Stratified Medicine in Cancer: The Role of HistopathologistDr. Shubhi Saxena
This document discusses stratified medicine approaches for cancer treatment. It describes how cellular pathologists classify gene mutations in cancer, including whether they are germline or somatic, synonymous or non-synonymous, activating or inactivating. Certain mutations can predict treatment response or resistance. Key driver mutations are discussed for lung cancer, including EGFR mutations and ALK translocations. EGFR mutant cancers may respond to EGFR tyrosine kinase inhibitors, while ALK rearrangements are targeted by crizotinib. Histopathology plays an important role in mutation detection and molecular testing to guide targeted therapies.
The document describes the development of over 200 TaqMan assays to detect mutations in the CFTR gene that cause cystic fibrosis. Key mutations were selected from databases and assays were designed to target single nucleotide variants, insertions/deletions, and large deletions. Assays were tested on various sample types and plate formats, with some assays requiring redesign to robustly detect mutations. The developed assays and analysis workflow provide a high-throughput method for CFTR mutation detection to help research efforts.
This study compared the effects of pre-storage vs post-storage pathogen reduction treatment on plasma constituents in fresh frozen plasma units. Biochemical, immune, and hemostatic parameters were analyzed in both groups. Results showed no clinically significant differences between the two groups in final plasma protein levels or activity of coagulation factors. Post-storage treated plasma remained high quality and could be effectively and safely inactivated prior to clinical use, providing an option for inactivating previously frozen units.
This document discusses cancer drug targets and profiling key genes related to cancer treatment. It begins with an overview of actively investigated cancer drug target genes across various categories like growth factors and receptors, protein kinases, apoptosis genes, and more. It then discusses profiling gene expressions using RT2 Profiler PCR Arrays which allow analyzing 84 pathway genes along with controls. The document also discusses detecting gene mutations using Cancer Mutation PCR Arrays designed around clinically relevant mutations. Finally, it discusses analyzing histone modifications of drug target genes using epigenetic approaches, as histone modifications influence gene transcription and cell response to drug regimens.
The EpiTect Methyl II PCR Array System provides a simple and reliable method to screen DNA methylation levels of multiple genes simultaneously without bisulfite conversion. It uses selective digestion of genomic DNA with methylation-sensitive and methylation-dependent restriction enzymes, followed by quantitative PCR to determine the relative amounts of methylated and unmethylated DNA for each targeted region. As little as 1 microgram of DNA can profile methylation status of up to 94 genes. The system yields comparable data to bisulfite sequencing and beadchip assays but does not require bisulfite conversion. It is a useful tool for high-throughput screening of DNA methylation biomarkers.
1. The ChampionChIP system allows for epigenetic analysis of histone modifications and transcription factor binding in a single day. It uses validated antibodies, primers, and qPCR arrays to analyze multiple genomic regions simultaneously.
2. The system was used to analyze dynamic bivalent histone modification patterns of pluripotency genes during mouse embryonic carcinoma cell differentiation induced by retinoic acid. Distinct patterns were observed for genes and heterochromatic regions.
3. The system correctly identified histone modification distribution and was used to map modifications around the CDKN1A gene. It also analyzed p53 binding and correlated gene expression changes in response to drug treatment in cancer cell lines.
1. Receptor tyrosine kinases (RTKs) drive key cancer pathways and can be exploited as therapeutic targets, as shown by drugs like imatinib that inhibit mutated kinases in cancers.
2. RTK inhibitors have shown efficacy against cancers dependent on single kinases, but resistance often emerges through secondary mutations or bypass pathways.
3. Effective combination therapies are needed to overcome resistance, such as combining RTK inhibitors with other drugs that block downstream or bypass pathways.
Stratified Medicine in Cancer: The Role of HistopathologistDr. Shubhi Saxena
This document discusses stratified medicine approaches for cancer treatment. It describes how cellular pathologists classify gene mutations in cancer, including whether they are germline or somatic, synonymous or non-synonymous, activating or inactivating. Certain mutations can predict treatment response or resistance. Key driver mutations are discussed for lung cancer, including EGFR mutations and ALK translocations. EGFR mutant cancers may respond to EGFR tyrosine kinase inhibitors, while ALK rearrangements are targeted by crizotinib. Histopathology plays an important role in mutation detection and molecular testing to guide targeted therapies.
The document describes the development of over 200 TaqMan assays to detect mutations in the CFTR gene that cause cystic fibrosis. Key mutations were selected from databases and assays were designed to target single nucleotide variants, insertions/deletions, and large deletions. Assays were tested on various sample types and plate formats, with some assays requiring redesign to robustly detect mutations. The developed assays and analysis workflow provide a high-throughput method for CFTR mutation detection to help research efforts.
This study compared the effects of pre-storage vs post-storage pathogen reduction treatment on plasma constituents in fresh frozen plasma units. Biochemical, immune, and hemostatic parameters were analyzed in both groups. Results showed no clinically significant differences between the two groups in final plasma protein levels or activity of coagulation factors. Post-storage treated plasma remained high quality and could be effectively and safely inactivated prior to clinical use, providing an option for inactivating previously frozen units.
This document discusses cancer drug targets and profiling key genes related to cancer treatment. It begins with an overview of actively investigated cancer drug target genes across various categories like growth factors and receptors, protein kinases, apoptosis genes, and more. It then discusses profiling gene expressions using RT2 Profiler PCR Arrays which allow analyzing 84 pathway genes along with controls. The document also discusses detecting gene mutations using Cancer Mutation PCR Arrays designed around clinically relevant mutations. Finally, it discusses analyzing histone modifications of drug target genes using epigenetic approaches, as histone modifications influence gene transcription and cell response to drug regimens.
The EpiTect Methyl II PCR Array System provides a simple and reliable method to screen DNA methylation levels of multiple genes simultaneously without bisulfite conversion. It uses selective digestion of genomic DNA with methylation-sensitive and methylation-dependent restriction enzymes, followed by quantitative PCR to determine the relative amounts of methylated and unmethylated DNA for each targeted region. As little as 1 microgram of DNA can profile methylation status of up to 94 genes. The system yields comparable data to bisulfite sequencing and beadchip assays but does not require bisulfite conversion. It is a useful tool for high-throughput screening of DNA methylation biomarkers.
1. The ChampionChIP system allows for epigenetic analysis of histone modifications and transcription factor binding in a single day. It uses validated antibodies, primers, and qPCR arrays to analyze multiple genomic regions simultaneously.
2. The system was used to analyze dynamic bivalent histone modification patterns of pluripotency genes during mouse embryonic carcinoma cell differentiation induced by retinoic acid. Distinct patterns were observed for genes and heterochromatic regions.
3. The system correctly identified histone modification distribution and was used to map modifications around the CDKN1A gene. It also analyzed p53 binding and correlated gene expression changes in response to drug treatment in cancer cell lines.
1. Receptor tyrosine kinases (RTKs) drive key cancer pathways and can be exploited as therapeutic targets, as shown by drugs like imatinib that inhibit mutated kinases in cancers.
2. RTK inhibitors have shown efficacy against cancers dependent on single kinases, but resistance often emerges through secondary mutations or bypass pathways.
3. Effective combination therapies are needed to overcome resistance, such as combining RTK inhibitors with other drugs that block downstream or bypass pathways.
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
We identified mutations in eleven cell free
(cf) DNA samples by next generation
sequencing (NGS) using the Ion AmpliSeq™
Colon & Lung Cancer Research Panel and
the Ion PGM™ System. Since detection of
low frequency mutant alleles may not always
be called confidently in NGS, we verified
results by rare mutation analysis using
digital PCR on the QuantStudio™ 3D Digital
PCR System as an independent method.
We show that frequencies detected are
consistent for both methods for low
frequency mutant alleles at and below 1%.
Inhibition of the mtorc pathway in the antiphospholipidSaris Arango
The document discusses the mTOR pathway and its relationship to the antiphospholipid syndrome. It provides background on mTOR and its role in regulating cell growth. Studies have shown that in patients with the antiphospholipid syndrome, IgG antibodies activate the mTOR pathway through PI3K-AKT signaling in endothelial cells. Inhibition of the mTOR pathway may help prevent complications in these patients such as thrombosis and graft loss after kidney transplantation. The document reviews several studies that demonstrate activation of mTOR and how it contributes to conditions common in antiphospholipid syndrome.
This document discusses tyrosine kinase inhibitors (TKIs), a class of targeted cancer drugs. It begins by introducing protein kinases and their role in cell signaling. There are two main categories of protein kinases - those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. Tyrosine kinases function as on/off switches in many cellular functions by adding phosphate groups to tyrosine residues on proteins. The document then discusses the different types of tyrosine kinases and how they can become mutated and cause unregulated cell growth leading to cancer. It describes targeted therapy and TKIs as targeted drugs that block specific molecules needed for tumor growth. The final sections provide examples of approved TKIs
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
A tyrosine kinase is an enzyme that transfers a phosphate group from ATP to tyrosine residues on proteins. This phosphorylation regulates protein activity and signal transduction within cells. Tyrosine kinase inhibitors, like nilotinib, are drugs that bind to and inhibit tyrosine kinases. Nilotinib was approved to treat chronic myeloid leukemia and research found it was effective against drug-resistant forms of the disease. It works by binding the inactive form of the Abl kinase to prevent phosphorylation and cancer cell growth.
Strong reversal of the lung fibrosis disease signature by autotaxin inhibitor...Maté Ongenaert
Strong reversal of the lung fibrosis disease signature by autotaxin inhibitor GLPG1690 in a mouse model for IPF
Maté Ongenaert (Mechelen, Belgium), Maté Ongenaert, Sonia Dupont, Roland Blanqué, Reginald Brys, Ellen van der Aar, Bertrand Heckmann
ERS - European Respiratory Society International Congress 2016. Session: Therapeutic horizons: novel targets and pharmacological models
Background and objectives
GLPG1690 is a novel potent autotaxin (ATX) inhibitor shown to be efficacious in the mouse bleomycin (BLM) lung fibrosis model. Here, we analyze the impact of GLPG1690 on the gene expression signature in mouse fibrotic lung tissue.
Methods
Lung fibrosis was induced by intranasal administration of BLM. Animals were treated with GLPG1690 or vehicle.Whole superior right lung was used for RNA extraction. Full transcriptome analysis was performed using the Agilent SurePrint G3 mouse chip. Analysis was performed using empirical Bayes methods and linear models. Public human IPF expression data were re-analyzed.
Results
GLPG1690 strongly reduced lung fibrosis as shown by reduction of Ashcroft scores and collagen content. Microarray analysis of the lungs revealed that GLPG1690 strongly reversed the impact of gene expression caused by BLM (367 out of the 2375 probes). As GLPG1690 treatment affects 395 probes, this treatment effect is highly relevant in the model. Gene clusters affected by BLM treatment and reverted by GLPG1690 are related to extracellular matrix (such as Tnc and Spp1), collagen (Col3a1) and cytokines/chemokines (Cxcl12). Several of the affected genes are known to be involved in the development or progression of lung fibrosis in IPF patients.
Conclusions
These data provide further mechanistic understanding of the efficacy of ATX inhibition in a pre-clinical lung fibrosis model, highlighting a role for extracellular matrix and inflammation biology. These data strongly suggest that GLPG1690 may be beneficial in treating IPF patients and support its evaluation in a clinical study
This review article discusses mTOR inhibitors and their use in cancer and transplantation. It outlines the mTOR signaling pathway and how mTOR inhibitors like sirolimus, everolimus and temsirolimus work. It describes how mTOR inhibitors are used to prevent organ rejection after transplantation. It also discusses how dysregulation of the PI3K/Akt/mTOR pathway promotes cancer and how mTOR inhibitors have shown efficacy in renal cell carcinoma and mantle cell lymphoma. The article concludes that biomarkers are needed to identify cancers sensitive to mTOR inhibition and that combination targeted therapies may help overcome resistance.
A novel platform for in situ, multiomic, hyper-plexed analyses of systems bio...Rafael Casiano
The document describes a novel platform called MultiOmyx that enables simultaneous imaging of protein and nucleic acid biomarkers at a subcellular level in tissue samples. It allows for quantification of biomarkers in individual cells while preserving spatial architecture. Analysis of a colon cancer cohort revealed extensive immune cell heterogeneity between patients and unexpected patterns of signal transduction pathway activation at single cell levels. This highlights the unique capability of MultiOmyx to provide novel insights into biological mechanisms through multiomic, hyper-plexed tissue analysis.
This document discusses challenges and recommendations for miRNA profiling from blood samples. It outlines that circulating miRNA in blood can serve as noninvasive biomarkers but presents purification challenges due to nucleases and other interfering components. The document recommends using optimized kits to purify miRNA from whole blood, blood cell fractions, or serum/plasma. It also recommends using miScript PCR systems for reliable miRNA quantification and normalization from blood samples via reverse transcription and real-time RT-PCR.
Epidermal growth factor (EGF) is a protein that binds to EGF receptors on epithelial and epidermal cells and initiates the EGF signaling pathway. When EGF binds to EGF receptors, it causes them to dimerize and activate their tyrosine kinase activity, leading to phosphorylation and activation of downstream proteins in the MAPK pathway. Mutations that cause constitutive activation of this pathway can lead to uncontrolled cell growth and cancer. Potential cancer treatments discussed include RGD-based peptides that target the αvβ3 integrin receptor involved in angiogenesis, and a conjugate of low molecular weight heparin and suramin that may inhibit tumor growth by blocking vascular endothelial growth factor (VEGF).
This technical article describes three case studies using RT2 Profiler PCR Arrays to analyze gene expression changes in different research areas: toxicology, oncology, and immunology. In the toxicology study, liver cells were treated with three drugs known to cause liver toxicity and the PCR Array identified distinct gene expression patterns for each drug, suggesting different mechanisms of toxicity. In the oncology study, gene expression in breast tumor samples was compared to normal tissue and a common set of upregulated genes was discovered in two independent tumor samples. In the immunology study, stimulated immune cells showed good correlation between cytokine gene and protein expression levels. The article concludes the PCR Array System is a reliable and accurate tool for pathway-focused gene expression profiling across
study of EGFR protein expression and mutation premvarma064
This document discusses oral squamous cell carcinoma (OSCC), which represents 90% of oral cancers and is characterized by a poor prognosis and lower survival rate. It notes that 0.7 million new cases and 0.3 million deaths occur annually from OSCC globally. In India, approximately 30-40% of all cancer cases are oral cancers, which is much higher than in Western countries. Risk factors for oral cancer include smoking, tobacco use, HPV infection, oral submucous fibrosis, and certain other conditions. The document discusses using immunohistochemistry to examine EGFR expression levels in oral cancer tissue samples and using PCR and restriction enzyme digestion to analyze for EGFR mutations, which can indicate prognosis and treatment options.
This document describes a study that used protein microarrays to systematically measure interactions between SH2/PTB domains and sites of tyrosine phosphorylation on receptor tyrosine kinases (RTKs) and adaptor proteins. They found that adaptor proteins, like RTKs, have many high affinity interactions with other adaptor proteins, demonstrating a high degree of connectivity. Additionally, proteins known to drive cancer through aberrant signaling, including both RTKs and adaptor proteins, tend to have more interaction partners than non-oncogenic proteins. This suggests that connectivity within signaling networks may help identify new potential drug targets for cancer treatment.
1) GRK5 regulates prostate cancer cell migration and invasion in vitro and tumor growth and metastasis in vivo.
2) GRK5 phosphorylates the cytoskeletal protein moesin, regulating its subcellular distribution and localization to the cell periphery.
3) Phosphorylation of moesin at threonine 66 by GRK5 is important for cell spreading, and mutation of this site reduces cell spreading.
Cardiotoxicity is unfortunately a common side effect of many modern chemotherapeutic agents. The mechanisms that underlie these detrimental effects on heart muscle, however, remain unclear. The Drug Toxicity Signature Generation Center at ISMMS aims to address this unresolved issue by providing a bridge between molecular changes in cells and the prediction of pathophysiological effects. I will discuss ongoing work in which we use next-generation sequencing to quantify changes in gene expression that occur in cardiac myocytes after they are treated with potentially toxic chemotherapeutic agents. I will focus in particular on the computational pipeline we are developing that integrates sophisticated sequence alignment, statistical and network analysis, and dynamical mathematical models to develop novel predictions about the mechanisms underlying drug-induced cardiotoxicity.
Jaehee Shim is a Ph.D candidate in the Biophysics and Systems Pharmacology Program at Icahn School of Medicine at Mount Sinai (ISMMS). As a part of her Ph.D. studies, she is building dynamical prediction models based on analysis of gene expression data generated by the Drug Toxicity Signature Generation Center at ISMMS. She received her B.S in Biochemistry from the University of Michigan-Dearborn. Prior to starting her Ph.D, Jaehee worked at the ISMMS Genomics Core with a team of senior scientists and gained experience in improving and troubleshooting RNA sequencing protocols using Next Generation Sequencing Platforms.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Rare Mutation Analysis Using Digital PCR on QuantStudio™ 3D to Verify Ion Amp...Thermo Fisher Scientific
We identified mutations in eleven cell free
(cf) DNA samples by next generation
sequencing (NGS) using the Ion AmpliSeq™
Colon & Lung Cancer Research Panel and
the Ion PGM™ System. Since detection of
low frequency mutant alleles may not always
be called confidently in NGS, we verified
results by rare mutation analysis using
digital PCR on the QuantStudio™ 3D Digital
PCR System as an independent method.
We show that frequencies detected are
consistent for both methods for low
frequency mutant alleles at and below 1%.
Inhibition of the mtorc pathway in the antiphospholipidSaris Arango
The document discusses the mTOR pathway and its relationship to the antiphospholipid syndrome. It provides background on mTOR and its role in regulating cell growth. Studies have shown that in patients with the antiphospholipid syndrome, IgG antibodies activate the mTOR pathway through PI3K-AKT signaling in endothelial cells. Inhibition of the mTOR pathway may help prevent complications in these patients such as thrombosis and graft loss after kidney transplantation. The document reviews several studies that demonstrate activation of mTOR and how it contributes to conditions common in antiphospholipid syndrome.
This document discusses tyrosine kinase inhibitors (TKIs), a class of targeted cancer drugs. It begins by introducing protein kinases and their role in cell signaling. There are two main categories of protein kinases - those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. Tyrosine kinases function as on/off switches in many cellular functions by adding phosphate groups to tyrosine residues on proteins. The document then discusses the different types of tyrosine kinases and how they can become mutated and cause unregulated cell growth leading to cancer. It describes targeted therapy and TKIs as targeted drugs that block specific molecules needed for tumor growth. The final sections provide examples of approved TKIs
The document summarizes qBiomarker Somatic Mutation PCR Arrays, which are PCR-based assays that can rapidly and accurately detect somatic mutations in cancer samples. The arrays can detect mutations present at as little as 0.01% of DNA in a sample. They have been validated to work reliably with different sample types, including archived samples. The arrays cover a wide range of clinically relevant cancer mutations and allow screening of many mutations simultaneously in a single PCR run. The assays and content were selected based on published data on mutation frequencies and functional significance. The arrays provide a simple method for sensitive somatic mutation profiling to aid cancer research.
A tyrosine kinase is an enzyme that transfers a phosphate group from ATP to tyrosine residues on proteins. This phosphorylation regulates protein activity and signal transduction within cells. Tyrosine kinase inhibitors, like nilotinib, are drugs that bind to and inhibit tyrosine kinases. Nilotinib was approved to treat chronic myeloid leukemia and research found it was effective against drug-resistant forms of the disease. It works by binding the inactive form of the Abl kinase to prevent phosphorylation and cancer cell growth.
Strong reversal of the lung fibrosis disease signature by autotaxin inhibitor...Maté Ongenaert
Strong reversal of the lung fibrosis disease signature by autotaxin inhibitor GLPG1690 in a mouse model for IPF
Maté Ongenaert (Mechelen, Belgium), Maté Ongenaert, Sonia Dupont, Roland Blanqué, Reginald Brys, Ellen van der Aar, Bertrand Heckmann
ERS - European Respiratory Society International Congress 2016. Session: Therapeutic horizons: novel targets and pharmacological models
Background and objectives
GLPG1690 is a novel potent autotaxin (ATX) inhibitor shown to be efficacious in the mouse bleomycin (BLM) lung fibrosis model. Here, we analyze the impact of GLPG1690 on the gene expression signature in mouse fibrotic lung tissue.
Methods
Lung fibrosis was induced by intranasal administration of BLM. Animals were treated with GLPG1690 or vehicle.Whole superior right lung was used for RNA extraction. Full transcriptome analysis was performed using the Agilent SurePrint G3 mouse chip. Analysis was performed using empirical Bayes methods and linear models. Public human IPF expression data were re-analyzed.
Results
GLPG1690 strongly reduced lung fibrosis as shown by reduction of Ashcroft scores and collagen content. Microarray analysis of the lungs revealed that GLPG1690 strongly reversed the impact of gene expression caused by BLM (367 out of the 2375 probes). As GLPG1690 treatment affects 395 probes, this treatment effect is highly relevant in the model. Gene clusters affected by BLM treatment and reverted by GLPG1690 are related to extracellular matrix (such as Tnc and Spp1), collagen (Col3a1) and cytokines/chemokines (Cxcl12). Several of the affected genes are known to be involved in the development or progression of lung fibrosis in IPF patients.
Conclusions
These data provide further mechanistic understanding of the efficacy of ATX inhibition in a pre-clinical lung fibrosis model, highlighting a role for extracellular matrix and inflammation biology. These data strongly suggest that GLPG1690 may be beneficial in treating IPF patients and support its evaluation in a clinical study
This review article discusses mTOR inhibitors and their use in cancer and transplantation. It outlines the mTOR signaling pathway and how mTOR inhibitors like sirolimus, everolimus and temsirolimus work. It describes how mTOR inhibitors are used to prevent organ rejection after transplantation. It also discusses how dysregulation of the PI3K/Akt/mTOR pathway promotes cancer and how mTOR inhibitors have shown efficacy in renal cell carcinoma and mantle cell lymphoma. The article concludes that biomarkers are needed to identify cancers sensitive to mTOR inhibition and that combination targeted therapies may help overcome resistance.
A novel platform for in situ, multiomic, hyper-plexed analyses of systems bio...Rafael Casiano
The document describes a novel platform called MultiOmyx that enables simultaneous imaging of protein and nucleic acid biomarkers at a subcellular level in tissue samples. It allows for quantification of biomarkers in individual cells while preserving spatial architecture. Analysis of a colon cancer cohort revealed extensive immune cell heterogeneity between patients and unexpected patterns of signal transduction pathway activation at single cell levels. This highlights the unique capability of MultiOmyx to provide novel insights into biological mechanisms through multiomic, hyper-plexed tissue analysis.
This document discusses challenges and recommendations for miRNA profiling from blood samples. It outlines that circulating miRNA in blood can serve as noninvasive biomarkers but presents purification challenges due to nucleases and other interfering components. The document recommends using optimized kits to purify miRNA from whole blood, blood cell fractions, or serum/plasma. It also recommends using miScript PCR systems for reliable miRNA quantification and normalization from blood samples via reverse transcription and real-time RT-PCR.
Epidermal growth factor (EGF) is a protein that binds to EGF receptors on epithelial and epidermal cells and initiates the EGF signaling pathway. When EGF binds to EGF receptors, it causes them to dimerize and activate their tyrosine kinase activity, leading to phosphorylation and activation of downstream proteins in the MAPK pathway. Mutations that cause constitutive activation of this pathway can lead to uncontrolled cell growth and cancer. Potential cancer treatments discussed include RGD-based peptides that target the αvβ3 integrin receptor involved in angiogenesis, and a conjugate of low molecular weight heparin and suramin that may inhibit tumor growth by blocking vascular endothelial growth factor (VEGF).
This technical article describes three case studies using RT2 Profiler PCR Arrays to analyze gene expression changes in different research areas: toxicology, oncology, and immunology. In the toxicology study, liver cells were treated with three drugs known to cause liver toxicity and the PCR Array identified distinct gene expression patterns for each drug, suggesting different mechanisms of toxicity. In the oncology study, gene expression in breast tumor samples was compared to normal tissue and a common set of upregulated genes was discovered in two independent tumor samples. In the immunology study, stimulated immune cells showed good correlation between cytokine gene and protein expression levels. The article concludes the PCR Array System is a reliable and accurate tool for pathway-focused gene expression profiling across
study of EGFR protein expression and mutation premvarma064
This document discusses oral squamous cell carcinoma (OSCC), which represents 90% of oral cancers and is characterized by a poor prognosis and lower survival rate. It notes that 0.7 million new cases and 0.3 million deaths occur annually from OSCC globally. In India, approximately 30-40% of all cancer cases are oral cancers, which is much higher than in Western countries. Risk factors for oral cancer include smoking, tobacco use, HPV infection, oral submucous fibrosis, and certain other conditions. The document discusses using immunohistochemistry to examine EGFR expression levels in oral cancer tissue samples and using PCR and restriction enzyme digestion to analyze for EGFR mutations, which can indicate prognosis and treatment options.
This document describes a study that used protein microarrays to systematically measure interactions between SH2/PTB domains and sites of tyrosine phosphorylation on receptor tyrosine kinases (RTKs) and adaptor proteins. They found that adaptor proteins, like RTKs, have many high affinity interactions with other adaptor proteins, demonstrating a high degree of connectivity. Additionally, proteins known to drive cancer through aberrant signaling, including both RTKs and adaptor proteins, tend to have more interaction partners than non-oncogenic proteins. This suggests that connectivity within signaling networks may help identify new potential drug targets for cancer treatment.
1) GRK5 regulates prostate cancer cell migration and invasion in vitro and tumor growth and metastasis in vivo.
2) GRK5 phosphorylates the cytoskeletal protein moesin, regulating its subcellular distribution and localization to the cell periphery.
3) Phosphorylation of moesin at threonine 66 by GRK5 is important for cell spreading, and mutation of this site reduces cell spreading.
Cardiotoxicity is unfortunately a common side effect of many modern chemotherapeutic agents. The mechanisms that underlie these detrimental effects on heart muscle, however, remain unclear. The Drug Toxicity Signature Generation Center at ISMMS aims to address this unresolved issue by providing a bridge between molecular changes in cells and the prediction of pathophysiological effects. I will discuss ongoing work in which we use next-generation sequencing to quantify changes in gene expression that occur in cardiac myocytes after they are treated with potentially toxic chemotherapeutic agents. I will focus in particular on the computational pipeline we are developing that integrates sophisticated sequence alignment, statistical and network analysis, and dynamical mathematical models to develop novel predictions about the mechanisms underlying drug-induced cardiotoxicity.
Jaehee Shim is a Ph.D candidate in the Biophysics and Systems Pharmacology Program at Icahn School of Medicine at Mount Sinai (ISMMS). As a part of her Ph.D. studies, she is building dynamical prediction models based on analysis of gene expression data generated by the Drug Toxicity Signature Generation Center at ISMMS. She received her B.S in Biochemistry from the University of Michigan-Dearborn. Prior to starting her Ph.D, Jaehee worked at the ISMMS Genomics Core with a team of senior scientists and gained experience in improving and troubleshooting RNA sequencing protocols using Next Generation Sequencing Platforms.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cel...Qiagen - Egypt
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
Cell-based Reporter Assays: Measure 45 Signaling Pathway Activity in Any Cell...QIAGEN
Would you like to measure signaling pathway activity in your favorite cell? Learn how to successfully apply convenient and robust reporter assays to your RNA interference, gene over-expression, protein, or small molecule studies. The Cignal Reporter Assays are an excellent tool for studying pathway signaling activity in cells that are amenable to transfection, available for studying numerous pathways including (ROS, Wnt, NF-kB, Notch, cAMP/PKA, TGFbeta, and the Cignal Lenti Reporter Assays combines the power of a lentiviral delivery system with our robust transcription factor reporter technology, enabling you to study signal pathways in virtually any cell type. You can find a technology overview, protocol tutorial, and application examples in the following presentation.
BioMAP<sup>®</sup> Primary Human Cell-Based Systems for Drug DiscoveryBioMAP® Systems
The document discusses BioMAP, a platform using primary human cell-based disease models to characterize compounds and discover their mechanisms of action and toxicity. It can assess over 2000 compounds across diverse pathways and targets. BioMAP generates biological profiles for compounds and uses these to classify compounds by similarity of mechanism. It has been used in collaborations to efficiently profile EPA ToxCast compounds and identify unexpected targets. The platform bridges molecular and cellular data to help validate targets and indications and connect to in vivo studies.
This document provides an overview of using gene expression profiling to evaluate drug metabolism-induced toxicity. It discusses how drug metabolism can lead to toxicity and the need to systematically evaluate this in pre-clinical studies. It then describes using Qiagen's RT2 Profiler PCR Arrays, which allow profiling of 84 drug metabolizing enzyme genes, to detect abnormalities in drug metabolism and identify mechanisms of toxicity using human hepatocytes treated with different compounds as an example application. The results showed induction and inhibition of various metabolizing enzyme genes in response to the compounds tested.
This is the Powerpoint presentation from my recent presentation at the TTP LabTech US Acumen Users Group Meeting (UGM) held at the British Consulate-General in Cambridge, MA on May 18, 2010
This document discusses using gene and miRNA expression profiling to develop biomarkers for monitoring genotoxicity. It describes:
1. Developing gene-based in vitro biomarkers for genotoxicity using RT2 Profiler PCR Arrays to analyze expression of DNA damage and p53 pathway genes in HepG2 cells exposed to genotoxic and non-genotoxic compounds. 11 genes were identified as classifiers.
2. Identifying miRNA-based in vivo biomarkers by profiling miRNA expression in mouse liver after exposure to the carcinogen ENU using miScript PCR Arrays. The mir-34 family showed temporal changes and clustering analysis identified differentially expressed miRNAs.
3. miRNA profiles have potential to serve as biomarkers for genotoxicity
Next generation sequencing of the whole transcriptome enables high resolution measurement of gene expression activity in different tissue and cell types. This methodology provides an in depth study of known transcripts and depending on the data analysis, allows identification of additional transcript types such as transcript variants, fusion transcripts, and small and long ncRNAs.
In this study we performed RNA-Seq using the Ion Torrent™ sequencing platform to compare the expression profile of testicular germ cell cancers (seminoma type, n=3) and normal testis (n=3). Using Partek Flow® 3.0 and TopHat/BowTie or Star aligners, we aligned the reads to the human genome and mapped sequences to the RefSeq database. Differentially expressed genes were identified and screened with additional germ cell tumors.
PCA analysis showed clear separation of the two sample types indicating biological differences. List of differentially expressed genes generated from TopHat/Bowtie and Star were similar. We identified a large number of genes that were up and down regulated with high degree of significance (p<0.01,>2X FC (fold change)). These included genes related to testicular tissue type, stem cell pluripotency (NANOG; POU5F1) and proliferation (KRAS, CCND2).
In addition, a number of differentially expressed noncoding RNAs were identified (SNORD12B, XIST). The method was validated on a small set of genes (n=20) using qPCR (TaqMan® Assays) and were found to be correlated. We used the OpenArray® platform to quickly and quantitatively screen 102 differentially expressed genes and 10 endogenous control genes across a number of different testicular germ cell cancer types.
We used a complete work flow solution from sample prep to NGS to qPCR to compare the expression profile of normal testis and seminoma type germ cell tumors. From the NGS experiments we identified a large number of differentially expressed genes for qPCR screening with samples from different types of germ cell tumors. Results from these screening studies will be presented.
Predictive Models for Mechanism of Action Classification from Phenotypic Assa...Ellen Berg
Predictive Models for Mechanism of Action Classification from Phenotypic Assay Data – Application to Phenotypic Drug Discovery
Presentation at SLAS 2014 conference in San Diego, 21 January 2014
The document discusses using microRNA (miRNA) expression analysis to understand the effects of the testicular toxicant 2,5-hexanedione in rats. Rats were dosed for 14 days, and 8 miRNAs were found to be differentially expressed in the testis. Predicted miRNA targets and known targets were analyzed, both suggesting involvement of the FSH signaling pathway, important for Sertoli cell function and germ cell maturation. The results indicate miRNAs play important roles in regulating testicular function, and integrating miRNA profiling with pathway analysis can provide insights into miRNA-mRNA interactions and potential mechanistic biomarkers of toxicity.
Please note: This presentation accompanies a recorded webinar at:
https://www1.gotomeeting.com/register/347794241
Biomarkers for studying gene regulation and cell function can be efficiently analyzed by multiplexed methods. Dr. Jim Lazar from OriGene Technologies will provide an overview of four different but related detection technologies that can be used to analyze genetic variants, microRNA expression, transcription factor binding, and protein expression on the Luminex xMAP platform. OriGene’s broad panel of assays and tools for discovery, analysis and validation of multiple classes of important biomarkers will allow researcher to develop more accurate descriptions of biologically complex systems.
This document summarizes a study that evaluated gene expression profiling of 84 human drug metabolizing enzymes using a pathway-focused PCR array. Researchers treated human hepatocytes with 3 drugs and used the PCR array to measure mRNA expression. They found high intra- and inter-laboratory reproducibility of the PCR array results based on correlation of ΔCT and ΔΔCT values and overlap of differentially expressed genes between experiments. Comparison to TaqMan data also showed high concordance, validating the PCR array as a reliable tool for quantifying drug metabolizing gene expression.
Translational Genomics and Prostate Cancer: Meet the NGS Experts Series Part 2QIAGEN
Advanced prostate cancer is highly heterogeneous but this inter-patient heterogeneity has until recently not been understood. We have through an international research effort dissected the molecular landscape of advanced castration resistant prostate, elucidating key molecular targets in this group of diseases. We have also shown that PARP inhibitors have antitumor activity against a significant proportion of these cancers, mainly in men whose cancers harbor DNA repair defects.
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration. The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will surely provide cost effective alternative to mammalian system forrt-PA production
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the tissue plasminogen activator gene was cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed expression of rt-PA at 66 kDa on SDS-PAGE. Size exclusion chromatography also showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers were able to successfully clone and express rt-PA in the Pichia pastoris system.
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
This document summarizes research conducted to clone and express recombinant tissue plasminogen activator (rt-PA) using the methylotrophic yeast Pichia pastoris. Specifically, the full-length tPA gene was amplified by PCR and cloned into the pICZαA expression vector. This construct was then transformed into Pichia pastoris X33 cells. One transformant, named Pichia tPA-3, showed extracellular expression of rt-PA at 66 kDa on SDS-PAGE after induction with methanol over 144 hours. Size exclusion chromatography of samples from this transformant showed a product peak at the same retention time as a reference tPA standard. Thus, the researchers successfully achieved cloning and extracellular expression of
Cloning and Extracellular Expression of Recombinant Tissue Plasminogen Activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZαA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+
phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
CLONING AND EXTRACELLULAR EXPRESSION OF RECOMBINANT TISSUE PLASMINOGEN ACTIVA...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZαA expression vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a way to get native tPA protein sequence in subsequent purification steps
A next generation sequencing based sample-to-result pharmacogenomics research...Thermo Fisher Scientific
Pharmacogenomics (PGx) is the study of genetic variations in terms of their response to drugs. Variations in gene sequence or copy numbers may result in complete loss of function, partial decrease or increase in enzyme activity, or an altered affinity for substrates, which may in turn significantly impact drug efficacy. PGx studies are becoming increasingly important for precision medicine. We have developed a next generation sequencing (NGS) PGx research solution with increased flexibility on the assay targets and combined detection of SNP/INDEL genotyping and CNV using Ion AmpliSeq™ technology for low to medium throughput laboratories. With this highly multiplexed PGx research panel we can profile a set of 136 genetic markers in 40 known PGx related genes (Table 1) and determine CYP2D6 copy number variation (CNV, Figure 1) in a single reaction using Ion Torrent™ semiconductor sequencing.
RT2 Profiler PCR Arrays allow researchers to analyze gene expression profiles for focused panels of genes involved in biological pathways or disease states. The arrays provide laboratory-verified gene assays, integrated controls, and free data analysis software to generate a gene expression profile from a sample in less than 3 hours. Popular arrays analyze pathways such as extracellular matrix and adhesion molecules, WNT signaling, and cancer-related genes. The complete workflow begins with sample preparation and ends with data interpretation and publication of results.
Similar to Mechanisms of Plaque Rupture in Advanced Atherosclerosis (20)
FAIR Data Knowledge Graphs–from Theory to PracticeTom Plasterer
This document discusses building FAIR data knowledge graphs from theory to practice. It begins by outlining what R&D researchers want to do with data, such as understanding disease mechanisms and using patient data, but that currently data is fragmented across systems. It then introduces the FAIR data principles and describes building a knowledge graph that incorporates data from multiple sources using standards like the Data Catalog vocabulary. The key challenges discussed are determining canonical representations for entities and linking data to public vocabularies through an enrichment process.
Making Data FAIR (Findable, Accessible, Interoperable, Reusable)Tom Plasterer
What to do About FAIR…
In the experience of most pharma professionals, FAIR remains fairly abstract, bordering on inconclusive. This session will outline specific case studies – real problems with real data, and address opportunities and real concerns.
·
Why making data Findable, Actionable, Interoperable and Reusable is important.
Talk presented at the Data Driven Drug Development (D4) conference on March 20th, 2019.
FAIR data has flown up the hype curve without a clear sense of return from the required data stewardship investment. The killer use case for FAIR data is a science knowledge graph. It enables you to richly address novel questions of your and the world’s data. We started with data catalogues (findability) which exploited linked/referenced data using a few focused vocabularies (interoperability), for credentialed users (accessibility), with provenance and attribution (reusability) to make this happen.
This talk was presented at The Molecular Medicine Tri-Conference/Bio-IT West on March 11, 2019.
Dataset Catalogs as a Foundation for FAIR* DataTom Plasterer
BioPharma and the broader research community is faced with the challenge of simply finding the appropriate internal and external datasets for downstream analytics, knowledge-generation and collaboration. With datasets as the core asset, we wanted to promote both human and machine exploitability, using web-centric data cataloguing principles as described in the W3C Data on the Web Best Practices. To do so, we adopted DCAT (Data CATalog Vocabulary) and VoID (Vocabulary of Interlinked Datasets) for both RDF and non-RDF datasets at summary, version and distribution levels. Further, we’ve described datasets using a limited set of well-vetted public vocabularies, focused on cross-omics analytes and clinical features of the catalogued datasets.
BioPharma and FAIR Data, a Collaborative AdvantageTom Plasterer
The concept of FAIR (Findable, Accessible, Interoperable and Reusable) data is becoming a reality as stakeholders from industry, academia, funding agencies and publishers are embracing this approach. For BioPharma being able to effectively share and reuse data is a tremendous competitive advantage, within a company, with peer organizations, key opinion leaders and regulatory agencies. A few key drivers, success stories and preliminary results of an industry data stewardship survey are presented.
Edge Informatics and FAIR (Findable, Accessible, Interoperable and Reusable) ...Tom Plasterer
Edge Informatics is an approach to accelerate collaboration in the BioPharma pipeline. By combining technical and social solutions knowledge can be shared and leveraged across the multiple internal and external silos participating in the drug development process. This is accomplished by making data assets findable, accessible, interoperable and reusable (FAIR). Public consortia and internal efforts embracing FAIR data and Edge Informatics are highlighted, in both preclinical and clinical domains.
This talk was presented at the Molecular Medicine Tri-Conference in San Francisco, CA on February 20, 2017
Harnessing Edge Informatics to Accelerate Collaboration in BioPharma (Bio-IT ...Tom Plasterer
As scientists in the life sciences we are trained to pursue singular goals around a publication or a validated target or a drug submission. Our failure rates are exceedingly high especially as we move closer to patients in the attempt to collect sufficient clinical evidence to demonstrate the value of novel therapeutics. This wastes resources as well as time for patients depending upon us for the next breakthrough.
Edge Informatics is an approach to ameliorate these failures. Using both technical and social solutions together knowledge can be shared and leveraged across the drug development process. This is accomplished by making data assets discoverable, accessible, self-described, reusable and annotatable. The Open PHACTS project pioneered this approach and has provided a number of the technical and social solutions to enable Edge Informatics. A number of pre-competitive consortia and some content providers have also embraced this approach, facilitating networks of collaborators within and outside a given organization. When taken together more accurate, timely and inclusive decision-making is fostered.
As BioPharma adapts to incorporate nimble networks of suppliers, collaborators, and regulators the ability to link data is critical for dynamic interoperability. Adoption of linked data paradigm allows BioPharma to focus on core business: delivering valuable therapeutics in a timely manner.
Enabling Discovery in High-Risk Plaque using Semantic Web ApproachesTom Plasterer
Enabling Discovery in High-Risk Plaque using Semantic Web Approaches
The HRP initiative (HRP) is a joint research and development effort to advance the understanding, recognition and management of high-risk plaque for the benefit of multiple stakeholders in the healthcare system. As the primary underlying cause of heart attacks, high-risk, or vulnerable plaque is the number one cause of death in the Western world. There are currently no methods of screening, diagnosis or treatment for high-risk plaque.
The HRP initiative leverages recent advances in biology and information technology to design and optimize a care-cycle for high-risk plaque, promising to reduce morbidity, mortality and cost associated with cardiovascular disease. This Initiative is being led by the world’s foremost scientists in the fields of cardiology, pathology, and imaging, and is made possible through funding by leading pharmaceutical and medical technology entities.
HRP takes advantages of semantic web technologies for physician and researcher-lead data analysis and data interoperability. One of the key applications is a web tool linking patient demographics, clinical chemistries, physical measurements and cardiovascular imaging modalities. This empowers scientists to rapidly compare multiple clinical parameters to find patients of interest, assisting greatly in defining high-risk plaque.
11. Distribution of Plaque Markers Distinguish Advanced-Stable from Ruptured Plaque 479 235 714 1,190 Px 3,892 3,771 7,663 23,719 Total 3,301 3,499 6,800 22,184 Tx 92 1 93 191 Lipid 20 29 49 118 GC/MS 0 7 7 36 AAA Up in Ruptured Up in Stable Number of markers Number of analytes Platform
21. Canonical Signaling Pathway Analysis : Cellular immune response Highest Tx significance: Role of NFAT in Regulation of the Immune Response (p=3.11E-10)
30. Acute Phase Response Signaling: Proteins Only Likely binding partners are integrins ITGAM & ITGB2 Origin of many of these proteins likely hepatic (outside of plaques)
KEY POINT: Majority of pathway analysis from the pink box; some specific pathway observations checked against cell type analysis
KEY POINT: Sets up integrin story Observations from comparative analysis: Vervain All Modalities
KEY POINT: starting slide for integrin story, sets up slides for the three sub-stories (integrin receptor, cytoskeletal rearrangements, ILK/PI3K/WNT signaling) MD/Maastricht: they have published on the integrins before; good candidates for imaging
KEY POINT: genes/proteins generally agree (down in R/S) Observations from comparative analysis: Vervain All Modalities
KEY POINT: differential use of alpha/beta subunits in ruptured vs. stable plaque indicate differential upstream & downstream signaling Observations from comparative analysis: Vervain All Modalities
KEY POINT: likely loss of cytoskeletal structures in ruptured plaque, loss of internal focal adhesion. Consistent in Px & Tx Observations from comparative analysis: Vervain All Modalities Striking agreement among both proteins and transcripts for actin, alpha-actinin, talin (all complexes) and vinculin. This is suggestive of a weakening of the actin cytoskeleton in ruptured plaques, possibly due to integrin signaling.
KEY POINT: Differential integrin receptor use and secondary messenger use promotes WNT signaling in plaques while demoting PI3K signaling & growth factor signaling. Again both Px & Tx story. WNT signaling, however, is generally downregulated despite contributions from GSK3B. Observations from comparative analysis: Vervain All Modalities
KEY POINT: majority of response in transcripts—many of these are low copy number analytes Observations from comparative analysis: Vervain All Modalities NFAT = Nuclear factor of activated T-cells
Observations from comparative analysis: Vervain All Modalities Many may be lymphocyte specific
Observations from comparative analysis: Vervain All Modalities
KEY POINT: Dominated by transcripts; TNF receptor 11B (Osteoprotegerin) is a possible decoy receptor but downregulation may contribute to calcification. Other TNF receptors (1A, 1B) are pro-apoptic. All three are part of the “TNFR” cluster. MD/Maastricht: t-cells also invade from circulation and proliferate in the adventitia (low numbers relative to other cells would explain lack of Px)
Observations from comparative analysis: Vervain All Modalities
Many proteins here from serum, hence synthesized by liver. MD/Maastricht: also proteins of local/plaque origin!
KEY POINT: proteomics response driven by hepatic origin
Observations from comparative analysis: Vervain All Modalities
Observations from comparative analysis: Vervain All Modalities
KEY POINT: C3 has the integrin a/b partners ITGAM & ITGB2. Proteomics response driven by hepatic origin; cytoplasmic representation of proteins likely out of context; Observations from comparative analysis: Vervain All Modalities
Observations from comparative analysis: Vervain All Modalities
KEY POINT: differential response from the inflammatory (IL-1) route from the TGFb route (SMC migration). Mostly a Tx response due to low copy numbers. Note differential enrichment: IL-1 route (inflammation) up, TGFb route (vascular SMC migration) down! Most obsevation are for transcripts only (low expression levels).
NOT a strong protein response. Many of the transcripts seen are low-intensity transcripts (where is the crossover point?) –OR—they’re not made into proteins—OR—proteomics sample prep isn’t capturing everything that Tx sample prep is.
Observations from comparative analysis: Vervain All Modalities
KEY POINT: actin signaling down in ruptured; also implicated from integrin pathways. Observations from comparative analysis: Vervain All Modalities MD/Maastricht: may be cell specific; not a general abnormality rendering stable more vulnerable
Observations from comparative analysis: Vervain All Modalities Note other than actin and PFN, hardly any of these proteins are components of the cytoskeleton