction of the human ‘proteome’ remains uncharacterized, and this paper shows how chemical approaches can be used to uncover proteins of a given functionality that have eluded classification based on sequence or predicted structure,” said co-senior author Benjamin F. Cravatt, chair of TSRI’s Department of Chemical Physiology.

“In this study, we found two genes that control levels of lipids with anti-diabetic and anti-inflammatory activity, suggesting exciting targets for diabetes and inflammatory diseases,” said co-senior author Alan Saghatelian, who holds the Dr. Frederik Paulsen Chair at the Salk Institute. The study, which appeared as a Nature Chemical Biology Advance Online Publication on March 28, 2016, began as an effort in the Cravatt laboratory to discover and characterize new serine/threonine hydrolases using fluorophosphonate (FP) probes—molecules that selectively bind and, in effect, label the active sites of these enzymes.

Pulling FP-binding proteins out of the entire proteome of test cells and identifying them using mass spectrometry techniques, the team matched nearly all to known hydrolases. The major outlier was a protein called androgen-induced gene 1 protein (AIG1). The only other one was a distant cousin in terms of sequence, a protein called ADTRP. “Neither of these proteins had been characterized as an enzyme; in fact, there had been little functional characterization of them at all,” said William H. Parsons, a research associate in the Cravatt laboratory who was co-first author of the study.

Experiments on AIG1 and ADTRP revealed that they do their enzymatic work in a unique way. “It looks like they have an active site that is novel—it had never been described in the literature,” said Parsons. Initial tests with panels of different enzyme inhibitors showed that AIG1 and ADTRP are moderately inhibited by inhibitors of lipases—enzymes that break down fats and other lipids. But on what specific lipids do these newly discovered outlier enzymes normally work?

At the Salk Institute, the Saghatelian laboratory was investigating a class of lipids it had discovered in 2014. Known as fatty acid esters of hydroxy fatty acids (FAHFAs), these molecules showed strong therapeutic potential. Saghatelian and his colleagues had found that boosting the levels of one key FAHFA lipid normalizes glucose levels in diabetic mice and also reduces inflammation.

“[Ben Cravatt’s] lab was screening panels of lipids to find the ones that their new enzymes work on,” said Saghatelian, who is a former research associate in the Cravatt laboratory. “We suggested they throw FAHFAs in there—and these turned out to be very good substrates.” The Cravatt laboratory soon developed powerful inhibitors of the newly discovered enzymes, and the two labs began working together, using the inhibitors and genetic techniques to explore the enzymes’ functions in vitro and in cultured cells.

Co-first author Matthew J. Kolar, an MD-PhD student, performed most of the experiments in the Saghatelian lab. The team concluded that AIG1 and ADTRP, at least in the cell types tested, appear to work mainly to break down FAHFAs and not any other major class of lipid. In principle, inhibitors of AIG1 and ADTRP could be developed into FAHFA-boosting therapies.

“Our prediction,” said Saghatelian, “is that if FAHFAs do what we think they’re doing, then using an enzyme inhibitor to block their degradation would make FAHFA levels go up and should thus reduce inflammation as well as improve glucose levels and insulin sensitivity.” The two labs are now collaborating on further studies of the new enzymes—and the potential benefits of inhibiting them—in mouse models of diabetes, inflammation and autoimmune disease.

“One of the neat things this study shows,” said Cravatt, “is that even for enzyme classes as well studied as the hydrolases, there may still be hidden members that, presumably by convergent evolution, arrived at that basic enzyme mechanism despite sharing no sequence or structural homology.”

Other co-authors of the study, “AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs,” were Siddhesh S. Kamat, Armand B. Cognetta III, Jonathan J. Hulce and Enrique Saez, of TSRI; and co-senior author Barbara B. Kahn of Beth Israel Deaconess Medical Center and Harvard Medical School

 

New Weapon Against Breast Cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says.

Harvard Stem Cell Institute (HSCI) researchers at Dana-Farber Cancer Institute (DFCI) and collaborators at Brigham and Women’s Hospital (BWH) have identified a molecular marker in normal breast tissue that can predict a woman’s risk for developing breast cancer, the leading cause of death in women with cancer worldwide.

The work, led by HSCI principal faculty member Kornelia Polyak and Rulla Tamimi of BWH, was published in an early online release and in the April 1 issue of Cancer Research.

The study builds on Polyak’s earlier research finding that women already identified as having a high risk of developing cancer — namely those with a mutation called BRCA1 or BRCA2 — or women who did not give birth before their 30s had a higher number of mammary gland progenitor cells.

In the latest study, Polyak, Tamimi, and their colleagues examined biopsies, some taken as many as four decades ago, from 302 participants in the Nurses’ Health Study and the Nurses’ Health Study II who had been diagnosed with benign breast disease. The researchers compared tissue from the 69 women who later developed cancer to the tissue from the 233 women who did not. They found that women were five times as likely to develop cancer if they had a higher percentage of Ki67, a molecular marker that identifies proliferating cells, in the cells that line the mammary ducts and milk-producing lobules. These cells, called the mammary epithelium, undergo drastic changes throughout a woman’s life, and the majority of breast cancers originate in these tissues.

Doctors already test breast tumors for Ki67 levels, which can inform decisions about treatment, but this is the first time scientists have been able to link Ki67 to precancerous tissue and use it as a predictive tool.

“Instead of only telling women that they don’t have cancer, we could test the biopsies and tell women if they were at high risk or low risk for developing breast cancer in the future,” said Polyak, a breast cancer researcher at Dana-Farber and co-senior author of the paper.

“Currently, we are not able to do a very good job at distinguishing women at high and low risk of breast cancer,” added co-senior author Tamimi, an associate professor at the Harvard T.H. Chan School of Public Health and Harvard Medical School. “By identifying women at high risk of breast cancer, we can better develop individualized screening and also target risk reducing strategies.”

To date, mammograms are the best tool for the early detection, but there are risks associated with screening. False positive and negative results and over-diagnosis could cause psychological distress, delay treatment, or lead to overtreatment, according to the National Cancer Institute (NCI).

Mammography machines also use low doses of radiation. While a single mammogram is unlikely to cause harm, repeated screening can potentially cause cancer, though the NCI writes that the benefits “nearly always outweigh the risks.”

“If we can minimize unnecessary radiation for women at low risk, that would be good,” said Tamimi.

Screening for Ki67 levels would “be easy to apply in the current setting,” said Polyak, though the researchers first want to reproduce the results in an independent cohort of women.

 

AIG1 and ADTRP are atypical integral membrane hydrolases that degrade bioactive FAHFAs

William H ParsonsMatthew J Kolar, …., Barbara B KahnAlan Saghatelian & Benjamin F Cravatt

Nature Chemical Biology 28 March 2016                    http://dx.doi.org:/10.1038/nchembio.2051

Enzyme classes may contain outlier members that share mechanistic, but not sequence or structural, relatedness with more common representatives. The functional annotation of such exceptional proteins can be challenging. Here, we use activity-based profiling to discover that the poorly characterized multipass transmembrane proteins AIG1 and ADTRP are atypical hydrolytic enzymes that depend on conserved threonine and histidine residues for catalysis. Both AIG1 and ADTRP hydrolyze bioactive fatty acid esters of hydroxy fatty acids (FAHFAs) but not other major classes of lipids. We identify multiple cell-active, covalent inhibitors of AIG1 and show that these agents block FAHFA hydrolysis in mammalian cells. These results indicate that AIG1 and ADTRP are founding members of an evolutionarily conserved class of transmembrane threonine hydrolases involved in bioactive lipid metabolism. More generally, our findings demonstrate how chemical proteomics can excavate potential cases of convergent or parallel protein evolution that defy conventional sequence- and structure-based predictions.

Figure 1: Discovery and characterization of AIG1 and ADTRP as FP-reactive proteins in the human proteome.

 

http://www.nature.com/nchembio/journal/vaop/ncurrent/carousel/nchembio.2051-F1.jpg

(a) Competitive ABPP-SILAC analysis to identify FP-alkyne-inhibited proteins, in which protein enrichment and inhibition were measured in proteomic lysates from SKOV3 cells treated with FP-alkyne (20 μM, 1 h) or DMSO using the FP-biotin…

 

  1. Willems, L.I., Overkleeft, H.S. & van Kasteren, S.I. Current developments in activity-based protein profiling. Bioconjug. Chem. 25, 11811191 (2014).
  2. Niphakis, M.J. & Cravatt, B.F. Enzyme inhibitor discovery by activity-based protein profiling.Annu. Rev. Biochem. 83, 341377 (2014).
  3. Berger, A.B., Vitorino, P.M. & Bogyo, M. Activity-based protein profiling: applications to biomarker discovery, in vivo imaging and drug discovery. Am. J. Pharmacogenomics 4,371381 (2004).
  4. Liu, Y., Patricelli, M.P. & Cravatt, B.F. Activity-based protein profiling: the serine hydrolases.Proc. Natl. Acad. Sci. USA 96, 1469414699 (1999).
  5. Simon, G.M. & Cravatt, B.F. Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J. Biol. Chem. 285, 1105111055 (2010).
  6. Bachovchin, D.A. et al. Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening. Proc. Natl. Acad. Sci. USA 107, 2094120946 (2010).
  7. Jessani, N. et al. A streamlined platform for high-content functional proteomics of primary human specimens. Nat. Methods 2, 691697 (2005).
  8. Higa, H.H., Diaz, S. & Varki, A. Biochemical and genetic evidence for distinct membrane-bound and cytosolic sialic acid O-acetyl-esterases: serine-active-site enzymes. Biochem. Biophys. Res. Commun. 144, 10991108 (1987).

Academic cross-fertilization by public screening yields a remarkable class of protein phosphatase methylesteras-1 inhibitors

Proc Natl Acad Sci U S A. 2011 Apr 26; 108(17): 6811–6816.    doi:  10.1073/pnas.1015248108
National Institutes of Health (NIH)-sponsored screening centers provide academic researchers with a special opportunity to pursue small-molecule probes for protein targets that are outside the current interest of, or beyond the standard technologies employed by, the pharmaceutical industry. Here, we describe the outcome of an inhibitor screen for one such target, the enzyme protein phosphatase methylesterase-1 (PME-1), which regulates the methylesterification state of protein phosphatase 2A (PP2A) and is implicated in cancer and neurodegeneration. Inhibitors of PME-1 have not yet been described, which we attribute, at least in part, to a dearth of substrate assays compatible with high-throughput screening. We show that PME-1 is assayable by fluorescence polarization-activity-based protein profiling (fluopol-ABPP) and use this platform to screen the 300,000+ member NIH small-molecule library. This screen identified an unusual class of compounds, the aza-β-lactams (ABLs), as potent (IC50 values of approximately 10 nM), covalent PME-1 inhibitors. Interestingly, ABLs did not derive from a commercial vendor but rather an academic contribution to the public library. We show using competitive-ABPP that ABLs are exquisitely selective for PME-1 in living cells and mice, where enzyme inactivation leads to substantial reductions in demethylated PP2A. In summary, we have combined advanced synthetic and chemoproteomic methods to discover a class of ABL inhibitors that can be used to selectively perturb PME-1 activity in diverse biological systems. More generally, these results illustrate how public screening centers can serve as hubs to create spontaneous collaborative opportunities between synthetic chemistry and chemical biology labs interested in creating first-in-class pharmacological probes for challenging protein targets.

Protein phosphorylation is a pervasive and dynamic posttranslational protein modification in eukaryotic cells. In mammals, more than 500 protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues on proteins (1). A much more limited number of phosphatases are responsible for reversing these phosphorylation events (2). For instance, protein phosphatase 2A (PP2A) and PP1 are thought to be responsible together for > 90% of the total serine/threonine phosphatase activity in mammalian cells (3). Specificity is imparted on PP2A activity by multiple mechanisms, including dynamic interactions between the catalytic subunit (C) and different protein-binding partners (B subunits), as well as a variety of posttranslational chemical modifications (2, 4). Within the latter category is an unusual methylesterification event found at the C terminus of the catalytic subunit of PP2A that is introduced and removed by a specific methyltransferase (leucine carbxoylmethyltransferase-1 or LCMT1) (5, 6) and methylesterase (protein phosphatase methylesterase-1 or PME-1) (7), respectively (Fig. 1A). PP2A carboxymethylation (hereafter referred to as “methylation”) has been proposed to regulate PP2A activity, at least in part, by modulating the binding interaction of the C subunit with various regulatory B subunits (810). A predicted outcome of these shifts in subunit association is the targeting of PP2A to different protein substrates in cells. PME-1 has also been hypothesized to stabilize inactive forms of nuclear PP2A (11), and recent structural studies have shed light on the physical interactions between PME-1 and the PP2A holoenzyme (12).

There were several keys to the success of our probe development effort. First, screening for inhibitors of PME-1 benefited from the fluopol-ABPP technology, which circumvented the limited throughput of previously described substrate assays for this enzyme. Second, we were fortunate that the NIH compound library contained several members of the ABL class of small molecules. These chiral compounds, which represent an academic contribution to the NIH library, occupy an unusual portion of structural space that is poorly accessed by commercial compound collections. Although at the time of their original synthesis (23) it may not have been possible to predict whether these ABLs would show specific biological activity, their incorporation into the NIH library provided a forum for screening against many proteins and cellular targets, culminating in their identification as PME-1 inhibitors. We then used advanced chemoproteomic assays to confirm the remarkable selectivity displayed by ABLs for PME-1 across (and beyond) the serine hydrolase superfamily. That the mechanism for PME-1 inhibition involves acylation of the enzyme’s conserved serine nucleophile (Fig. 3) suggests that exploration of a more structurally diverse set of ABLs might uncover inhibitors for other serine hydrolases. In this way, the chemical information gained from a single high-throughput screen may be leveraged to initiate probe development programs for additional enzyme targets.

Projecting forward, this research provides an example of how public small-molecule screening centers can serve as a portal for spawning academic collaborations between chemical biology and synthetic chemistry labs. By continuing to develop versatile high-throughput screens and combining them with a small-molecule library of expanding structural diversity conferred by advanced synthetic methodologies, academic biologists and chemists are well-positioned to collaboratively deliver pharmacological probes for a wide range of proteins and pathways in cell biology.

 

New weapon against breast cancer

Molecular marker in healthy tissue can predict a woman’s risk of getting the disease, research says

April 6, 2016 | Popular
BRC_Cancer605

 

New Group of Aging-Related Proteins Discovered

http://www.genengnews.com/gen-news-highlights/new-group-of-aging-related-proteins-discovered/81252599/

Scientists have discovered a group of six proteins that may help to divulge secrets of how we age, potentially unlocking new insights into diabetes, Alzheimer’s, cancer, and other aging-related diseases.

The proteins appear to play several roles in our bodies’ cells, from decreasing the amount of damaging free radicals and controlling the rate at which cells die to boosting metabolism and helping tissues throughout the body respond better to insulin. The naturally occurring amounts of each protein decrease with age, leading investigators to believe that they play an important role in the aging process and the onset of diseases linked to older age.

The research team led by Pinchas Cohen, M.D., dean and professor of the University of Southern California Leonard Davis School of Gerontology, identified the proteins and observed their origin from mitochondria and their game-changing roles in metabolism and cell survival. This latest finding builds upon prior research by Dr. Cohen and his team that uncovered two significant proteins, humanin and MOTS-c, hormones that appear to have significant roles in metabolism and diseases of aging.

Unlike most other proteins, humanin and MOTS-c are encoded in mitochondria. Dr. Cohen’s team used computer analysis to see if the part of the mitochondrial genome that provides the code for humanin was coding for other proteins as well. The analysis uncovered the genes for six new proteins, which were dubbed small humanin-like peptides, or SHLPs, 1 through 6 (pronounced “schlep”).

After identifying the six SHLPs and successfully developing antibodies to test for several of them, the team examined both mouse tissues and human cells to determine their abundance in different organs as well as their functions. The proteins were distributed quite differently among organs, which suggests that the proteins have varying functions based on where they are in the body. Of particular interest is SHLP 2, according to Dr. Cohen.  The protein appears to have insulin-sensitizing, antidiabetic effects as well as neuroprotective activity that may emerge as a strategy to combat Alzheimer’s disease. He added that SHLP 6 is also intriguing, with a unique ability to promote cancer cell death and thus potentially target malignant diseases.

Proteins That May Protect Against Age Related Illnesses Discovered

 

The cell proliferation antigen Ki-67 organises heterochromatin

 Michal Sobecki, 

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression.

 

A protein called Ki-67 is only produced in actively dividing cells, where it is located in the nucleus – the structure that contains most of the cell’s DNA. Researchers often use Ki-67 as a marker to identify which cells are actively dividing in tissue samples from cancer patients, and previous studies indicated that Ki-67 is needed for cells to divide. However, the exact role of this protein was not clear. Before cells can divide they need to make large amounts of new proteins using molecular machines called ribosomes and it has been suggested that Ki-67 helps to produce ribosomes.

Now, Sobecki et al. used genetic techniques to study the role of Ki-67 in mice. The experiments show that Ki-67 is not required for cells to divide in the laboratory or to make ribosomes. Instead, Ki-67 alters the way that DNA is packaged in the nucleus. Loss of Ki-67 from mice cells resulted in DNA becoming less compact, which in turn altered the activity of genes in those cells.

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BioMEMS The Market aspects of Oligonucleotide-Chips, Products and Applications, Competition, January 21, 2016

Curator: Gérard LOISEAU, ESQ

 

BioMEMS

The Market aspects of Oligonucleotide-Chips, Products, Applications, Competition 

January 21, 2016

2015-2020

The oligonucleotide synthesis market is expected to reach USD 1.918.6Billion at a CAGR of 10.1% by 2020 from USD 1.078.1Billion in 2015.

SOURCE

MARKETSANDMARKETS marketsandmarkets.com/

 

PLAYERS

  • Agilent Technologies Inc.
  • BioAutomation Corp.
  • Biosearch Technologies
  • Gen9 Inc.
  • GenScript Inc.
  • Illumina Inc.
  • Integrated DNA Technologies
  • New England Biolabs Inc.
  • Nitto Denko Avecia Inc.
  • OriGene Technologies Inc.
  • Sigma-Aldrich Corporation
  • Thermo Fisher Scientific Inc.
  • TriLink Biotechnologies

 

Agilent Technologies
 CA NYSE :A


http://www.agilent.com/

  • Agilent was created as a spin off from Hewlett-Packard Company in 1999.
  • Agilent Technologies Inc. is engaged in the life sciences, diagnostics and applied chemical markets. The Company provides application focused solutions that include instruments, software, services and consumables for the entire laboratory workflow. The Company has three business segments:

the life sciences and applied markets business,

the diagnostics and genomics business, and

the Agilent Cross Lab business

  • The Company’s life sciences and applied markets business segment brings together the Company’s analytical laboratory instrumentation and informatics.
  • The Company’s diagnostics and genomics business segment consists of three businesses: the Dako business, the genomics business and the nucleic acid solutions business.
  • The Company’s Agilent Cross Lab business segment combines its analytical laboratory services and consumables business

SOURCE

http://reuters.com/

PRODUCTS AND SERVICES

https://www.agilent.com/en-us/default#collapse-0

  • October 09, 2015 03:21 PM Eastern Daylight Time
  • CARPINTERIA, Calif.–(BUSINESS WIRE)–Dako, an Agilent Technologies company and a worldwide provider of cancer diagnostics, today announced the U.S. Food and Drug Administration has approved a new test that can identify PD-L1 expression levels on the surface of non-small cell lung cancer tumor cells and provide information on the survival benefit with OPDIVO® (nivolumab) for patients with non-squamous NSCLC.

SOURCE

BUSINESS WIRE busibesswire.com/

 

BioAutomation Corp.

 TX


 

http://bioautomation.com/

          PRODUCTS AND SERVICES

  • DNA and RNA synthesis reagents for the MerMades

 

Note: The MerMade 192E Oligonucleotide synthesizer is designed to synthesize DNA, RNA & LNA oligonucleotides in a column format

          PARTNERSHIPS

  • HONGENE BIOTECH : BIOAUTOMATION is the exclusive distributor for the Americas
  • EMD MILLIPORE
  • BIOSEARCH TECHNOLOGIES

 

DISTRIBUTORS

  • LINK TECHNOLOGIES : UK
  • AME BIOSCIENCE : UK
  • BOSUNG SCIENCE : KOREA
  • DNA CHEM : CHINA
  • WAKO : JAPAN
  • ACE PROBE : INDIA

SOURCE

bioautomation.com/

 

Biosearch Technologies
 CA


http://biosearchtech.com/

          PRODUCTS

  • qPCR & SNP Genotyping
  • Custom Oligonucleotides
  • – highly sophisticated oligonucleotides
  • – simple PCR primers
  • Oligos in Plates
  • RNA FISH
  • Synthesis Reagents
  • Immunochemicals
  • Primers
  • Probes
  • Large-Scale Synthesis Oligos
  • Intermediate-Scale Synthesis Oligos

          SERVICES

  • GMP & Commercial Services
  • OEM & Kit Manufacturing
  • qPCR Design Collaborations

          DISTRIBUTORS

Argentina | Australia | Austria | Brazil | Canada |Chile | China | Colombia | Czech Republic | Denmark | Ecuador | Finland | Germany |Hong Kong | Israel | Italy | Japan | Korea | Malaysia | Mexico | New Zealand | Norway | Paraguay | Peru| Philippines | Poland | Romania | Singapore | South Africa | Spain | Sweden |Switzerland | Taiwan ROC | Thailand | Turkey | United Kingdom | Uruguay | Vietnam

SOURCE

biosearchtech.com/

 

Gen9 Inc.
 MA 


http://www.gen9bio.com/

          PRODUCTS

Gen9 is building on advances in synthetic biology to power a scalable fabrication capability that will significantly increase the world’s capacity to produce DNA content. The privately held company’s next-generation gene synthesis technology allows for the high-throughput, automated production of DNA constructs at lower cost and higher accuracy than previous methods on the market. Founded by world leaders in synthetic biology, Gen9 aims to ensure the constructive application of synthetic biology in industries ranging from enzyme and chemical production to pharmaceuticals and biofuels.

          SERVICES

  • Synthetic Biology
  • Gene Synthesis Services
  • Variant Libraries
  • Gene Sequence Design Services

         INVESTORS

  • Agilent Technologies : Private Equity
  • CAMBRIDGE, Mass. and SANTA CLARA, Calif. — April 24, 2013 —Gen9 Receives $21 Million Strategic Investment from Agilent Technologies

SOURCE

gen9bio.com/

 

GenScript Inc.
 NJ 


http://www.genscript.com/

  • GenScript is the largest gene synthesis provider in the USA
  • GenScript Corporation, a biology contract research organization, provides biological research and drug discovery services to pharmaceutical companies, biotech firms, and research institutions in the United States, Europe, and Japan. It offers bio-reagent, custom molecular biology, custom peptide, protein production, custom antibody production, drug candidates testing, assay development and screening, lead optimization, antibody drug development, gene synthesis, and assay-ready cell line production services.
  • The company also offers molecular biology, peptide, protein, immunoassay, chemicals, and cell biology products. It offers its products through distributors in Tokyo, Japan; and Seoul, Korea. GenScript Corporation has a strategic partnership with Immunologix, Inc. The company was founded in 2002 and is based in Piscataway, New Jersey. It has subsidiaries in France, Japan, and China.

 

Note: As of October 24, 2011, Immunologix, Inc. was acquired by Intrexon Corporation. Immunologix, Inc. develops and produces antibody-based therapeutics for various biological targets. It produces human monoclonal antibodies against viral, bacterial, and tumor antigens, as well as human auto antigens.

Intrexon Corporation, founded in 1998, is a leader in synthetic biology focused on collaborating with companies in Health, Food, Energy, Environment and Consumer sectors to create biologically based products that improve quality of life and the health of the planet.

 

 

             PRODUCTS AND SERVICES

  • Gene synthesis
  • Antibody services
  • Protein Services
  • Peptide services

 

               INVESTORS


Note: The Balloch Group (‘TBG’) was established in 2001 by Howard Balloch (Canada‘s ambassador to China from 1996 to 2001). TBG has since grown from a market-entry consultancy working with North American clients in China to a leading advisory and merchant banking firm serving both domestic Chinese companies and multinational corporations. TBG was ranked as the number one boutique investment bank in China by ChinaVenture in 2008.

Kleiner, Perkins, Caufield and Byers

 

Illumina
Inc. CA


http://illumina.com/

 

Monica Heger : SAN FRANCISCO (GenomeWeb) – Illumina today announced two new next-generation sequencing platforms, a targeted sequencing system called MiniSeq and a semiconductor sequencer that is still under development.

Illumina disclosed the initiatives during a presentation at the JP Morgan Healthcare conference held here today. During the presentation, Illumina CEO Jay Flatley also announced a new genotyping array called Infinium XT; a partnership with Bio-Rad to develop a single-cell sequencing workflow; preliminary estimates of its fourth-quarter 2015 revenues; and an update on existing products. The presentation followed the company’s announcement on Sunday that it has launched a new company called Grail to develop a next-generation sequencing test for early cancer detection from patient blood samples.

The MiniSeq system, which is based on Illumina’s current sequencing technology, will begin shipping early this quarter and has a list price of $49,500. It can perform a variety of targeted DNA and RNA applications, from single-gene to pathway sequencing, and promises “all-in” prices, including library prep and sequencing, of $200 to $300 per sample, Flatley said during the JP Morgan presentation.

SOURCES

https://www.genomeweb.com/sequencing-technology/illumina-unveils-mini-targeted-sequencer-semiconductor-sequencing-project-jp

http://investor.biospace.com/biospace/quote?Symbol=ILMN

 

              PRODUCTS AND SERVICES

  •               Mid to large scale manufacturing assets
  •               Analytical Labs
  •               Pre-clinical
  •               Clinical
  •               Launched products

 

              COMPETITORS

https://finance.yahoo.com/q/co?s=ILMN+Competitors Tue, Feb 2, 2016, 2:16pm EST – US Markets

ILMN PVT1 AFFX LMNX Industry
Market Cap: 22.75B N/A 1.13B 835.66M 134.14M
Employees: 3,700 10,000 1,200 745 45.00
Qtrly Rev Growth (yoy): 0.14 N/A -0.01 0.07 0.18
Revenue (ttm): 2.14B 3.80B1 357.74M 235.37M 8.47M
Gross Margin (ttm): 0.73 N/A 0.63 0.71 0.58
EBITDA (ttm): 770.84M N/A 46.64M 52.99M -12.31M
Operating Margin (ttm): 0.30 N/A 0.08 0.17 -1.62
Net Income (ttm): 510.36M 430.90M1 11.22M 39.29M N/A
EPS (ttm): 3.42 N/A 0.13 0.93 -0.34
P/E (ttm): 45.43 N/A 104.40 20.91 25.33
PEG (5 yr expected): 2.68 N/A 4.66 0.55 N/A
P/S (ttm): 10.87 N/A 3.13 3.45 13.65

 

Pvt1 = Life Technologies Corporation (privately held)

AFFX = Affymetrix Inc.

LMNX = Luminex Corporation

 

 

Integrated DNA Technologies (IDT)
IOWA + CA

http://www.com/

 

Integrated DNA Technologies, Inc. (IDT), the global leader in nucleic acid synthesis, serving all areas of life sciences research and development, offers products for a broad range of genomics applications. IDT’s primary business is the production of custom, synthetic nucleic acids for molecular biology applications, including qPCR, sequencing, synthetic biology, and functional genomics. The company manufactures and ships an average of 44,000 custom nucleic acids per day to more than 82,000 customers worldwide. For more information, visit idtdna.com.

 

               PRODUCTS AND SERVICES

               https://eu.idtdna.com/site

  • DNA & RNA Synthesis
  • Custom DNA Oligos 96- & 384-Well Plates Ultramer Oligos Custom RNA Oligos SameDay Oligos HotPlates ReadyMade Primers Oligo Modifications Freedom
  • Dyes GMP for Molecular Diagnostics Large Scale Oligo Synthesis

 

Note : Skokie, IL – December 1, 2015. Integrated DNA Technologies Inc. (“IDT”), the global leader in custom nucleic acid synthesis, has entered into a definitive agreement to acquire the oligonucleotide synthesis business of AITbiotech Pte. Ltd. in Singapore (“AITbiotech”). With this acquisition, IDT expands its customer base across Southeast Asia making it possible for these additional customers to now have access to its broad range of products for genomic applications. AITbiotech will continue operations in its other core business areas.

 

New England Biolabs Inc.
 MA 


http://www.neb.com/

 

                PRODUCTS AND SERVICES

  •                 Restriction Endonucleases
  •                 PCR, Polymerases & Amplification Technologies
  •                 DNA Modifying Enzymes
  •                 Library Preparation for Next Generation Sequencing
  •                 Nucleic Acid Purification
  •                 Markers & Ladders
  •                 RNA Reagents
  •                 Gene Expression
  •                 Cellular Analysis

SOURCE

neb.com/

 

Nitto Denko Avecia Inc.
 MA


http://avecia.com/

 

With over 20 years of experience in oligonucleotide development and production, and over 1000 sequences manufactured, Avecia has played an integral role in the advancing oligo therapeutic market. Our mission is to continue to build value for our customers, as they progress through drug development into commercialization. And as a member of the Nitto Denko Corporation (nitto.com), Avecia is committed to the future of the oligonucleotide market. We are driven by innovative ideas and flexible solutions, designed to provide our customers with the best in service, quality, and technology.

 

SOURCE

http://avecia.com/

 

Note : 1918 Nitto Electric Industrial Co., Ltd. forms in Ohsaki, Tokyo, to produce electrical insulating materials in Japan.

2011 Acquires Avecia Biotechnology Inc. in the U.S.A.

 

 

OriGene Technologies Inc.
 CA

http://www.com/

 

OriGene Technologies, Inc. develops, manufactures, and sells genome wide research and diagnostic products for pharmaceutical, biotechnology, and academic research applications. The company offers cDNA clones, including TrueORF cDNA, viral ORF, destination vectors, TrueClones (human), TrueClones (mouse), organelle marker plasmids, MicroRNA tools, mutant and variant clones, plasmid purification kits, transfection reagents, and gene synthesis service; and HuSH shRNA, siRNA, miRNA, qPCR reagents, plasmid purification products, transfection reagents, PolyA+ and total RNA products, first-strand cDNA synthesis, and CRISPR/Cas9 genome products. It also provides proteins and lysates, such as purified human proteins, over-expression cell lysates, mass spectrometry standard proteins, and protein purification reagents; UltraMAB IHC antibodies, TrueMAB primary antibodies, anti-tag and fluorescent proteins, ELISA antibodies, luminex antibodies, secondary antibodies, and controls and others; and anatomic pathology products, including IHC antibodies, detection systems, and IHC accessories

The company offers luminex and ELISA antibody pairs, autoantibody profiling arrays, ELISA kits, cell assay kits, assay reagents, custom development, and fluorogenic cell assays; TissueFocus search tools; tissue sections; tissue microarrays, cancer protein lysate arrays, TissueScan cDNA arrays, tissue blocks, and quality control products, as well as tissue RNA, DNA, and protein lysates; and lab essentials. Its research areas include cancer biomarker research, RNAi, pathology IHC, stem cell research, ion channels, and protein kinase products. The company provides gene synthesis and molecular biology services, genome editing, custom cloning, custom shRNA, purified protein, monoclonal antibody development, and assay development. It sells its products through distributors worldwide, as well as online. OriGene Technologies, Inc. was incorporated in 1995 and is based in Rockville, Maryland.

SOURCE

http://BLOOMBERG.com

               PRODUCTS AND SERVICES

  •                cDNA Clones
Human, mouse, rat
Expression validated
  •                RNAi
shRNA, siRNA
microRNA & 3’UTR clones
  •                Gene Synthesis
Codon optimization
Variant libraries
  •                Real-time PCR
Primer pairs, panels
SYBR green reagents
  •                Lab Essentials
DNA/RNA purification kits
Transfection reagents
  •                Anatomic Pathology
UltraMAB antibodies
Specificity validated
  •                Recombinant Proteins
10,000 human proteins
from mammalian system
  •                Antibodies
TrueMAB primary antibodies
Anti-tag antibodies
  •                Assays and Kits
ELISA & Luminex antibodies
Autoantibody Profiling Array
  •                Cancer & Normal Tissues
Pathologist verified
gDNA, RNA, sections, arrays

SOURCE

origene.com/

 

Sigma-Aldrich Corporation 
MI 


http://www.sigmaaldrich.com/

Louis, MO – November 18, 2015 Merck KGaA, Darmstadt, Germany, Completes Sigma-Aldrich Acquisition

Merck KGaA today announced the completion of its $17 billion acquisition of Sigma-Aldrich, creating one of the leaders in the $130 billion global industry to help solve the toughest problems in life science.

Press Release: 18-Nov-2015

Letter to our Life Science Customers from Dr. Udit Batra

The life science business of Merck KGaA, Darmstadt, Germany brings together the world-class products and services, innovative capabilities and exceptional talent of EMD Millipore and Sigma-Aldrich to create a global leader in the life science industry.

Everything we do starts with our shared purpose – to solve the toughest problems in life science by collaborating with the global scientific community. 

This combination is built on complementary strengths, which will enable us to serve you even better as one organization than either company could alone.

This means providing a broader portfolio with a catalog of more than 300,000 products, including many of the most respected brands in the industry, greater geographic reach, and an unmatched combination of industry-leading capabilities.

                PRODUCTS AND SERVICES

                http://www.sigmaaldrich.com/configurator/servlet/DesignCenter?btnOpen_0.x=1

                http://www.sigmaaldrich.com/content/dam/sigma-aldrich/common/quality-products.jpg

 

Thermo Fisher Scientific Inc.
 MA 
NYSE :TMO


http://thermofisher.com/

Thermo Fisher Scientific Inc. is a provider of analytical instruments, equipment, reagents and consumables, software and services for research, manufacturing, analysis, discovery and diagnostics. The company operates through four segments: Life Sciences Solutions, provides reagents, instruments and consumables used in biological and medical research, discovery and production of new drugs and vaccines as well as diagnosis of disease; Analytical Instruments, provides instruments, consumables, software and services that are used in the laboratory; Specialty Diagnostics, offers diagnostic test kits, reagents, culture media, instruments and associated products, and Laboratory Products and Services, offers self-manufactured and sourced products for the laboratory.

SOURCE

http://REUTERS.com

 

                PRODUCTS AND SERVICES

  •                 Oligos Value – Standard – Plate
  •                 Primers
  •                 Probes
  •                 Nucleotides

 

                BRANDS

  1.                THERMO SCIENTIFIC
  2.                 APPLIED BIOSYSTEMS
  3.                 INVITROGEN
  4.                 FISHER SCIENTIFIC
  5.                 UNITY LAB SERVICES

 

                 PARTNERSHIPS

AFFYMETRIX : NASDAQ : AFFX : affymetrix.com/

WALTHAM, Mass. & SANTA CLARA, Calif.–(BUSINESS WIRE)–Jan. 8, 2016– Thermo Fisher Scientific Inc. (NYSE:TMO), the world leader in serving science, and Affymetrix Inc. (NASDAQ:AFFX), a leading provider of cellular and genetic analysis products, today announced that their boards of directors have unanimously approved Thermo Fisher’s acquisition of Affymetrix for $14.00 per share in cash. The transaction represents a purchase price of approximately $1.3 billion.

SOURCE

http://BUSINESSWIRE.com

 

TriLink Biotechnologies
 CA 


http://www.com/

 

              PRODUCTS

              Oligonucleotides

  •               DNA Oligos
  •               RNA Oligos
  •               Modified Oligos
  •               Specialty Oligos

              Nucleotides

  •               NTPs (Nucleoside Triphosphates)
  •               Biphosphates
  •               Monophosphates

 

              SERVICES

  •              Custom Chemistry
  •              Reagents
  •              Aptamers

 

             PARTNERSHIPS

  • LIFE TECHNOLOGIES,
  • TERMO FISHER SCIENTIFIC since July 2015 thermofisher.com/
  • GENMARK genmarkdx.com/

SOURCE

http://trilinkbiotech.com/

 

Other related articles published in this Open Access Online Scientific Journal include the following:

Gene Editing: The Role of Oligonucleotide Chips

https://pharmaceuticalintelligence.com/2016/01/07/gene-editing-the-role-of-oligonucleotide-chips/

Gene Editing for Exon 51: Why CRISPR Snipping might be better than Exon Skipping for DMD

https://pharmaceuticalintelligence.com/2016/01/23/gene-editing-for-exon-51-why-crispr-snipping-might-be-better-than-exon-skipping-for-dmd/

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Read Full Post »


Antibody alternatives in specific aptamer 3-D scaffold binding

Curator: Larry H. Bernstein, MD, FCAP

 

 

New Proteomics Tools to Open Up Drug Discovery

Dr. Paul Ko Ferrigno, Chief Scientific Officer, and Dr. Jane McLeod, of Avacta Life Sciences

http://www.dddmag.com/articles/2016/01/new-proteomics-tools-open-drug-discovery

 

Size matters: Smaller molecules allow a tighter packing density on solid surfaces for improved signal-to-noise in assays, such as SPR or ELISA.

http://www.dddmag.com/sites/dddmag.com/files/AvactaAffimer%20stills_00006.jpg

Size matters: Smaller molecules allow a tighter packing density on solid surfaces for improved signal-to-noise in assays, such as SPR or ELISA.

 

In the current genomic era of very accurate DNA analyses by in situ hybridization, DNA chip analyses, and deep sequencing, it is often assumed that antibodies have an analogous ability to identify molecular targets accurately. Nothing could be further from the truth. Proteomics as a field is still lagging behind its genomic counterpart in the level of detail we can achieve, the level of data we can collect and the overall levels of accuracy and reliability that the collected data represent.

From the estimated 20,000 human genes 100,000 different possible proteins have been predicted. What is more, the variation achieved via the post-translational modification of these proteins brings another layer of complexity to cellular signalling. All this means that while studying the genetic blueprint can offer insights into the cell, it is only through examining the functional protein units that we can comprehensively map the dynamic interactions that occur within the cell to drive an organism or disease process.

 

Why not use antibodies?

Antibodies form the basis of molecular recognition in proteomics, whether this is to identify a protein within a complex mixture or label a specific protein within a cell. Both their target specificity and high binding capacity have made these molecules fantastically useful tools within diagnostics. However, the large majority of commercially available antibodies are for use as reagents in research and development, where they are simply not as well validated and issues with their manufacture have created problems that have hindered drug development.

It has proven next to impossible to develop antibodies to certain targets. This may be because of their high homology to the host protein, so the immune system fails to recognise them as different, or due to antigen processing resulting in the loss of post-translational modifications or discontinuous epitopes. However, without the necessary antibodies to investigate these targets the corresponding research avenues have remained closed and key drug targets may have been missed.

Almost worse than lacking the necessary research tools is the problem of antibody reproducibility. Matthias Uhlen revealed that of the 5,436 antibodies tested as part of the Protein Atlas project over 50 percent failed to recognize their target in at least one of two commonly used assays. Antibodies that are not specific to their target or do not recognize their target at all are responsible for increasing the cost of biological research, with an estimated $800 million spent globally every year on bad antibodies. Many published studies have had to be retracted due to antibody-derived error and those that remain unidentified in the literature will continue to lead researchers down blind alleys. This level of misinformation in the published literature is not just hindering the progression of the field, but possibly even sending it backwards and costing more than is needed — an issue not to be taken lightly when so many research budgets are coming under the knife.

More recently there has been a call from a number of leading scientists for the use of polyclonal antibodies, considered to be the worst offenders in terms of batch-to-batch irreproducibility, to be abandoned. They suggest a move towards recombinant systems of production, which would remove the restrictions of the immune system on antibody production. Yet, they state that $1 billion dollars investment would be required to re-route antibody production down this path and suggest that a period of five to ten years may be required to bring about these changes.

Simply producing recombinant antibodies rather than animal-derived affinity reagents will still leave us with a number of problems regarding their use. Antibodies are simply too large to target many smaller, hidden epitopes and the presence of key disulphide bonds within their structure makes them all too susceptible to reduction within the cell, rendering them useless for applications such as live cell imaging of molecules within the cytoplasm. Moreover, can we afford to wait another decade for a solution to this problem before we pursue protein targets for basic understanding and drug development?

 

 

 

 

Antibody alternatives for new targets and techniques

Antibody alternatives are already available to researchers in the life sciences field. They are produced either from nucleic acid or protein molecules. Aptamers are short, single-stranded RNA or DNA molecules that fold to form 3D scaffolds, which can present a specific interaction surface to allow specific binding to its target molecule. Protein scaffolds are formed from parts of or whole proteins modified to present a peptide sequence. This peptide sequence works in a similar manner to present a specific interaction surface for specific binding to a desired target.

These antibody alternatives are produced in recombinant systems, minimizing batch-to-batch variation and allowing them to be produced to theoretically any target. Additionally, as they do not use animals in their production they are generally less expensive to produce than traditional antibodies.

 

 

 

 

While companies such as Affibody and Avacta Life Sciences are aiming to open up the drug pipeline, by offering these alternative affinity reagents to previously inaccessible targets for use in research and development, many have moved into exploiting their therapeutic potential. Noxxon produce an RNA-based scaffold, Spiegelmers, which are currently in phase 2 clinical trials for diabetic nephropathy, and Molecular Partners have reached phase 3 clinical trials with their protein scaffold DARPins, for wet age-related macular degeneration.

These new antibody alternatives are smaller than traditional antibodies. This opens up the use of new technologies, such as super resolution microscopy. While the diffraction limit remained at about 250 nmm the length of the antibody at 15 nm was of little importance, tagging your molecule as accurately as necessary. Removing this limit in super resolution microscopy has meant that antibodies are now too large to provide the required level of accuracy. Instead, using an antibody alternative of only 2nm to tag your protein of interest opens up this new technique offering greater precision to intracellular localization.

 

 

 

As these scaffolds have been engineered to be fit-for-purpose many contain no intramolecular disulphide bonds and so are not sensitive to the reducing environment of the cell. This function enables their use as intracellular reporters of molecular conformation, as well as in standard assays like IHC or western blotting, so allowing scientists to use the same reagent across both intracellular and biochemical assays, thus bridging the gap between cell biology and biochemical studies.

Offering increased reproducibility, access to an increased range of applications, and the opportunity to hit previously inaccessible targets, antibody alternatives are opening up potential new avenues of drug discovery.

 

 

About the Authors

Dr. Paul Ko Ferrigno is Chief Scientific Officer at Avacta Life Sciences and a Visiting Professor at the University of Leeds. He has been working on peptide aptamers since 2001 in Leeds and at the MRC Cancer Cell Unit in Cambridge, UK where his team developed the Affimer scaffold technology.

Dr. Jane McLeod is a Scientific Writer at Avacta Life Sciences.

 

 

 

 

Read Full Post »


Development of Chemoresistance to Targeted Therapies: Alterations of Cell Signaling, & the Kinome [11.4.1.2]

 

Curator, Reporter: Stephen J. Williams, Ph.D.

The advent of molecular targeted therapies like Imatinib (Gleevec), and other tyrosine kinase inhibitors (TKI) has been transformative to cancer therapy. However, as with all chemotherapeutics, including radiation therapy, the development of chemo-resistance toward personalized, molecular therapies has been disastrous to the successful treatment of cancer. The fact that chemo-resistance develops to personalized therapies was a serious disappointment to clinicians (although most expected this to be the case) but more surprisingly it was the rapidity of onset and speed of early reported cases which may have been the biggest shocker.

A post on resistance to other TKIs (to EGFR and ALK) can be seen here: https://pharmaceuticalintelligence.com/2013/11/01/resistance-to-receptor-of-tyrosine-kinase/

History of Development of Resistance to Imatinib (Gleevec)

The Melo group published a paper in Blood showing that short exposure to STI571 (imatinib; trade name Gleevec®) could result in drug resistant clones

Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor STI571: diverse mechanisms of resistance. Blood. 2000 Aug 1;96(3):1070-9.

Mahon FX1, Deininger MW, Schultheis B, Chabrol J, Reiffers J, Goldman JM, Melo JV.

Abstract

Targeting the tyrosine kinase activity of Bcr-Abl with STI571 is an attractive therapeutic strategy in chronic myelogenous leukemia (CML). A few CML cell lines and primary progenitors are, however, resistant to this compound. We investigated the mechanism of this resistance in clones of the murine BaF/3 cells transfected with BCR-ABL and in 4 human cell lines from which sensitive (s) and resistant (r) clones were generated by various methods. Although the resistant cells were able to survive in the presence of STI571, their proliferation was approximately 30% lower than that of their sensitive counterparts in the absence of the compound. The concentration of STI571 needed for a 50% reduction in viable cells after a 3-day exposure was on average 10 times higher in the resistant (2-3 micromol/L) than in the sensitive (0.2-0.25 micromol/L) clones. The mechanism of resistance to STI571 varied among the cell lines. Thus, in Baf/BCR-ABL-r, LAMA84-r, and AR230-r, there was up-regulation of the Bcr-Abl protein associated with amplification of the BCR-ABL gene. In K562-r, there was no Bcr-Abl overexpression, but the IC(50) for the inhibition of Bcr-Abl autophosphorylation was increased in the resistant clones. Sequencing of the Abl kinase domain revealed no mutations. The multidrug resistance P-glycoprotein (Pgp) was overexpressed in LAMA84-r, indicating that at least 2 mechanisms of resistance operate in this cell line. KCL22-r showed neither Bcr-Abl up-regulation nor a higher threshold for tyrosine kinase inhibition by STI571. We conclude that BCR-ABL-positive cells can evade the inhibitory effect of STI571 by different mechanisms, such as Bcr-Abl overexpression, reduced intake mediated by Pgp, and, possibly, acquisition of compensatory mutations in genes other than BCR-ABL.

mellobcrablresistamplification

FISH analysis of AR230 and LAMA84 sensitive and resistant clones, with probes for the ABL (red signal) and theBCR (green signal) genes. BCR-ABL is identified as a red–green or yellow fused signal. Adapted from Mahon et al., Blood 2000; 96(3):1070-9.

This rapid onset of imatinib resistance also see in the clinic and more prominent in advance disease

From NCCN 2nd Annual Congress: Hematologic Malignancies – Update on Primary Therapy, Second-Line Therapy, and New Agents for Chronic Myelogenous Leukemia (Slides with Transcript)

http://www.medscape.org/viewarticle/564097

There is some evidence that even looking earlier makes some sense in determining what the prognosis is. This is from Timothy Hughes’ group in Adelaide, and he is looking at an earlier molecular time point, 3 months after initiation of therapy. And what you have done here is you have taken the 3-month mark and you have said, “Well, based on your response at 3 months, what is your likelihood that in the future you will either get a major molecular response or become resistant?”

3monthimitanibresist

If you look at the accumulation of imatinib resistance to find if it is either initially not responding or becoming resistant after a good response, it goes up with type of disease and phase of disease. So if you look at patients who have early chronic phase disease — that is, they start getting imatinib less than a year from the diagnosis — their chance of failure is pretty low. With later disease — they are in a chronic phase but they have had disease more than a year before they get imatinib — it is higher. If you see patients with accelerated phase or blast crisis, the chances are that they will fail sometime in the future.

speed of imitinib resistance

Therefore, because not all resistant samples show gene amplification of Bcr/Abl and the rapidity of onset of resistance, many feel that there are other mechanisms of resistance at play, like kinome plasticity.

Kinome Plasticity Contributes to TKI resistance

Beyond gene amplification, other mechanisms of imitanib and other tyrosine kinase inhibitors (TKI) include alterations in compensatory signaling pathways. This can be referred to as kinome plasticity and is explained in the following abstracts from the AACR 2015 meeting.

Systems-pharmacology dissection of a drug synergy in imatinib-resistant CML

Georg E Winter, Uwe Rix, Scott M Carlson, Karoline V Gleixner, Florian Grebien, Manuela Gridling, André C Müller, Florian P Breitwieser, Martin Bilban, Jacques Colinge, Peter Valent, Keiryn L Bennett, Forest M White & Giulio Superti-Furga. Nature Chemical Biology 8,905–912(2012)

Occurrence of the BCR-ABLT315I gatekeeper mutation is among the most pressing challenges in the therapy of chronic myeloid leukemia (CML). Several BCR-ABL inhibitors have multiple targets and pleiotropic effects that could be exploited for their synergistic potential. Testing combinations of such kinase inhibitors identified a strong synergy between danusertib and bosutinib that exclusively affected CML cells harboring BCR-ABLT315I. To elucidate the underlying mechanisms, we applied a systems-level approach comprising phosphoproteomics, transcriptomics and chemical proteomics. Data integration revealed that both compounds targeted Mapk pathways downstream of BCR-ABL, resulting in impaired activity of c-Myc. Using pharmacological validation, we assessed that the relative contributions of danusertib and bosutinib could be mimicked individually by Mapk inhibitors and collectively by downregulation of c-Myc through Brd4 inhibition. Thus, integration of genome- and proteome-wide technologies enabled the elucidation of the mechanism by which a new drug synergy targets the dependency of BCR-ABLT315I CML cells on c-Myc through nonobvious off targets.

nchembio.1085-F2kinomegleevecresistance

Please see VIDEO and SLIDESHARE of a roundtable Expert Discussion on CML

Curated Content From the 2015 AACR National Meeting on Drug Resistance Mechanisms and tyrosine kinase inhibitors

Session Title: Mechanisms of Resistance: From Signaling Pathways to Stem Cells
Session Type: Major Symposium
Session Start/End Time: Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
Location: Terrace Ballroom II-III (400 Level), Pennsylvania Convention Center
CME: CME-Designated
CME/CE Hours: 2
Session Description: Even the most effective cancer therapies are limited due to the development of one or more resistance mechanisms. Acquired resistance to targeted therapies can, in some cases, be attributed to the selective propagation of a small population of intrinsically resistant cells. However, there is also evidence that cancer drugs themselves can drive resistance by triggering the biochemical- or genetic-reprogramming of cells within the tumor or its microenvironment. Therefore, understanding drug resistance at the molecular and biological levels may enable the selection of specific drug combinations to counteract these adaptive responses. This symposium will explore some of the recent advances addressing the molecular basis of cancer cell drug resistance. We will address how tumor cell signaling pathways become rewired to facilitate tumor cell survival in the face of some of our most promising cancer drugs. Another topic to be discussed involves how drugs select for or induce the reprogramming of tumor cells toward a stem-like, drug resistant fate. By targeting the molecular driver(s) of rewired signaling pathways and/or cancer stemness it may be possible to select drug combinations that prevent the reprogramming of tumors and thereby delay or eliminate the onset of drug resistance.
Presentations:
Chairperson
Tuesday, Apr 21, 2015, 10:30 AM -12:30 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA
Introduction
Tuesday, Apr 21, 2015, 10:30 AM -10:40 AM
Resistance to tyrosine kinase inhibitors: Heterogeneity and therapeutic strategies.
Tuesday, Apr 21, 2015, 10:40 AM -10:55 AM
Jeffrey A. Engelman. Massachusetts General Hospital, Boston, MA
Discussion
Tuesday, Apr 21, 2015, 10:55 AM -11:00 AM
NG04: Clinical acquired resistance to RAF inhibitor combinations in BRAF mutant colorectal cancer through MAPK pathway alterations
Tuesday, Apr 21, 2015, 11:00 AM -11:15 AM
Ryan B. Corcoran, Leanne G. Ahronian, Eliezer Van Allen, Erin M. Coffee, Nikhil Wagle, Eunice L. Kwak, Jason E. Faris, A. John Iafrate, Levi A. Garraway, Jeffrey A. Engelman. Massachusetts General Hospital Cancer Center, Boston, MA, Dana-Farber Cancer Institute, Boston, MA
Discussion
Tuesday, Apr 21, 2015, 11:15 AM -11:20 AM
SY27-02: Tumour heterogeneity and therapy resistance in melanoma
Tuesday, Apr 21, 2015, 11:20 AM -11:35 AM
Claudia Wellbrock. Univ. of Manchester, Manchester, United Kingdom
Discussion
Tuesday, Apr 21, 2015, 11:35 AM -11:40 AM
SY27-03: Breast cancer stem cell state transitions mediate therapeutic resistance
Tuesday, Apr 21, 2015, 11:40 AM -11:55 AM
Max S. Wicha. University of Michigan, Comprehensive Cancer Center, Ann Arbor, MI
Discussion
Tuesday, Apr 21, 2015, 11:55 AM -12:00 PM
SY27-04: Induction of cancer stemness and drug resistance by EGFR blockade
Tuesday, Apr 21, 2015, 12:00 PM -12:15 PM
David A. Cheresh. UCSD Moores Cancer Center, La Jolla, CA
Discussion
Tuesday, Apr 21, 2015, 12:15 PM -12:20 PM
General Discussion
Tuesday, Apr 21, 2015, 12:20 PM -12:30 PM

Targeting Macromolecular Signaling Complexes 
Room 115, Pennsylvania Convention Center

Drug Resistance 
Hall A (200 Level), Pennsylvania Convention Center
Resistance to Pathway-Targeted Therapeutics 1 
Section 33

Molecular Mechanisms of Sensitivity or Resistance to Pathway-Targeted Agents 
Room 118, Pennsylvania Convention Center

Targeting Signaling Pathways in Cancer 
Room 204, Pennsylvania Convention Center
Exploiting the MAPK Pathway in Cancer 
Room 115, Pennsylvania Convention Center

PLEASE see the attached WORD file which includes ALL abstracts, posters, and talks on this subject from the AACR 2015 national meeting BELOW

 AACR2015resistancekinome

Other posts related to, Cancer, Chemotherapy, Gleevec and Resistance on this Open Access Journal Include

Imatinib (Gleevec) May Help Treat Aggressive Lymphoma: Chronic Lymphocytic Leukemia (CLL)

Treatments for Acute Leukemias [2.4.4A]

Therapeutic Implications for Targeted Therapy from the Resurgence of Warburg ‘Hypothesis’

Hematologic Malignancies [6.2]

Overview of Posttranslational Modification (PTM)

Novel Modeling Methods for Genomic Data Analysis & Evolutionary Systems Biology to Design Dosing Regimens to Minimize Resistance

Mechanisms of Drug Resistance

Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia

An alternative approach to overcoming the apoptotic resistance of pancreatic cancer

Resistance to Receptor of Tyrosine Kinase

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Heroes in Basic Medical Research – Leroy Hood

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 4.5

Leroy Hood, MD, PhD

Dr. Hood created the technological foundation for the sciences of genomics (study of genes) and proteomics (study of proteins) through the invention of five groundbreaking instruments and by explicating the potentialities of genome and proteome research into the future through his pioneering of the fields of systems biology and systems medicine. Hood’s instruments not only pioneered the deciphering of biological information, but also introduced the concept of high throughput data accumulation through automation and parallelization of the protein and DNA chemistries.

The first four instruments were commercialized by Applied Biosystems, Inc., a company founded by Dr. Hood in 1981, and the ink-jet technology was commercialized by Agilent Technologies, thus making these instruments immediately available to the world-community of scientists.

The first two instruments transformed the field of proteomics. The protein sequencer allowed scientists to read and analyze proteins that had not previously been accessible, resulting in the characterization of a series of new proteins whose genes could then be cloned and analyzed. These discoveries led to significant ramifications for biology, medicine, and pharmacology. The second instrument, the protein synthesizer, synthesized proteins and peptides in sufficient quantities to begin characterizing their functions. The DNA synthesizer, the first of three instruments for genomic analyses, was used to synthesize DNA fragments for DNA mapping and gene cloning. The most notable of Hood’s inventions, the automated DNA sequencer developed in 1986, made possible high-speed sequencing of human genomes and was the key technology enabling the Human Genome Project.

In the early 1990s Hood and his colleagues developed the ink-jet DNA synthesis technology for creating DNA arrays with tens of thousands of gene fragments, one of the first of the so-called DNA chips, which enabled measuring the levels of 10,000s of expressed genes. This instrument has also transformed genomics, biology, and medicine.

In 1992, Hood created the first cross-disciplinary biology department, Molecular Biotechnology, at the University of Washington. In 2000, he left the UW to co-found Institute for Systems Biology, the first of its kind. He has pioneered systems medicine the years since ISB’s founding.

In 2000, Hood and two colleagues founded the Institute for Systems Biology (ISB), a nonprofit research institute integrating biology, technology, computation and medicine to take a systems (holistic) approach to studying the complexity of biology and medicine by analyzing all elements in a biological system rather than studying them one gene or protein at a time (an atomistic approach).

Hood has made many seminal discoveries in the fields of immunology, neurobiology and biotechnology and, most recently, has been a leader in the development of systems biology, its applications to cancer, neurodegenerative disease, and the linkage of systems biology to personalized medicine.

Hood’s efforts in a systems approach to disease have led him to pioneer a new approach to medicine that he coined P4 Medicine in 2003. His view is that P4 medicine will transform the practice of medicine over the next decade, moving it from a largely reactive discipline to a proactive one.

Dr. Hood’s outstanding contributions have had a resounding effect on the advancement of science since the 1960s. Throughout his career, he has adhered to the advice of his mentor, Dr. William J. Dreyer: “If you want to practice biology, do it on the leading edge, and if you want to be on the leading edge, invent new tools for deciphering biological information.”

 

Hood is now pioneering new approaches to P4 medicine

Co-founder and Chairman P4 Medicine institute

—predictive, preventive, personalized and participatory, and most recently, has embarked on creating a P4 pilot project on 100,000 well individuals, that is transforming healthcare.

In addition to his ground-breaking research, Hood has published 750 papers, received 36 patents, 17 honorary degrees and more than 100 awards and honors. He is one of only 15 individuals elected to all three National Academies—the National Academy of Science, the National Academy of Engineering, and the Institute of Medicine. Hood has founded or co-founded 15 different biotechnology companies.

 

http://www.youtube.com/watch%3Fv%3D5aE8tgbsl9U Feb 18, 2015 Dr. Leroy Hood, President and Co-founder, Institute for Systems Biology, gives a talk entitled “Systems Medicine and a Longitudinal, …

http://www.youtube.com/watch%3Fv%3DaYGTLj02sx0  Nov 19, 2014 … of Healthcare? A Personal View of Biological Complexity, Paradigm Changes, Systems Biology and Systems Medicine .Speaker: Leroy Hood.

http://www.youtube.com/watch%3Fv%3DnT1MvnH6j8Q Sep 26, 2014 Dr. Leroy Hood discusses how P4 (Predictive, Preventive, … EMBC 2014 Theme Keynote Lecture with Dr. Emery Brown – Duration: 58:49. by …

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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle


Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD

 

Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at

http://www.amazon.com/dp/B012BB0ZF0.

This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:

https://pharmaceuticalintelligence.com/biomed-e-books/

Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 

 

Summary 

Epilogue

 

 

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Biomarker Guided Therapy

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

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer

Liping Chung, K Moore, L Phillips, FM Boyle, DJ Marsh and RC Baxter
Breast Cancer Research 2014, 16:R63
http://breast-cancer-research.com/content/16/3/R63

Introduction: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed  proteins in sera from BC and healthy volunteers (HV), with the
goal  of developing a new prognostic biomarker panel.
Methods: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated.
Results: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up.
Conclusions: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Variants of uncertain significance in BRCA: a harbinger of ethical and policy issues to come?

Jae Yeon Cheon, Jessica Mozersky and Robert Cook-Deegan
Genome Medicine 2014, 6:121
http://genomemedicine.com/content/6/12/121

After two decades of genetic testing and research, the BRCA1 and BRCA2 genes are two of the most well-characterized genes in the human genome. As a result, variants of uncertain significance (VUS; also called variants of unknown significance) are reported less frequently than for genes that have been less thoroughly studied. However, VUS continue to be uncovered, even for BRCA1/2. The increasing use of multi-gene panels and whole-genome and whole-exome sequencing will lead to higher rates of VUS detection because more genes are being tested, and most genomic loci have been far less intensively characterized than BRCA1/2. In this article, we draw attention to ethical and policy-related issues that will emerge. Experience garnered from BRCA1/2 testing is a useful introduction to the challenges of detecting VUS in other genetic testing contexts, while features unique to BRCA1/2 suggest key differences between the BRCA experience and the current challenges of multi-gene panels in clinical care. We propose lines of research and policy development, emphasizing the importance of pooling data into a centralized open-access database for the storage of gene variants to improve VUS interpretation. In addition, establishing ethical norms and regulated practices for sharing and curating data, analytical algorithms, interpretive frameworks and patient re-contact are important policy areas.

The Significance of Normal Pretreatment Levels of CA125 (<35 U/mL) in Epithelial Ovarian Carcinoma

Joseph Menczer,  Erez Ben-Shem,  Abraham Golan, and Tally Levy
Rambam Maimonides Med J 2015;6 (1):e0005. http://dx.doi.org:/10.5041/RMMJ.10180

Objective: To assess the association between normal CA125 levels at diagnosis of epithelial ovarian carcinoma (EOC) with prognostic factors and with outcome.
Methods: The study group consisted of histologically confirmed EOC patients with normal pretreatment CA125 levels, and the controls consisted of EOC patients with elevated (≥35 U/mL) pretreatment CA125 levels, diagnosed and treated between 1995 and 2112. Study and control group patients fulfilled the following criteria: 1) their pretreatment CA125 levels were assessed; 2) they had full standard primary treatment, i.e. cytoreductive surgery and cisplatin-based chemotherapy; and 3) they were followed every 2–4 months during the first two years and every 4–6 months thereafter.
Results: Of 114 EOC patients who fulfilled the inclusion criteria, 22 (19.3%) had normal pretreatment CA125 levels. The control group consisted of the remaining 92 patients with ≥35 U/mL serum CA125 levels pretreatment. The proportion of patients with early-stage and low-grade disease, with optimal cytoreduction, and with platin-sensitive tumors was significantly higher in the study group than in the control group. The progression-free survival (PFS) and overall survival (OS) were significantly higher in the study group than in the control group on univariate analysis but not on multivariate analysis.

Higher gene expression variability in the more aggressive subtype of chronic lymphocytic leukemia

Simone Ecker, Vera Pancaldi, Daniel Rico and Alfonso Valencia
Genome Medicine (2015) 7:8 http://dx.doi.org:/10.1186/s13073-014-0125-z

Background: Chronic lymphocytic leukemia (CLL) presents two subtypes which have drastically different clinical outcomes, IgVH mutated (M-CLL) and IgVH unmutated (U-CLL). So far, these two subtypes are not associated to clear differences in gene expression profiles. Interestingly, recent results have highlighted important roles for heterogeneity, both at the genetic and at the epigenetic level in CLL progression.
Methods: We analyzed gene expression data of two large cohorts of CLL patients and quantified expression variability across individuals to investigate differences between the two subtypes using different measures and statistical tests. Functional significance was explored by pathway enrichment and network analyses. Furthermore, we implemented a random forest approach based on expression variability to classify patients into disease subtypes.
Results: We found that U-CLL, the more aggressive type of the disease, shows significantly increased variability of gene expression across patients and that, overall, genes that show higher variability in the aggressive subtype are related to cell cycle, development and inter-cellular communication. These functions indicate a potential relation between gene expression variability and the faster progression of this CLL subtype. Finally, a classifier based on gene expression variability was able to correctly predict the disease subtype of CLL patients.
Conclusions: There are strong relations between gene expression variability and disease subtype linking significantly increased expression variability to phenotypes such as aggressiveness and resistance to therapy in CLL.

The Emerging Roles of Thyroglobulin

Yuqian Luo, Yuko Ishido, Naoki Hiroi, Norihisa Ishii, and Koichi Suzuki
Advances in Endocrinology 2014, Article ID 189194, 7 pages http://dx.doi.org/10.1155/2014/189194

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

The GUIDE-IT trial will help doctors find a new standard of care for heart failure.

Heart failure affects more than 25 million people worldwide, including 5.8 million in the United States and 6.9 million in Europe. About one to two percent of adults in developed countries have been diagnosed with heart failure; this increases to more than 10 percent in people over age 70. Moreover, heart failure accounts for more than 17 percent of Medicare spending and about 5 percent of total US healthcare spending. The cost to society in the US is about 30 billion dollars a year—and rising.

For people hospitalized due to heart failure, the outlook isn’t encouraging. Following discharge, one in four patients is likely to be back in the hospital in less than a month. With every acute heart failure event that requires readmission, the chances of dying from the disease increase.

Heart failure occurs when the heart is unable to fill with or pump sufficient blood to meet the needs of the body. Some heart failure symptoms—shortness of breath, fatigue and fluid buildup—which are present in other health problems. Heart failure may develop from coronary artery disease, high blood pressure, cardiomyopathy, heart valve disease, arrhythmias, viral or bacterial infections, and congenital heart defects. As a consequence, these patients often have additional diseases (comorbidities) and managing heart failure can be extremely challenging.

There have been no new drugs for heart failure in more than a decade. The last breakthrough was cardiac resynchronization therapy, a device and not a drug. The goals of therapy are to treat heart failure’s underlying causes, reduce symptoms, improve the patient’s quality of life and keep the disease from getting worse.

More than a pump

The heart isn’t just a muscle pumping blood through the body. It is also an endocrine gland that secretes peptides and hormones. When the heart is failing, its stressed cells release larger amounts of substances known as natriuretic peptides, including N-terminal prohormone brain natriuretic peptide, or NT-proBNP.

Roche’s NT-proBNP test measures the levels of this peptide and helps doctors to determine whether patients are suffering from heart failure and to assess their prognosis. Most recently, NT-proBNP has also been shown to help physicians guide and adjust the patient’s drug therapy. The objective of the pivotal GUIDE-IT trial is to demonstrate the efficacy and safety of NT-proBNP guided heart failure therapy.

Sponsored by the National Institutes of Health (NIH), the GUIDE-IT trial will help doctors answer important questions about NT-proBNP’s impact on medical care. About 1100 patients are enrolled in this robustly powered, randomized controlled trial comparing NT-proBNP guided therapy on top of standard care versus standard care alone in high-risk heart failure patients. Its primary endpoint is time to cardiovascular death or first heart failure hospitalization.

With the NT-proBNP biomarker, doctors can create personalized treatment plans for patients to substantially reduce mortality and morbidity. It can be viewed as a companion diagnostic that works with all the drugs recommended by the major guidelines.

Finding new answers

GUIDE-IT will last five years and involve approximately 45 trial sites in the United States. The first group of patients will be enrolled by the end of 2012.

“We need to take a more strategic approach if we are going to meet the AHA/ASA’s 2020 goal of reducing heart failure hospitalizations by 20 percent,” Dr. O’Connor, Chief of the Division of Cardiovascular Medicine at Duke Heart Center in Durham, North Carolina, said at a media briefing held in October at Roche Diagnostics International in Rotkreuz, Switzerland.
The relative and combined ability of: high-sensitivity cardiac troponin T, and N-terminal pro-B-type natriuretic Peptide – to predict cardiovascular events and death in patients with type 2 diabetes.

Hillis GS; Welsh P; Chalmers J; Perkovic V; Chow CK; Li Q; Jun M; Neal B; et al.
http://reference.medscape.com/medline/abstract/24089534?src=wnl_ref_prac_diab

OBJECTIVE Current methods of risk stratification in patients with type 2 diabetes are suboptimal. The current study assesses the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and high-sensitivity cardiac troponin T (hs-cTnT) to improve the prediction of cardiovascular events and death in patients with type 2 diabetes.
RESEARCH DESIGN AND METHODS A nested case-cohort study was performed in 3,862 patients who participated in the Action in Diabetes and Vascular Disease: Preterax and Diamicron Modified Release Controlled Evaluation (ADVANCE) trial. RESULTS Seven hundred nine (18%) patients experienced a major cardiovascular event (composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke) and 706 (18%) died during a median of 5 years of follow-up. In Cox regression models, adjusting for all established risk predictors, the hazard ratio for cardiovascular events for NT-proBNP was 1.95 per 1 SD increase (95% CI 1.72, 2.20) and the hazard ratio for hs-cTnT was 1.50 per 1 SD increase (95% CI 1.36, 1.65). The hazard ratios for death were 1.97 (95% CI 1.73, 2.24) and 1.52 (95% CI 1.37, 1.67), respectively. The addition of either marker improved 5-year risk classification for cardiovascular events (net reclassification index in continuous model, 39% for NT-proBNP and 46% for hs-cTnT). Likewise, both markers greatly improved the accuracy with which the 5-year risk of death was predicted. The combination of both markers provided optimal risk discrimination.
CONCLUSIONS NT-proBNP and hs-cTnT appear to greatly improve the accuracy with which the risk of cardiovascular events or death can be estimated in patients with type 2 diabetes.

Genetics and Heart Failure: A Concise Guide for the Clinician

Cécile Skrzynia, Jonathan S. Berg, Monte S. Willis and Brian C. Jensen
Current Cardiology Reviews, 2013; 9.

Abstract: The pathogenesis of heart failure involves a complex interaction between genetic and environmental factors. Genetic factors may influence the susceptibility to the underlying etiology of heart failure, the rapidity of disease progression, or the response to pharmacologic therapy. The genetic contribution to heart failure is relatively minor in most multifactorial cases, but more direct and profound in the case of familial dilated cardiomyopathy. Early studies of genetic risk for heart failure focused on polymorphisms in genes integral to the adrenergic and renin-angiotensin-aldosterone system. Some of these variants were found to increase the risk of developing heart failure, and others appeared to affect the therapeutic response to neurohormonal antagonists. Regardless, each variant individually confers a relatively modest increase in risk and likely requires complex interaction with other variants and the environment for heart failure to develop. Dilated cardiomyopathy frequently leads to heart failure, and a genetic etiology increasingly has been recognized in cases previously considered to be “idiopathic”. Up to 50% of dilated cardiomyopathy cases without other cause likely are due to a heritable genetic mutation. Such mutations typically are found in genes encoding sarcomeric proteins and are inherited in an autosomal dominant fashion. In recent years, rapid advances in sequencing technology have improved our ability to diagnose familial dilated cardiomyopathy and those diagnostic tests are available widely. Optimal care for the expanding population of patients with heritable heart failure involves counselors and physicians with specialized training in genetics, but numerous online genetics resources are available to practicing clinicians.

Cardiac Troponin Testing Is Overused after the Rule-In or Rule-Out of Myocardial Infarction

Olaia Rodriguez Fraga, Y Sandoval, SA Love, ZJ McKinney, MAM Murakami, SW Smith, FS Apple
Clinical Chemistry 2015; 61:2 http://dx.doi.org:/10.1373/clinchem.2014.232694

No good studies have systematically evaluated appropriate clinical utilization of cardiac troponin testing in the clinical setting of the rule-in and rule-out of myocardial infarction (MI). Our collective 100-plus years of clinical and laboratory experience suggested that provider test ordering and use of cardiac troponin has been excessive after a diagnosis of MI or no MI has been determined. There is no evidence that supports continuation of cardiac troponin testing after a diagnosis is made.

Number of cTnI results demonstrating excessive orders by diagnosis

Number of cTnI results demonstrating excessive orders by diagnosis

Time and Frequency Domain Analysis of Heart Rate Variability and their orrelations in Diabetes Mellitus
T. Ahamed Seyd, V. I. Thajudin Ahamed, Jeevamma Jacob, Paul Joseph K
Intl J Biolog and Life Sciences 2008; 4(1)

Diabetes mellitus (DM) is frequently characterized by autonomic nervous dysfunction. Analysis of heart rate variability (HRV) has become a popular noninvasive tool for assessing the activities of autonomic nervous system (ANS). In this paper, changes in ANS
activity are quantified by means of frequency and time domain analysis of R-R interval variability. Electrocardiograms (ECG) of 16 patients suffering from DM and of 16 healthy volunteers were recorded. Frequency domain analysis of extracted normal to normal interval (NN interval) data indicates significant difference in very low frequency (VLF) power, low frequency (LF) power and high frequency (HF) power, between the DM patients and control group. Time domain measures, standard deviation of NN interval (SDNN), root mean square of successive NN interval differences (RMSSD), successive NN intervals differing more than 50 ms (NN50 Count), percentage value of NN50 count (pNN50), HRV triangular index and triangular interpolation of NN intervals (TINN) also show significant difference between the DM patients and control group.

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering

Power Spectral Density of the RR interval of a 62 year old woman suffering
from diabetes for the last 15 years

HRV analysis has gained much importance in recent years, as a technique employed to explore the activity of ANS, and as an important early marker for identifying different pathological conditions. DM is a disease in which the cardiac autonomic activity is progressively compromised. Our investigation indicates that different time domain and frequency domain measures of HRV would be able to provide valuable information regarding the autonomic dysfunction to DM.

Time domain and frequency domain analysis of the RR interval variability of diabetic and normal subjects shows that there is significant difference in these measures for DM patients with respect to normal subjects. Variation of the HRV parameters indicates changes in ANS activity of DM patients. This can provide valid information regarding autonomic neuropathy in people with diabetes. It may be noted that these methods can detect changes before clinical signs appear. So we can expect that these measures enable early detection and treatment/subsequent management of patients and thus can avoid acute and chronic complications.

Multiparametric diagnostics of cardiomyopathies by microRNA signatures

Christine S. Siegismund & Maria Rohde & Uwe Kühl & Dirk Lassner
Microchim Acta 2014   http://dx.doi.org:/10.1007/s00604-014-1249-y

The diagnosis of cardiomyopathies by endomyocardial biopsy analysis is the gold standard for confirmation of causative reasons but is failing if a sample does not contain the area of interest due to focal pathology. Biopsies are revealing an extract of the current situation of the heart muscle only, and the need for global organ-specific or systemic markers is obvious in order to minimize sampling errors. Global markers like specific gene expression signatures in myocardial tissue may therefore reflect the focal situation or condition of the whole myocardium. Besides gene expression profiles, microRNAs (miRNAs) represent a new group of stable biomarkers that are detectable both in tissue and body fluids. Such miRNAs may serve as cardiological biomarkers to characterize inflammatory processes, to confirm viral infections, and to differentiate various forms of infection.
The predictive power of single miRNAs for diagnosis of complex diseases may be further increased if several distinctly deregulated candidates are combined to form a specific miRNA signature. Diagnostic systems that generate disease related miRNA profiles are based on microarrays, bead-based oligo sorbent assays, or on assays based on real-time polymerase chain reactions and placed on microfluidic cards or nanowell plates. Multiparametric diagnostic systems that can measure differentially expressed miRNAs may become the diagnostic tool of the future due to their predictive value with respect to clinical course, therapeutic decisions, and therapy monitoring. We discuss here specific merits, limitations and the potential of currently available analytical platforms for diagnostics of heart muscle diseases based on miRNA profiling.

Predictive value of plasma galectin-3 levels in heart failure with reduced and preserved ejection fraction

Rudolf A. de Boer, DJA Lok, T Jaarsma, P van der Meer, AA Voors, et al.
Annals Med, 2011; 43: 60–68 http://dx.doi.org:/10.3109/07853890.2010.538080

We studied the prognostic value of base-line galectin-3 in a large HF cohort, with preserved and reduced left ventricular ejection fraction (LVEF), and compared this to other biomarkers.
Methods. We studied 592 HF patients who had been hospitalized for HF and were followed for 18 months. The primary end-point was a composite of all-cause mortality and HF hospitalization.
Results. A doubling of galectin-3 levels was associated with a hazard ratio (HR) of 1.97 (1.62–2.42) for the primary outcome (P= 0.001). After correction for age, gender, BNP, eGFR, and diabetes the HR was 1.38 (1.07–1.78; P= 0.015). Galectin-3 levels were correlated with higher IL -6 and CRP levels (P= 0.002). Changes of galectin-3 levels after 6 months did not add prognostic information to the base-line value (n= 291); however, combining plasma galectin-3 and BNP levels increased prognostic value over either biomarker alone (ROC analysis, P = 0.05). The predictive value of galectin-3 was stronger in patients with preserved LVEF (n= 114) compared to patients with reduced LVEF (P= 0.001).
Conclusions. Galectin-3 is an independent marker for outcome in HF and appears to be particularly useful in HF patients with preserved LVEF.

Criteria for the use of omics-based predictors in clinical trials

Lisa M. McShane, MM Cavenagh, TG Lively, DA Eberhard, et al.
Nature  17 Oct 2013; 502: 317-320. http://dx.doi.org:/10.1038/nature12564

The US National Cancer Institute (NCI), in collaboration with scientists representing multiple areas of expertise relevant to ‘omics’-based test development, has developed a checklist of criteria that can be used to determine the readiness of omics-based tests for guiding patient care in clinical trials. The checklist criteria cover issues relating to specimens, assays, mathematical modelling, clinical trial design, and ethical, legal and regulatory aspects. Funding bodies and journals are encouraged to consider the checklist, which they may find useful for assessing study quality and evidence strength. The checklist will be used to evaluate proposals for NCI-sponsored clinical
trials in which omics tests will be used to guide therapy.

M-Atrial Natriuretic Peptide and Nitroglycerin in a Canine Model of Experimental Acute Hypertensive Heart Failure: Differential Actions of 2 cGMP Activating Therapeutics.

Paul M McKie, Alessandro Cataliotti, Tomoko Ichiki, S Jeson Sangaralingham, Horng H Chen, John C Burnett
J Am Heart Assoc 01/2014; 3(1):e000206. http://dx.doi.org:/10.1161/JAHA.113.000206

Systemic hypertension is a common characteristic in acute heart failure (HF). This increasingly recognized phenotype is commonly associated with renal dysfunction and there is an unmet need for renal enhancing therapies. In a canine model of HF and acute vasoconstrictive hypertension we characterized and compared the cardiorenal actions of M-atrial natriuretic peptide (M-ANP), a novel particulate guanylyl cyclase (pGC) activator, and nitroglycerin, a soluble guanylyl cyclase (sGC) activator.
HF was induced by rapid RV pacing (180 beats per minute) for 10 days. On day 11, hypertension was induced by continuous angiotensin II infusion. We characterized the cardiorenal and humoral actions prior to, during, and following intravenous M-ANP (n=7), nitroglycerin (n=7), and vehicle (n=7) infusion. Mean arterial pressure (MAP) was reduced by M-ANP (139±4 to 118±3 mm Hg, P<0.05) and nitroglycerin (137±3 to 116±4 mm Hg, P<0.05); similar findings were recorded for pulmonary wedge pressure (PCWP) with M-ANP (12±2 to 6±2 mm Hg, P<0.05) and nitroglycerin (12±1 to 6±1 mm Hg, P<0.05). M-ANP enhanced renal function with significant increases (P<0.05) in glomerular filtration rate (38±4 to 53±5 mL/min), renal blood flow (132±18 to 236±23 mL/min), and natriuresis (11±4 to 689±37 mEq/min) and also inhibited aldosterone activation (32±3 to 23±2 ng/dL, P<0.05), whereas nitroglycerin had no significant (P>0.05) effects on these renal parameters or aldosterone activation.
Our results advance the differential cardiorenal actions of pGC (M-ANP) and sGC (nitroglycerin) mediated cGMP activation. These distinct renal and aldosterone modulating actions make M-ANP an attractive therapeutic for HF with concomitant hypertension, where renal protection is a key therapeutic goal.

Genome-Wide Association Study of a Heart Failure Related Metabolomic Profile Among African Americans in the Atherosclerosis Risk in Communities (ARIC) Study

Bing Yu, Y Zheng, D Alexander, TA Manolio, A Alonso, JA Nettleton, & E Boerwinkle
Genet Epidemiol 2013; 00:1–6, http://dx.doi.org:/10.1002/gepi.21752

Both the prevalence and incidence of heart failure (HF) are increasing, especially among African Americans, but no large-scale, genome-wide association study (GWAS) of HF-related metabolites has been reported. We sought to identify novel genetic variants that are associated with metabolites previously reported to relate to HF incidence. GWASs of three metabolites identified previously as risk factors for incident HF (pyroglutamine, dihydroxy docosatrienoic acid, and X-11787, being either hydroxy-leucine or hydroxy-isoleucine) were performed in 1,260 African Americans free of HF at the baseline examination of the Atherosclerosis Risk in Communities (ARIC) study. A significant association on chromosome 5q33 (rs10463316, MAF = 0.358, P-value = 1.92 × 10−10) was identified for pyroglutamine. One region on chromosome 2p13 contained a nonsynonymous substitution in N-acetyltransferase 8 (NAT8) was associated with X-11787 (rs13538, MAF = 0.481, P-value = 1.71 × 10−23). The smallest P-value for dihydroxy docosatrienoic acid was rs4006531 on chromosome 8q24 (MAF = 0.400, P-value = 6.98 × 10−7). None of the above SNPs were individually associated with incident HF, but a genetic risk score (GRS) created by summing the most significant risk alleles from each metabolite detected 11% greater risk of HF per allele. In summary, we identified three loci associated with previously reported HF-related metabolites. Further use of metabolomics technology will facilitate replication of these findings in independent samples.

Global Left Atrial Strain Correlates with CHADS2 Risk Score in Patients with Atrial Fibrillation

SK Saha, PL Anderson, G Caracciolo, A Kiotsekoglou, S Wilansky, S Govind, et al.
J Am Soc Echocardiogr 2011; 24(5): 506-512.
http://dx.doi.org:/10.1016/j.echo.2011.02.012

Background: The aim of this cross-sectional study was to explore the association between echocardiographic parameters and CHADS2 score in patients with nonvalvular atrial fibrillation (AF).
Methods: Seventy-seven subjects (36 patients with AF, 41 control subjects) underwent standard twodimensional, Doppler, and speckle-tracking echocardiography to compute regional and global left atrial (LA) strain.
Results: Global longitudinal LA strain was reduced in patients with AF compared with controls (P < .001) and was a predictor of high risk for thromboembolism (CHADS2 score $ 2; odds ratio, 0.86; P = .02). LA strain indexes showed good interobserver and intraobserver variability. In sequential Cox models, the prediction of hospitalization and/or death was improved by addition of global LA strain and indexed LA volume to CHADS2 score (P = .003).
Conclusions: LA strain is a reproducible marker of dynamic LA function and a predictor of stroke risk and cardiovascular outcomes in patients with AF.

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin, EV Dolmatova, MP Morley, KL Lunetta, et al.
Heart Rhythm, HRTHM5533. PII: S1547-5271(13)01226-5
http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrial fibrillation (AF). However, the molecular events related to structural and functional changes that enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Methods— We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function. Gene expression was profiled using the Affymetrix GeneChip Human Genome U133A Array. Genetic variation was profiled using the Affymetrix Genome-Wide Human SNP Array 6.0.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Atrial Natriuretic Peptide Single Nucleotide Polymorphisms in Patients with Nonfamilial Structural Atrial Fibrillation

Pietro Francia, A Ricotta, A Frattari, R Stanzione, A Modestino, et al.
Clinical Medicine Insights: Cardiology 2013:7 153–159
http://dx.doi.org:/10.4137/CMC.S12239

Background: Atrial natriuretic peptide (ANP) has antihypertrophic and antifibrotic properties that are relevant to AF substrates. The −G664C and rs5065 ANP single nucleotide polymorphisms (SNP) have been described in association with clinical phenotypes, including hypertension and left ventricular hypertrophy. A recent study assessed the association of early AF and rs5065 SNPs in low-risk subjects. In a Caucasian population with moderate-to-high cardiovascular risk profile and structural AF, we conducted a case-control study to assess whether the ANP −G664C and rs5065 SNP associate with nonfamilial structural AF.
Methods: 168 patients with nonfamilial structural AF and 168 age- and sex-matched controls were recruited. The rs5065 and −G664C ANP SNPs were genotyped.
Results: The study population had a moderate-to-high cardiovascular risk profile with 86% having hypertension, 23% diabetes, 26% previous myocardial infarction, and 23% left ventricular systolic dysfunction. Patients with AF had greater left atrial diameter (44 ± 7
vs. 39 ± 5 mm; P , 0.001) and higher plasma NTproANP levels (6240 ± 5317 vs. 3649 ± 2946 pmol/mL; P , 0.01). Odds ratios (ORs)
for rs5065 and −G664C gene variants were 1.1 (95% confidence interval [CI], 0.7–1.8; P = 0.71) and 1.2 (95% CI, 0.3–3.2; P = 0.79), respectively, indicating no association with AF. There were no differences in baseline clinical characteristics among carriers and noncarriers of the −664C and rs5065 minor allele variants.
Conclusions: We report lack of association between the rs5065 and −G664C ANP gene SNPs and AF in a Caucasian population of patients with structural AF. Further studies will clarify whether these or other ANP gene variants affect the risk of different subphenotypes of AF driven by distinct pathophysiological mechanisms.

N-terminal proBNP and mortality in hospitalized patients with heart failure and preserved vs. reduced systolic function: data from the prospective Copenhagen Hospital Heart Failure Study (CHHF)

Kirk, M. Bay, J. Parnerc, K. Krogsgaard, T.M. Herzog, S. Boesgaard, et al.
Eur Journal Heart Failure 6 (2004) 335–341
http://dx.doi.org:/10.1016/j.ejheart.2004.01.002

Preserved systolic function among heart failure patients is a common finding, a fact that has only recently been fully appreciated. The aim of the present study was to examine the value of NT-proBNP to predict mortality in relation to established risk factors among consecutively hospitalised heart failure patients and secondly to characterise patients in relation to preserved and reduced systolic function. Material: At the time of admission 2230 consecutively hospitalised patients had their cardiac status evaluated through determinations of NT-proBNP, echocardiography, clinical examination and medical history. Follow-up was performed 1 year later in all patients. Results: 161 patients fulfilled strict diagnostic criteria for heart failure (HF). In this subgroup of patients 1-year mortality was approximately 30% and significantly higher as compared to the remaining non-heart failure population (approx. 16%). Using univariate analysis left ventricular ejection fraction (LVEF), New York Heart Association classification (NYHA) and plasma levels of NT-proBNP all predicted mortality independently. However, regardless of systolic function, age and NYHA class, risk-stratification was provided by measurements of NT-proBNP. Having measured plasma levels of NT-proBNP, LVEF did not provide any additional prognostic information on mortality among heart failure patients (multivariate analysis).
Conclusion: The results show that independent of LVEF, measurements of NT-proBNP add additional prognostic information. It is concluded that NT-proBNP is a strong predictor of 1-year mortality in consecutively hospitalised patients with heart failure with preserved as well as reduced systolic function.

N-terminal pro-B-type natriuretic peptide and the prediction of primary cardiovascular events: results from 15-year follow-up of WOSCOPS

Paul Welsh, Orla Doolin, Peter Willeit, Chris Packard, Peter Macfarlane, et al.
Eur Heart Journal 2014. http://eurheartj.oxfordjournals.org/

Aims: To test whether N-terminal pro-B-type natriuretic peptide (NT-proBNP) was independently associated with, and improved the prediction of, cardiovascular disease (CVD) in a primary prevention cohort.
Methods and results:  In the West of Scotland Coronary Prevention Study (WOSCOPS), a cohort of middle-aged men with hypercholesterolemia at a moderate risk of CVD, we related the baseline NT-proBNP (geometric mean 28 pg/mL) in 4801 men to the risk of CVD over 15 years during which 1690 experienced CVD events. Taking into account the competing risk of non-CVD death, NT-proBNP was associated with an increased risk of all CVD [HR: 1.17 (95% CI: 1.11–1.23) per standard deviation increase in log NT-proBNP] after adjustment for classical and clinical cardiovascular risk factors plus C-reactive protein. N-terminal pro-B-type natriuretic peptide was more strongly related to the risk of fatal [HR: 1.34 (95% CI: 1.19–1.52)] than non-fatal CVD [HR: 1.17 (95% CI: 1.10–1.24)] (P ¼ 0.022). The addition of NT-proBNP to traditional risk factors improved the C-index (+0.013; P , 0.001). The continuous net reclassification index improved with the addition of NT-proBNP by 19.8% (95% CI: 13.6–25.9%) compared with 9.8% (95% CI: 4.2–15.6%) with the addition of C-reactive protein. N-terminal pro-B-type natriuretic peptide correctly reclassified 14.7% of events, whereas C-reactive protein correctly reclassified 3.4% of events. Results were similar in the 4128 men without evidence of angina, nitrate prescription, minor ECG abnormalities, or prior cerebrovascular disease.
Conclusion: N-terminal pro-B-type natriuretic peptide predicts CVD events in men without clinical evidence of CHD, angina, or history of stroke, and appears related more strongly to the risk for fatal events. N-terminal pro-B-type natriuretic peptide also provides moderate risk discrimination, in excess of that provided by the measurement of C-reactive protein.

Effect of B-type natriuretic peptide-guided treatment of chronic heart failure on total mortality and hospitalization: an individual patient meta-analysis

Richard W. Troughton, Christopher M. Frampton, Hans-Peter Brunner-La Rocca,
Matthias Pfisterer, Luc W.M. Eurlings, Hans Erntell, Hans Persson, et al.
Eur Heart J 2014; 35: 1559–1567 http://dx.doi.org:/10.1093/eurheartj/ehu090

Aims Natriuretic peptide-guided (NP-guided) treatment of heart failure has been tested against standard clinically guided care in multiple studies, but findings have been limited by study size. We sought to perform an individual patient data metaanalysis to evaluate the effect of NP-guided treatment of heart failure on all-cause mortality.
Methods and results
Eligible randomized clinical trials were identified from searches of Medline andEMBASEdatabases and the Cochrane Clinical
Trials Register. The primary pre-specified outcome, all-cause mortality was tested using a Cox proportional hazards regression model that included study of origin, age (< 75 or ≥75 years), and left ventricular ejection fraction (LVEF, ≤45 or .45%) as covariates. Secondary endpoints included heart failure or cardiovascular hospitalization. Of 11 eligible studies, 9 provided individual patient data and 2 aggregate data. For the primary endpoint individual data from 2000 patients were included, 994 randomized to clinically guided care and 1006 to NP-guided care. All-cause mortality was significantly reduced by NP-guided treatment [hazard ratio = 0.62 (0.45–0.86);
P = 0.004] with no heterogeneity between studies or interaction with LVEF. The survival benefit from NP-guided therapy was seen in younger ( <75 years) patients [0.62 (0.45–0.85); P = 0.004] but not older (≥75 years) patients [0.98 (0.75–1.27); P = 0.96]. Hospitalization due to heart failure [0.80 (0.67–0.94); P = 0.009] or cardiovascular disease [0.82 (0.67–0.99); P = 0.048]was significantly lower in NP-guided patients with no heterogeneity between studies and no interaction with age or LVEF.
Conclusion: Natriuretic peptide-guided treatment of heart failure reduces all-cause mortality in patients aged < 75 years and overall reduces heart failure and cardiovascular hospitalization.

Diagnostic and prognostic evaluation of left ventricular systolic heart failure by plasma N-terminal pro-brain natriuretic peptide concentrations in a large sample of the general population

B A Groenning, I Raymond, P R Hildebrandt, J C Nilsson, M Baumann, F Pedersen
Heart 2004;90:297–303. http://dx.doi.org:/10.1136/hrt.2003.026021

Objective: To evaluate N-terminal pro-brain natriuretic peptide (NT-proBNP) as a diagnostic and prognostic marker for systolic heart failure in the general population.
Design: Study participants, randomly selected to be representative of the background population, filled in a heart failure questionnaire and underwent pulse and blood pressure measurements, electrocardiography, echocardiography, and blood sampling and were followed up for a median (range) period of 805 (6021171) days.
Setting: Participants were recruited from four randomly selected general practitioners and were examined in a Copenhagen university hospital.
Patients: 382 women and 290 men in four age groups (50259 (n = 174); 60269 (n = 204); 70279 (n = 174); > 80 years (n = 120)).
Main outcome measures: Value of NT-proBNP in evaluating patients with symptoms of heart failure and impaired left ventricular (LV) systolic function; prognostic value of NT-proBNP for mortality and hospital admissions.
Results: In 38 (5.6%) participants LV ejection fraction (LVEF) was (40%. NT-proBNP identified patients with symptoms of heart failure and LVEF (40% with a sensitivity of 0.92, a specificity of 0.86, positive and negative predictive values of 0.11 and 1.00, and area under the curve of 0.94. NT-proBNP was the strongest independent predictor of mortality (hazard ratio (HR) = 5.70, p = 0.0001), hospital admissions for heart failure (HR = 13.83, p = 0.0001), and other cardiac admissions (HR = 3.69, p = 0.0001). Mortality (26 v 6, p = 0.0003), heart failure admissions (18 v 2, p = 0.0002), and admissions for other cardiac causes (44 v 13, p = 0.0001) were significantly higher in patients with NTproBNP above the study median (32.5 pmol/l). Conclusions: Measurement of NT-proBNP may be useful as a screening tool for systolic heart failure in the general population.

Copeptin—Marker of Acute Myocardial Infarction

Martin Möckel & Julia Searle
Curr Atheroscler Rep 2014; 16:421 http://dx.doi.org:/10.1007/s11883-014-0421-5

The concentration of copeptin, the C-terminal part of pro-arginine vasopressin, has been shown to increase early after acute and severe events. Owing to complementary pathophysiology and kinetics, the unspecific marker copeptin, in combination with highly cardio-specific troponin, has been evaluated as an early-rule-out strategy for acute myocardial infarction in patients presenting with signs and symptoms of acute coronary syndrome. Overall, most studies have reported a negative predictive value between 97 and 100 % for the diagnosis of acute myocardial infarction in low- to intermediate-risk patients with suspected acute coronary syndrome. Additionally, a recent multicenter, randomized process study, where patients who tested negative for copeptin and troponin were discharged from the emergency department, showed that the safety of the new process was comparable to that of the current standard process. Further interventional trials and data from registries are needed to ensure the effectiveness and patient benefit of the new strategy.

The role of copeptin as a diagnostic and prognostic biomarker for risk stratification in the emergency department

Christian H Nickel1, Roland Bingisser and Nils G Morgenthaler
BMC Medicine 2012, 10:7 http://www.biomedcentral.com/1741-7015/10/7

The hypothalamic-pituitary-adrenal axis is activated in response to stress. One of the activated hypothalamic hormones is arginine vasopressin, a hormone involved in hemodynamics and osmoregulation. Copeptin, the C-terminal part of the arginine vasopressin precursor peptide, is a sensitive and stable surrogate marker for arginine vasopressin release. Measurement of copeptin levels has been shown to be useful in a variety of clinical scenarios, particularly as a prognostic marker in patients with acute diseases such as lower respiratory tract infection, heart disease and stroke. The measurement of copeptin levels may provide crucial information for risk stratification in a variety of clinical situations. As such, the emergency department appears to be the ideal setting for its potential use. This review summarizes the recent progress towards determining the prognostic and diagnostic value of copeptin in the emergency department.

Variability of the Transferrin Receptor 2 Gene in AMD

Daniel Wysokinski, Janusz Blasiak, Mariola Dorecka, Marta Kowalska, et al.
Disease Markers 2014, Article ID 507356, 8 pages http://dx.doi.org/10.1155/2014/507356

Oxidative stress is a major factor in the pathogenesis of age-related macular degeneration (AMD). Iron may catalyze the Fenton reaction resulting in overproduction of reactive oxygen species. Transferrin receptor 2 plays a critical role in iron homeostasis and variability in its gene may influence oxidative stress and AMD occurrence. To verify this hypothesis we assessed the association between  polymorphisms of the TFR2 gene and AMD. A total of 493AMDpatients and 171matched controls were genotyped for the two polymorphisms of the TFR2 gene: c.1892C>T (rs2075674) and c.−258+123T>C (rs4434553). We also assessed the modulation of some AMD risk factors by these polymorphisms.The CC and TT genotypes of the c.1892C>T were associated with AMD occurrence but the latter only in obese patients. The other polymorphism was not associated with AMD occurrence, but the CC genotype was correlated with an increasing AMD frequency in subjects with BMI < 26. The TT genotype and the T allele of this polymorphism decreased AMD occurrence in subjects above 72 years, whereas the TC genotype and the C allele increased occurrence of AMD in this group.The c.1892C>T and c.−258+123T>C polymorphisms of the TRF2 gene may be associated with AMD occurrence, either directly or by modulation of risk factors.

Urinary N-Acetyl-beta-D-glucosaminidase as an Early Marker for Acute Kidney Injury in Full-Term Newborns with Neonatal Hyperbilirubinemia

Bangning Cheng, Y Jin, G Liu, Z Chen, H Dai, and M Liu
Disease Markers 2014, Article ID 315843, 6 pages http://dx.doi.org/10.1155/2014/315843

Purpose. To investigate renal function estimated by markers in full-term newborns with hyperbilirubinemia.
Methods. A total of 332 full-term newborns with hyperbilirubinemia and 60 healthy full-term newborns were enrolled. Total serum bilirubin, serum creatinine (Cr), serum blood urea nitrogen (BUN), serum cystatin C (Cys-C), urinary beta-2-microglobulin (𝛽2MG) index, and urinary N-acetyl-beta-D-glucosaminidase (NAG) index were measured before and after treatment. All newborns were divided into three groups according to total serum bilirubin levels: group 1 (221-256), group 2 (256-342), and group 3 (>342). Results. The control group and group 1 did not differ significantly in regard to serum Cr, serum BUN, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index. Urinary NAG index in group 2 was significantly higher than that in control group (𝑃 < 0.001). Between control group and group 3, serum Cys-C, urinary 𝛽2MG index, and urinary NAG index differed significantly. The significant positive correlation between total serum bilirubin and urinary NAG index was found in newborns when total serum bilirubin level was more than 272 𝜇mol/L.
Conclusions. High unconjugated bilirubin could result in acute kidney injury in full-term newborns. Urinary NAG might be the suitable marker for predicting acute kidney injury in full-term newborns with hyperbilirubinemia.

Urinary C-peptide creatinine ratio detects absolute insulin deficiency in Type 2 diabetes.

S V Hope, A G Jones, E Goodchild, M Shepherd, R E J Besser, B Shields, T McDonald, B A Knight, A Hattersley

Department of Geriatrics, Royal Devon and Exeter NHS Foundation Trust; NIHR Exeter Clinical Research Facility, University of Exeter.

Diabetic Medicine (impact factor: 2.9). 05/2013; http://dx.doi.org:/10.1111/dme.12222

Source: PubMed

ABSTRACT AIMS: To determine the prevalence and clinical characteristics of absolute insulin deficiency in long-standing Type 2 diabetes, using a strategy based on home urinary C-peptide creatinine ratio measurement.
METHODS: We assessed the urinary C-peptide creatinine ratios, from urine samples taken at home 2 h after the largest meal of the day, in 191 insulin-treated subjects with Type 2 diabetes (diagnosis age ≥45 years, no insulin in the first year). If the initial urinary C-peptide creatinine ratio was ≤0.2 nmol/mmol (representing absolute insulin deficiency), the assessment was repeated. A standardized mixed-meal tolerance test with 90-min stimulated serum C-peptide measurement was performed in nine subjects with a urinary C-peptide creatinine ratio ≤ 0.2 nmol/mmol (and in nine controls with a urinary C-peptide creatinine ratio >0.2 nmol/mmol) to confirm absolute insulin deficiency.
RESULTS: A total of 2.7% of participants had absolute insulin deficiency confirmed by a mixed-meal tolerance test. They were identified initially using urinary C-peptide creatinine ratio: 11/191 subjects (5.8%) had two consistent urinary C-peptide creatinine ratios ≤ 0.2 nmol/mmol; 9/11 subjects completed a mixed-meal tolerance test and had a median stimulated serum C-peptide of 0.18nmol/l. Five out of nine subjects had stimulated serum C-peptide <0.2 nmol/l and 9/9 subjects with urinary C-peptide creatinine ratio >0.2 had endogenous insulin secretion confirmed by the mixed-meal tolerance test. Compared with subjects with a urinary C-peptide creatinine ratio >0.2 nmol/mmol, those with confirmed absolute insulin deficiency had a shorter time to insulin treatment (median 2.5 vs. 6 years, P=0.005) and lower BMI (25.1 vs. 29.1kg/m(2) , P=0.04). Two out of five patients were glutamic acid decarboxylase autoantibody-positive.
CONCLUSIONS: Absolute insulin deficiency may occur in long-standing Type 2 diabetes, and cannot be reliably predicted by clinical features or autoantibodies. Its recognition should help guide treatment, education and management. The urinary C-peptide creatinine ratio is a practical non-invasive method to aid detection of absolute insulin deficiency, with a urinary C-peptide creatinine ratio > 0.2 nmol/mmol being a reliable indicator of retained endogenous insulin secretion.

Unlocking Biomarkers’ Full Potential

David Daniels, Ph.D.     genengnews  Feb 1, 2013 (Vol. 33, No. 3)

http://www.genengnews.com/gen-articles/unlocking-biomarkers-full-potential/4700/

Biomarker research and development has evolved over the past years from looking for a single marker (e.g., PSA) linked to a disease state to looking for a panel of markers that can capture the heterogeneity inherent in both the disease and the impacted patient population.

That is one of the key messages to be delivered at GTC’s “Biomarkers Summit” next month. Across the board, resources are being focused on the delivery of more precise, quantifiable biomarkers with predictive value in therapeutic decisions and for the prognosis of illness.

“Our focus on biomarker development is the recognition that the new products need to provide cost savings for the already strapped healthcare systems rather than just be cost effective,” shares Paul Billings, M.D., Ph.D., CMO at Life Technologies.

“We have built a new medical sciences group to address the needs of the multiple delivery systems in the world—from the sophisticated medical clinics in the developed world to the nurse-run shanty clinic in the third world. Providing tools for equitable access to quality diagnosis, on assay platforms that can provide care for all patients, is our goal.”

Life Tech’s medical sciences division has been built by acquisition of Pinpoint Genomics, Navigenics, and Compendia, and collaborations with partners such as Ingenuity Systems and CollabRx. The division is focused on taking the tools that have been used in the life science laboratories and providing molecular diagnostic data to the clinic. The intent is to deliver data in a valuable format that can be used by the molecular pathologist or the treating physician.

The division is developing the Pervenio™ Lung RS assay, a 14-gene expression profile that serves as a risk stratifier that uses a weighted algorithm for the expressed biomarkers within the tumor biopsy, a first-of-its-kind prognostic test for lung cancer, the firm reports.

Initially, tests will be offered as a service through Life Tech’s CLIA laboratory. Then, from the performance lessons learned, Life Tech’s will develop a simpler assay platform, with FDA approval, that can be dispersed globally without reduction of the essential content in the biomarker panel. The focus is on the workflow—screening for known mutations using established easy-to-use assay platforms, like RT-PCR. Should the screen not produce useful results, clinicians can search for new mutations via discovery platforms like next-gen sequencing (NGS).

http://www.genengnews.com/Media/images/Article/thumb_Sequenom_LungCartaPanel1722631391.jpg

Sequenom’s LungCarta panel of 214 somatic mutations in 26 tumor suppressors and oncogenes covers highly mutated pathways in lung adenocarcinomas.

At Sequenom, the company provides both the tools (DNA mass spectrometry and reagents) for confirmatory biomarker development as well as serving on the front lines as a diagnostic service provider (CLIA lab). The beauty of DNA mass spec is that it can process multiplexed PCR samples (10–60 loci) in a method that is quantitative when used for profiling tumor biopsies that are either archival or fresh tissue.

Given a tumor sample with multiple somatic mutations, the instrument enables the determination of the homogeneity of the cells, in which case the mutations will have the same allele frequency. Accuracy, as measured by coefficient of variance, is less than 2%. Despite this level of sensitivity, the mass spec can only be used as a confirmatory tool looking for known mutations. Discovery is best done using DNA sequencing. DNA mass spec can also be used to study methylation in tumor samples.

“In the not-too-distant future, we will be looking for mutations in plasma samples rather than biopsies,” predicts Charles Cantor, Ph.D., CSO at Sequenom.

“The key is to look noninvasively for mutations within plasma samples such that we can potentially catch the disease state earlier, rather than after tumor formation. Regardless of the tumor type, this approach will enable us to monitor therapeutic response and metastatic potential noninvasively. DNA mass spec is an ultrasensitive detection product that can detect somatic mutations at levels of 1 per 1,000. This level of sensitivity is critical for the future of plasma screening. NGS technology is not that sensitive.”

Sequenom’s CLIA lab is using automated DNA mass spec to provide three different test protocols: (1) carrier screening for cystic fibrosis looking at more than 100 different mutations, (2) adult macular degeneration progression using an SNP test with 13 loci, and (3) a noninvasive test for Rh compatibility between a mother and her unborn fetus.

http://www.genengnews.com/Media/images/Article/thumb_Illumina_HiSeq_Scientist2141841107.jpg

Scientists are using Illumina’s HiSeq system to discover molecular biomarkers that may provide opportunities for early detection of a range of diseases.

Sequenom has also set up an NGS facility within a CLIA lab in San Diego using Illumina’s HiSEQ platform. The NGS platform has been set up for noninvasive aneuploidy detection of maternal plasma (10 cc sample) looking at chromosomes 13, 18, and 21. The lab says it has analyzed more than 40,000 samples this year and is planning to increase that volume up to 100,000 samples per year. Most of these samples come from the U.S., but given the development of a new blood collection tube that allows for 72-hour ambient shipping, the lab is looking to increase the number of samples from outside the U.S.

Drug Development

During drug development, biomarkers function as pharmacodynamic markers to help assess the mechanism of action of a drug candidate, to define the downstream biological pathway, and to determine whether the drug is engaging the target with the anticipated biological effect. Later, biomarkers help determine whether a drug is effective using the tested regime (route of delivery, dosage level, and length of exposure time).

Following early development, the second stage is to use biomarkers to help segment patients for clinical trials. Part of the consideration here is how heterogeneous the disease is; are there homogeneous subsets of patients that will respond differentially to the drug based on different mechanisms of the disease?

“Biomarker research is focused on on- target effects,” says Nick Dracopoli, Ph.D., vp, head of oncology biomarkers at Janssen Research and Development, a J&J company.

“We look at indications and at patients with those indications that are most likely to respond to the drug candidates we’re developing. For oncology biomarkers, germ-line effects are weaker indicators than somatic changes in the tumor. As a consequence, SNP-based, genome-wide association studies are not very useful. It is better to focus on molecular changes within the tumor and define gene expression profiles and epigenetic modifications that correlate with the tumor phenotype. We are increasingly tracking patient immune response, particularly as more immuno-oncology products are moving into the drug development pipeline.”

The number of biomarkers being developed varies from project to project. But it is very clear that to be successful in the clinic, the biomarkers and the assays need to be of low complexity. Of the 10 to 12 companion diagnostics that have been approved by the FDA to date, all measure the status of the drug target (on-target markers). For example, EGFR measures the level of receptor expression; Braf and Kras markers measure the presence of the mutation and translocation in the ALK gene measures gene knockout.

It is important to realize that molecular profiles for first-in-class drugs are not optimal because they are based on only a few patients. Consequently they have weak predictive value overall.

“Aside from that rule of thumb, if you have a greater than 50% response rate for your drug, it is unlikely that you need a biomarker to predict response. Biomarker utility is best for drugs that would have a difficult road to approval, where it is critical to enrich for the subpopulation of responders. For example, Pfizer’s crizontinib was approved for non-small-cell lung patients but is only effective for 5% of all patients. If Pfizer was unable to demonstrate the relationship between activation of the ALK gene and disease, this inhibitor would not have been approved,” says Dr. Dracopoli.

“Drugs that are more broadly active can come to market without a companion diagnostic test. There is always a balance between the predictive values of the biomarker test and the response rates to treatment. That is, we should not treat if the chance of response is only 3–5%, rather than if it were 50% where the patient would want to take the chance if the drug were safe.”

An important take-home message is that mutations are not unique to an indication. So if you find a driver mutation in indications for which the drug has not been approved, you could discover new indications for the drug.

“At the end of the day, this is what cancer is—heterogeneous,” says Dr. Dracopoli. “We’d all love to treat one cancer with one drug and at one dose, but the story is more complex. The future of oncology is around understanding the molecular heterogeneity or underlying molecular pathology of the disease and the diversity of it, and then treat each patient accordingly.”

Clinical Considerations

“Given the complexity of biology,” says Achim Plum, Ph.D., principal consultant, Siemens, “whether is it cancer, metabolic disease, or any other disease state, we have been forced to move away from the idea that a single biomarker can capture the entire ‘story’ or mechanistic view of any disease. Hence newly developed biomarkers will be made up of a panel of markers that serve as a profile. In addition, with the sheer volume of DNA and protein analytics data, the clinic will need to employ software tools and algorithms to help the decision making.”

The task of getting broad profiling technologies that are analytical into a clinical setting and making them routine is difficult but not insurmontable. This will take a collaborative effort, something that Siemens among others are looking to develop. The key is to avoid technology hype and to establish good reliable software to process the data for decision making. “Data is not knowledge, and knowledge is not automatically decision making.”

As an academic, Daniel Chan, Ph.D., has a view of the whole value chain for biomarkers from discovery to development to use in the clinic. Dr. Chan holds the titles of professor in pathology, oncology, radiology, and urology, and is the director of the clinical chemistry division lab at Johns Hopkins Hospital.

Given his perspective from discovery to clinical use, Dr. Chan indicated that from the clinical point of view, “we need more markers.” He oversees the discovery of new biomarkers in his research lab, their validation in his translational research lab, and finally their utility in practice in his clinical chemistry lab. He is a strong advocate for collaboration of biomarker development from discovery to verification and validation to incorporation within the clinical practice.

Beyond the use of biomarkers for patient stratification and correlation between marker and therapeutic choice, as is the focus of the biopharma industry, for the clinic the use of biomarkers is for prevention and early detection. The earlier the detection, the better the outcome. That is, provide the “cure” before you need to initiate treatment.

To be successful in the future of biomarkers, we need to look beyond the biopharma focus and expand the horizon for early detection and monitor therapy later, says Dr. Chan. He describes a roadmap of developing bridges (to bridge the knowledge gaps), gates (decision gates for go/no go decisions as to whether a development path is viable), and partnerships (to collaborate with different points of view) for efficient new biomarker development.

According to Dr. Chan, we must define the intended use of the biomarker, which identifies the specific application and sets up the clinical study and study population to meet the clinical needs. We need to define specific assays to monitor biomarkers that will work within a clinical setting, not a research lab setting that uses disease models (tissue culture cells or small animals) and not real patient samples.

“The days when single markers are sufficient (PSA for prostate cancer or troponin for cardiovascular disease) are behind us. We need to develop a panel of markers or a profile pattern to address patient population heterogeneity and disease complexity that will guide our decision-making process,” remarks Dr. Chan. “Molecular biomarkers are giving way to protein biomarkers,” he adds.

Prevention and early detection will require the use of whole-body scans, so the sampling technology and analytical tools to be developed are critical to realize this goal. Assay ease of use, automation, and analytical performance that is suitable for the clinical lab are fundamental.

“An important future goal for biomarkers,” says Dr. Billings, “is to sample circulating tumor cells or circulating DNA in blood or plasma samples as a noninvasive measure of patient status. A decline in tumor biomarkers during chemotherapy, for example, could reflect the efficacy of the therapy. In contrast, an increase in tumor biomarkers, in a patient who had previously undergone surgery and therapy, might indicate disease recurrence, and is likely to do so before a tumor mass is detectable by imaging methods.”

STAT4 Gene Polymorphisms Are Associated with Susceptibility and ANA Status in Primary Biliary Cirrhosis

Satoru Joshita, T Umemura, M Nakamura, Y Katsuyama, S Shibata, et al.
Disease Markers  2014, Article ID 727393, 8 pages http://dx.doi.org/10.1155/2014/727393

Recent genome-wide association studies suggest that genetic factors contribute to primary biliary cirrhosis (PBC) susceptibility. Although several reports have demonstrated that the interleukin (IL) 12 signaling pathway is involved in PBC pathogenesis, its precise genetic factors have not been fully clarified. Here, we performed an association analysis between IL12A, IL12RB, and signal transducer and activator of transcription 4 (STAT4) genetic variations and susceptibility to PBC. Single nucleotide polymorphisms (SNPs) were genotyped in 395 PBC patients and 458 healthy subjects of Japanese ethnicity and evaluated for associations with PBC susceptibility, anti-nuclear antibody (ANA) status, and anti-mitochondrial antibody (AMA) status. We detected significant associations with PBC susceptibility for several STAT4 SNPs (rs10168266; p = 9.4 × 10−3, rs11889341; p = 1.2 × 10−3, rs7574865; p = 4.0 × 10−4, rs8179673; p = 2.0 × 10−4, and rs10181656; p = 4.2 × 10−5). Three risk alleles (rs7574865; p = 0.040, rs8179673; p = 0.032, and rs10181656; p = 0.031) were associated with ANA status, but not with AMA positivity. Our findings confirm that STAT4 is involved in PBC susceptibility and may play a role in ANA status in the Japanese population.

Serum Omentin-1 as a Disease Activity Marker for Crohn’s Disease

Yan Lu, Li Zhou, L Liu, Yan Feng, Li Lu, X Ren, X Dong, & W Sang
Disease Markers  2014, Article ID 162517, 5 pages   http://dx.doi.org/10.1155/2014/162517

Background and Aim. It remains challenging to determine the inflammatory activity in Crohn’s disease (CD) for lack of specific laboratory markers. Recent studies suggest that serum omentin-1 is associated with inflammatory response. We aimed to assess the potential of serum omentin-1 as a marker of disease activity in CD patients.
Methods. Serum omentin-1 concentrations were determined by enzyme-linked immunosorbent assay (ELISA) in patients with CD (n = 240), functional gastrointestinal disorders (FGDs, n = 120), and healthy controls (HC, n = 60) and evaluated for correlation with disease activity. Expression of omentin-1 in colonic tissues from patients with CD was also analyzed by real-time PCR and Western blotting. Serum omentin-1 levels as an activity index were evaluated using a receiver operating characteristic (ROC) curve.
Results. Serum omentin-1 concentrations were significantly decreased in active CD patients compared with patients in remission, FGDs, and HC (all p < 0.001). Expression of omentin-1 was decreased at mRNA and protein levels in inflamed colonic tissues in active CD than that in noninflamed colonic tissues. Serum omentin-1 levels were negatively correlated with disease activity in CD, better than C-reactive protein (CRP).
Conclusion. Our results indicate that serum and colonic omentin-1 expressions are decreased in active CD patients. The correlation of serum omentin-1 with disease activity in CD is superior to that of CRP. Serum omentin-1 is a potential marker for CD disease activity.
Serum Levels of Resistin, Adiponectin, and Apelin in Gastroesophageal Cancer Patients

Dorota Diakowska, K Markocka-Mdczka, P Szelachowski, and K Grabowski
Disease Markers 2014, Article ID 619649, 8 pages   http://dx.doi.org/10.1155/2014/619649

The aim of the study was the investigation of relationship between cachexia syndrome and serum resistin, adiponectin, and apelin in patients with gastroesophageal cancer (GEC).
Material and Methods. Adipocytokines concentrations were measured in sera of 85 GEC patients and 60 healthy controls. They were also evaluated in tumor tissue and appropriate normal mucosa of 38 operated cancer patients.
Results. Resistin and apelin concentrations were significantly higher in GEC patients than in the controls. The highest resistin levels were found in cachectic patients and in patients with distant metastasis. Serum adiponectin significantly decreased in GEC patients with regional and distant metastasis. Serum apelin was significantly higher in cachectic patients than in the controls. Apelin was positively correlated with hsCRP level. Resistin and apelin levels increased significantly in tumor tissues. Weak positive correlations between adipocytokines levels in serum and in tumor tissue were observed.
Conclusions. Resistin is associated with cachexia and metastasis processes of GEC. Reduction of serum adiponectin reflects adipose tissue wasting in relation to GEC progression. Correlation of apelin with hsCRP can reflect a presumable role of apelin in systemic inflammatory response in esophageal and gastric cancer.

Serum Level of HER-2 Extracellular Domain in Iranian Patients with Breast Cancer: A Follow-up Study

Mehrnoosh Doroudchi, Abdolrasoul Talei, Helmout Modjtahedi, et al.
IJI 2005; 2(4): 191-200

Background: A soluble form of HER-2/neu extracellular domain (sHER-2) is reported to be released in the sera of metastatic breast cancer patients.
Objective: To measure the level of sHER-2 in sera of 115 breast cancer patients. Methods: Serial samples of 27 patients with metastasis, 18 non-metastatic patients, 15 patients in stage 0/I and 14 patients with accompanying benign breast disease were also included in this study.
Results: No significant difference was observed between sHER-2 level in the pre-operative sera of breast cancer patients and that of healthy individuals. Only 8 out of 27 patients whom later developed metastasis showed elevated levels of sHER-2 in their first serum sample. However, a trend of increase in the level of sHER-2 was observed in 14 (51.8%) of 27 metastatic sera before clinical diagnosis of the metastasis. A significant association between sHER-2 positive status and vascular invasion of the tumor was observed (P = 0.02). In addition, significant correlation of sHER-2 level with CEA (highest r = 0.74) and CA 15.3 (highest r = 0.74) tumor marker levels in the serial sera were observed. The mean time from sHER-2 positivity to tumor metastasis was calculated to be 98 days (range = 29-174).  Conclusion: Our results indicate that a relatively high percentage of Iranian patients with breast cancer show an elevated level of sHER-2 in their sera before clinical diagnosis of the tumor metastasis. Therefore, measuring the level of this oncoprotein, not only helps physicians in monitoring the patients during HERCEPTIN therapy, but also can be helpful in choosing more aggressive treatments at the early satges of tumor metastasis.
B-type natriuretic peptide is a biomarker for pulmonary hypertension in preterm infants with bronchopulmonary dysplasia

Alain Cuna, Jegen Kandasamy, Naomi Fineberg, Brian Sims
Research and Reports in Neonatology 2013:3 33–36
http://dx.doi.org/10.2147/RRN.S42236

Background: B-type natriuretic peptide (BNP) is a cardiac biomarker useful in screening for pulmonary hypertension (PH) in adults. It is possible that BNP may also be useful in detecting PH among preterm infants with bronchopulmonary dysplasia (BPD).
Objective: To determine the utility of BNP for identification of PH among preterm infants with BPD.
Methods: We retrospectively identified preterm infants with BPD who underwent screening echocardiography for suspected PH and had serum BNP levels measured within 10 days before or after echocardiography. Eligible infants were classified based on echocardiographic diagnosis of either PH or no PH. Median and interquartile ranges (IQR) of BNP values were compared, and area under the curve (AUC) of receiver operator characteristic (ROC) analysis was used to determine the optimum threshold value for detection of PH.
Results: Twenty-five preterm infants with BPD (mean gestational age 26.5 ± 1.7 weeks, mean birth weight 747 ± 248 g) were identified. The median difference in days between echocardiography and BNP measurement was 1 day (IQR 0–3, range 0–10 days). Based on echocardiography, 16 were diagnosed with PH and nine without PH. No significant difference in terms of gestational age, birth weight, sex, race, or respiratory support was found between the two groups. Median (IQR) BNP values of those with PH were higher than those without PH (413 [212–1178] pg/mL versus 55 [21–84] pg/mL, P , 0.001). AUC of ROC analysis showed that a BNP value of 117 pg/mL had 93.8% sensitivity and 100% specificity for detecting PH.
Conclusion: BNP estimation may be useful for screening of PH in infants with BPD.

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