Discovery Of Novel Markers Research Articles

Identifying Candidate Circulating RNA Markers for Coronary Artery Disease by Deep RNA-Sequencing in Human Plasma.

Advances in RNA sequencing (RNA-Seq) have facilitated transcriptomic analysis of plasma for the discovery of new diagnostic and prognostic markers for disease. We aimed to develop a short-read RNA-Seq protocol to detect mRNAs, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in plasma for the discovery of novel markers for coronary artery disease (CAD) and heart failure (HF). Circulating cell-free RNA from 59 patients with stable CAD (half of whom developed HF within 3 years) and 30 controls was sequenced to a median depth of 108 paired reads per sample. We identified fragments from 3986 messenger RNAs (mRNAs), 164 long non-coding RNAs (lncRNAs), 405 putative novel lncRNAs and 227 circular RNAs in plasma. Circulating levels of 160 mRNAs, 10 lncRNAs and 2 putative novel lncRNAs were altered in patients compared with controls (absolute fold change >1.2, p < 0.01 adjusted for multiple comparisons). The most differentially abundant transcripts were enriched in mRNAs encoded by the mitochondrial genome. We did not detect any differences in the plasma RNA profile between patients who developed HF compared with those who did not. In summary, we show that mRNAs, lncRNAs and circular RNAs can be reliably detected in plasma by deep RNA-Seq. Multiple coding and non-coding transcripts were altered in association with CAD, including several mitochondrial mRNAs, which may indicate underlying myocardial ischaemia and oxidative stress. If validated, circulating levels of these transcripts could potentially be used to help identify asymptomatic individuals with established CAD prior to an acute coronary event.

Resolution of apolipoprotein A1 and A2 proteoforms: their cardiometabolic correlates and implications for future research.

A 'proteoform' is defined as one specific protein structural form that results from the combination of allelic variation, alternative RNA splicing, and/or posttranslational modifications (PTMs) in specific locations on the amino acid backbone. Apolipoproteins A1 and A2 are highly abundant apolipoproteins that mediate HDL structure and function. ApoA1 and apoA2 are known to undergo PTMs, which results in multiple proteoforms. However, the catalogue of apoA1 and apoA2 proteoforms as well as their associations with cardiometabolic health characteristics has not been described until recently. In this brief review, we discuss recent efforts to catalogue the spectrum of apoA1 and apoA2 proteoforms, to understand the relationships between the relative abundance of these proteoforms with cardiometabolic phenotypic characteristics, and we will discuss the implications of these findings to future research. A broad spectrum of apoA1 and apoA2 proteoforms has been characterized. Although, the types of apoA1 and A2 proteoforms are consistent across individuals, the relative abundances of proteoforms can vary substantially between individuals. Proteoform-specific associations with cardiometabolic characteristics in humans, independent of absolute apolipoprotein abundance, have been described. These recent findings suggest multiple levels of protein structural variation that arise from known and unknown metabolic pathways may be important markers or mediators of cardiometabolic health. Understanding the associations between apolipoprotein proteoforms and phenotype may lead to enhanced understanding of how apolipoproteins mediate lipid metabolism and affect atherosclerotic cardiovascular disease (ASCVD) risk, which may lead to discovery of novel markers of risk and/or key mechanistic insights that may drive further druggable targets for modifying lipid metabolism and reducing ASCVD risk.

Golgin Subfamily A Member 5 Is Essential for Production of Extracellular Matrix Proteins during TGF-β1-Induced Periodontal Ligament-Fibroblastic Differentiation.

Human periodontal ligament stem cells (hPDLSCs) can be differentiated into periodontal ligament- (PDL-) fibroblastic progenitors by treatment with low concentrations of transforming growth factor beta 1 (TGF-β1). Although much is known about the profibrotic effects of TGF-β1, the molecular mechanisms mediating the activation of fibroblasts in periodontal ligament-fibroblastic differentiation are not well known. Our study was to investigate the mechanism of the fibroblastic process in the periodontal ligament differentiation of hPDLSCs through the discovery of novel markers. One of the monoclonal antibodies previously established through decoy immunization was the anti-LG11 antibody, which recognized Golgi subfamily A member 5 (GOLGA5) as a PDL-fibroblastic progenitor-specific antigen. GOLGA5/LG11 was significantly upregulated in TGF-β1-induced PDL-fibroblastic progenitors and accumulated in the PDL region of the tooth root. GOLGA5 plays a role in vesicle tethering and docking between the endoplasmic reticulum and the Golgi apparatus. siRNA-mediated depletion of endogenous GOLGA5 upregulated in TGF-β1-induced PDL-fibroblastic progenitors resulted in downregulation of representative PDL-fibroblastic markers and upregulation of osteoblast markers. When the TGF-β1 signaling pathway was blocked or GOLGA5 was depleted by siRNA, the levels of extracellular matrix (ECM) proteins, such as type I collagen and fibronectin, decreased in PDL-fibroblastic progenitors. In addition, Golgi structures in the perinuclear region underwent fragmentation under these conditions. These results suggest that GOLGA5/LG11 is a PDL-fibroblastic marker with functional importance in ECM protein production and secretion, which are important processes in PDL-fibroblastic differentiation.

Lipid Metabolism Interplay in CRC-An Update.

Colorectal cancer (CRC) to date still ranks as one of the deadliest cancer entities globally, and despite recent advances, the incidence in young adolescents is dramatically increasing. Lipid metabolism has recently received increased attention as a crucial element for multiple aspects of carcinogenesis and our knowledge of the underlying mechanisms is steadily growing. However, the mechanism how fatty acid metabolism contributes to CRC is still not understood in detail. In this review, we aim to summarize our vastly growing comprehension and the accompanied complexity of cellular fatty acid metabolism in CRC by describing inputs and outputs of intracellular free fatty acid pools and how these contribute to cancer initiation, disease progression and metastasis. We highlight how different lipid pathways can contribute to the aggressiveness of tumors and affect the prognosis of patients. Furthermore, we focus on the role of lipid metabolism in cell communication and interplay within the tumor microenvironment (TME) and beyond. Understanding these interactions in depth might lead to the discovery of novel markers and new therapeutic interventions for CRC. Finally, we discuss the crucial role of fatty acid metabolism as new targetable gatekeeper in colorectal cancer.

Shotgun proteomics approaches for authentication, biological analyses, and allergen detection in feed and food-grade insect species

Untargeted proteomics can contribute to composition and authenticity analyses of highly processed mixed food and feed products. Here, we present the setup of an analytical flow tandem mass spectrometry method (AF-HPLC HR-MS) for analysis of insect meal from five different species. Data acquired were compared with previously published data employing spectra matching and standard bottom-up proteomics bioinformatics analyses. In addition, data were screened for insect species marker peptides and common allergens, respectively. The results obtained indicate that the performance of the newly established AF-HPLC HR-MS workflow is in line with previously published methods for insect species differentiation. Data obtained in the present study, also lead to the discovery of novel markers for the development of targeted MS analyses of insect species in food- and feed-mixes and highlighted that known allergen such as arginine kinase or tropomyosin were consistently detected across all five species tested.

Discovery of Novel Markers for Identifying Cognitive Decline Using Neuron-Derived Exosomes.

Alzheimer’s disease (AD), the predominant cause of late-life dementia, has a multifactorial etiology. Since there are few therapeutic options for symptomatic AD, research is increasingly focused on the identification of pre-symptomatic biomarkers. Recently, evaluation of neuron-derived exosomal markers has emerged as a promising novel approach for determining neuronal dysfunction. We aimed to identify novel neuron-derived exosomal markers that signify a transition from normal aging to Mild Cognitive Impairment (MCI) and then to clinically established AD, a sequence we refer to as AD progression. By using a Tandem Mass Tag-based quantitative proteomic approach, we identified a total of 360 neuron-derived exosomal proteins. Subsequent fuzzy c-means clustering revealed two clusters of proteins displaying trends of gradually increasing/decreasing expression over the period of AD progression (normal to MCI to AD), both of which were mainly involved in immune response-associated pathways, proteins within these clusters were defined as bridge proteins. Several differentially expressed proteins (DEPs) were identified in the progression of AD. The intersections of bridge proteins and DEPs were defined as key proteins, including C7 (Complement component 7), FERMT3 (Fermitin Family Member 3), CAP1 (Adenylyl cyclase-associated protein 1), ENO1 (Enolase 1), and ZYX (Zyxin), among which the expression patterns of C7 and ZYX were almost consistent with the proteomic results. Collectively, we propose that C7 and ZYX might be two novel neuron-derived exosomal protein markers, expression of which might be used to evaluate cognitive decline before a clinical diagnosis of AD is warranted.

Large-scale prospection of genes on caffeine-free Coffea arabica plants – Discovery of novel markers associated with development and secondary metabolism

Differential gene expression profiles and metabolic networks are valuable tools for the genetic characterization of agronomic traits. In this study, we used large-scale expression analyses to identify modified biological processes in caffeine-free coffee plants. The first step was the large-scale sequencing of RNA from young and developing tissues of caffeine-free plants (AC1) and plants with normal concentrations of the compound (MN). The resulting 65,000 sequences were analyzed in silico for identification of 171 genes with differential expression between treatments, and establishment of metabolic networks associated with levels of caffeine. Few genes were mapped onto metabolic pathways, indicating that low caffeine has no major effects on physiological processes. The differential expression observed in silico was validated for 12 selected genes in field experiments using qPCR. The expression profile of 5 genes differed on the analyses, and the rest confirmed the in silico profile. Among the validated genes two of them, FIG and LSM-l, may control other agronomic traits associated with low caffeine content in coffee tissues. These genes are potential markers for use in association with other current markers for assisted selection of low-caffeine coffee. Therefore, they may improve the efficiency and effectiveness of coffee breeding programs.

Proteomic analysis of microglia during LPS-induced acute neuroinflammation.

Abstract Dysregulation of microglia function is associated with neuroinflammation. A major limitation in understanding microglial contribution to cellular processes and their role in disease has been the lack of tools to specifically distinguish these cells from other myeloid cells. We generated and validated a rat monoclonal antibody against a selective microglia marker P2RY12. We utilized LPS administration as a model for neuroinflammation to assess alterations in P2RY12 expression, a homeostatic protein. Mice were injected with either PBS, low or high dosages of LPS daily for 4 days after which microglia were harvested from the brain. Isolated cells were co-stained with CD11b, CX3CR1, and P2RY12 as general markers for microglia. Flow cytometric quantification using live cells showed a significant increase in CD11b+ expression in both low and high dose LPS treated mice compared to the control untreated group. Low and high dose LPS injections led to a significant reduction in P2RY12 and CX3CR1 expression in these cells. These data are consistent with a decrease in P2RY12 expression under inflammatory conditions. We assessed microglial proteomic profile using BioLegend’s proprietary technology, TotalSeq™, which allows simultaneous analysis of both the transcriptome and proteome at the single-cell level. We utilized a panel of 200 antibody-oligonucleotide conjugates, including P2RY12, to analyze differentially expressed proteins under normal and LPS-induced inflammation in mice. These studies help elucidate the diversity and physiology of microglia, and potentially aid in the discovery of novel markers in response to neuroinflammation.

NGFR Increases the Chemosensitivity of Colorectal Cancer Cells by Enhancing the Apoptotic and Autophagic Effects of 5-fluorouracil via the Activation of S100A9.

Colorectal cancer (CRC) is currently the third leading cause of cancer-related deaths worldwide, and 5-fluorouracil (5-FU)-based chemotherapies serve as important adjuvant therapies before and after surgery for CRC. However, the efficacy of CRC chemotherapy is limited by chemoresistance, and therefore the discovery of novel markers to indicate chemosensitivity is essential. Nerve growth factor receptor (NGFR), a cell surface receptor, is involved in cell death and survival. Our previous study indicated that NGFR acts as a tumor suppressor, and high expression is associated with better outcomes in patients receiving 5-FU-based adjuvant chemotherapy after surgery. The aim of this study was to investigate the effect of NGFR on the chemotherapeutic response in CRC. Chemosensitivity was investigated using DLD1 and HCT8 cells after NGFR transfection. Apoptosis was investigated by flow cytometry. Autophagy was assessed using GFP-LC3B transient transfection. Gene expression was measured using an mRNA microarray. Beclin-1 and Bcl-2 protein expressions were assessed by western blot. NGFR and S100 calcium-binding protein A9 (S100A9) expressions in CRC patients were investigated by immunohistochemistry. The results showed that the half maximal inhibitory concentration of NGFR-transfected cells was lower than that of controls in DLD1 and HCT8 cells after 5-FU treatment, and cell viability was lower than in empty-vector cells. Tumor sizes were also smaller than in empty-vector cells in vivo. The percentages of apoptotic and autophagic cells were higher in NGFR-transfected cells. NGFR elevated the expression of S100A9 after 5-FU treatment. The combination of Bcl-2 and Beclin-1 was significantly suppressed by overexpressed NGFR. Five-year overall and disease-free survival in NGFR+/S100A9+ patients was better than in NGFR−/S100A9− patients. This study’s findings suggest that NGFR may serve as a marker predicting CRC patients’ chemosensitivity.

Preliminary discovery of novel markers for human cell line activation test (h-CLAT)

The human cell line activation test (h-CLAT) is an OECD approved (Test No. 442E) assay to identify novel skin sensitizers. h-CLAT simulates dendritic cell activation in the skin sensitization pathway and is based on the measurement of CD54 and CD86 overexpression on monocytic, leukemic THP-1 cells. However, the current h-CLAT markers show inconsistent results with moderate and weak sensitizers. Moreover, these markers have accessory roles in cell adhesion and signaling rather than a direct role in cellular inflammation. Therefore, we have explored other inflammation-related markers in this study. PBMCs comprises a mixture of cells that resemble the complex immunological milieu in adults and were primarily used to identify markers. PBMCs (n = 10) and THP-1 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB, strong) and NiCl2 (Ni, moderate) sensitizers or DMSO (control) and incubated for 24 h. The samples were subjected to RNA sequencing to obtain log2fold change in gene expression. DNCB and NiCl2 significantly upregulated 80 genes in both cell types. Of these, CD109, CD181, CD183, CLEC5A, CLEC8A & CD354 were experimentally validated. DNCB and Ni but not isopropyl alcohol (non-sensitizer) significantly induced the expression of all novel markers except CLEC8A. Moreover, the percentage induction of all novel markers except CLEC8A satisfied the OECD acceptance criteria. In summary, we identified five novel markers that may supplement the current repertoire of h-CLAT markers.

STEM-02. IN VIVO PHAGE DISPLAY IDENTIFIES PEPTIDE TARGETING N-CADHERIN ON GLIOMA STEM CELLS

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with high mortality rates and resistance to conventional therapy. Glioma stem cells (GSCs) comprise a sub-population of glioma tumor cells with the ability of self-renewal and tumor recapitulation, and may be responsible for GBM’s treatment resistant properties. Identification of surface receptors that are novel and specific to GSCs may be the key to the development of effective therapeutic strategies. We have selected a GSC specific targeting peptide isolated through in vitro and in vivo phage display biopanning. This screening technique allowed us to determine a peptide (GBM-IC2) which binds specifically to GSCs in vitro, and to GBM tissue in vivo. Although this screening process allows for isolation of cell specific targeting peptides, it does so without identification of the cellular binding partner. Given the specificity of the peptide, identification of the cellular receptor may allow for discovery of novel markers to identify GSCs. To identify the peptide binding partner of GBM-IC2, the biotinylated peptide was incubated with GSC protein lysate. The peptide, along with its binding partner, was isolated using streptavidin agarose resin. The binding partner protein was then identified using mass spectroscopy. This revealed N-cadherin (CDH2) as a potential binding partner for the GBM-IC2 peptide. GBM-IC2 demonstrated specificity for targeting CDH2 compared to control peptide using ELISA. Lentiviral induced overexpression of CDH2 in HEK293 cells allowed for GBM-IC2 peptide binding. Competition assay was performed by applying anti-CDH2 antibody to GBM-IC2 peptide and GSCs in culture. Application of anti-CDH2 antibody decreased peptide binding to GSCs, confirming CDH2 as the binding partner for GBM-IC2. These results demonstrate that cell specific targeting peptides isolated through phage display may lead to the isolation of novel cell specific proteins through immunoprecipitation isolation and mass spectroscopy analysis.

Transcriptomic responses associated with kidney injury and repair in acute decompensated heart failure

Abstract Background The discovery of new markers for acute kidney injury (AKI) in acute decompensated heart failure (ADHF) has been hampered by an incomplete understanding of the pathological processes underlying AKI in ADHF. Purpose In a sheep model of ADHF, we investigated changes in kidney gene expression in response to the development of, and recovery from, ADHF. Methods We collected serial kidney biopsies from 6 sheep prior to rapid cardiac pacing (day 0), after development of ADHF (pacing @220bpm for 14 days), and at the end of a 25-day (non-pacing) recovery period. Serial biopsies were supplemented with kidney samples collected post-mortem from animals undergoing a similar pacing/recovery protocol, giving a total of 11 “baseline” (B), 13 “heart failure” (HF) and 8 “recovery” (R) samples. We prepared RNA-Sequencing libraries using total RNA and Illumina TruSeq stranded mRNA library kits. Hormonal, haemodynamic, biochemical and urine measurements were also performed in all sheep before, during, and after development of ADHF. The study followed the principles of laboratory animal care and was approved by our institution's Animal Ethics Committee. Results We observed profound changes in hormonal, haemodynamic, biochemical and urine measures of cardio-renal injury in all sheep, confirming simulation of the peripheral consequences of ADHF, including clinically-relevant kidney dysfunction. This occurred in conjunction with altered kidney expression of 982 genes during ADHF development and 1,807 genes during ADHF recovery (p adj.&amp;lt;0.05, Fig 1). During ADHF development, changes in kidney gene expression were associated with activation of the pro-inflammatory p38 MAPK pathway and repression of several anti-inflammatory and reno-protective pathways, including eNOS signalling (all p adj.&amp;lt;0.001). In contrast, during ADHF recovery, changes in kidney gene expression were associated with reactivation of reno-protective pathways repressed during ADHF development, activation of anti-fibrotic pathways (including PTEN signalling) and repression of pathways that mediate inflammation and renal injury (including NF-kB signalling, all p adj.&amp;lt;0.001). Among 431 ADHF “responsive” genes (i.e. those that increased during ADHF development and decreased during ADHF recovery, or vice versa, Fig. 1), 37 genes encoded proteins detectable in plasma or urine and may represent markers of kidney repair in ADHF. Although most gene expression changes were transient, 192 genes remained altered after 4-weeks recovery (p adj.&amp;lt;0.05, Fig 1). Of these, 13 genes were predicted to encode proteins detectable in plasma or urine and may represent persistent markers of kidney injury in ADHF. Conclusion Our data provide the first insight into the gene pathways associated with kidney injury and repair in ADHF, in an established ovine model. Understanding the pathological processes underlying AKI in ADHF may enable discovery of novel markers for monitoring kidney injury and repair in ADHF. Figure 1. Genes altered in the kidney in ADHF Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Health Research Council of New Zealand, Heart Foundation of New Zealand

An association between right ventricular dysfunction and sudden cardiac death

The effectiveness of severely reduced left ventricular ejection fraction (LVEF <35%) as a predictor of sudden cardiac death (SCD) has diminished, and improvements in risk stratification await discovery of novel markers. Right ventricular (RV) abnormalities can be observed in conditions such as chronic obstructive pulmonary disease and sleep apnea, which have been linked to SCD. The purpose of this study was to evaluate whether RV abnormalities were associated with SCD after accounting for LVEF and other patient characteristics. In a large, prospective ongoing community-based study of SCD in the Portland, Oregon, metropolitan area, SCD cases (age ≥18 years; 2002-2014) were compared to controls with coronary artery disease but no SCD. Using a novel archive of digital echocardiograms, a standardized approach was used to evaluate RV basal diameter, RV end-diastolic area, and right ventricular fractional area change (RVFAC). A total of 350 subjects were studied, including 81 SCD cases (age 68.7 ± 13.6 years; 73% male) and 269 controls (age 66.5 ± 10.2 years; 69% male). In multivariate analysis, RVFAC was significantly associated with SCD (odds ratio 1.14 for each 5% decrease; 95% confidence interval 1.03-1.25; P = .01). When modeled with LVEF ≤35%, RVFAC ≤35% was significantly associated with increased risk of SCD. Individuals with both left ventricular and RV dysfunction had a 3× higher odds of SCD than those with neither (odds ratio 3.19; 95% confidence interval 1.33-7.68; P = .01). RV dysfunction was associated with a significantly increased risk of SCD independent of LVEF and, when combined with LVEF, had additive effects on SCD risk.

Abstract 2691: Discovery of novel markers in Barrett's-related esophageal adenocarcinoma

Abstract Despite the fact there are currently a dozen treatment options for esophageal adenocarcinoma (EAC), durable patient responses to anticancer therapies are hard to achieve in EAC patients. The rate of incidence of EAC is increasing faster than any cancer in our country, and the survival rates have remained very low for decades, even in the current era of molecular medicine. Therefore, our group designed an ambitious, large scale mass spectrometric experiment to cast a molecular dragnet across our patient samples to detect overexpression and downregulation patterns that contribute to the aggressiveness of EAC. We used FFPE tissue samples from esophagectomy surgery before any chemotherapy or radiation had been administered, analyzing unadulterated EAC tissue, Barrett's esophagus (BE) tissue, and normal esophageal squamous epithelium from 50 patients. Our platform consisted of pathologist-guided microdissection, a Liquid Tissue® process turning FFPE tissue into a digested lysate, discovery mass spectrometry (TripleTOF 6600) and extensive biostatistics. We discovered a perfect storm of pathogenic processes which explains from several perspectives the aggressiveness of this disease as well as EAC's proclivity to resist chemotherapeutic agents. We found 20 relevant proteomic events in which tumor suppressors are turned off, growth factors are overexpressed, chemoresistance markers are upregulated, chemosensitivity markers are downregulated as well as revealing eight new potential drug targets in which therapeutic antagonism could slow tumorigenesis in EAC. Of these 20 proteins we robustly investigated four markers (DAD1, ISG15, S100P and UBE2N) which all have mutual intersections with prognostic, diagnostic and therapeutic aspects of esophageal pathogenesis. Overexpression of DAD1 (9 out of 10), ISG15 (10/10), S100P (10/10) and UBE2N (10/10) was discovered in BE tissue. In the adjacent EAC tissue, we found significant overexpression of DAD1 (20 out of 20), ISG15 (20/20), S100P (20/20) and UBE2N (19/20) proteins. These expression levels spiked when transitioning from normal squamous epithelium to highly dysplastic BE tissue demonstrating that BE starts to act like cancer by using more glucose for energy (Warburg effect), increasing proliferative measures, and suppressing apoptosis. Also, overexpressed DAD1 protein levels contributes to cisplatin resistance which is striking since 95% of the 123 patients treated at CUMC for EAC received cisplatin in the first-line setting. S100P expression has been found to increase resistance to 5-FU in colorectal cancer. 5-FU was prescribed to 72% of the patients in our cohort (n=123), so if there is innate resistance to the two most prescribed drugs for EAC patients then alternative patient management strategies are needed. Here we've discovered four new prognostic and therapeutic biomarkers with potential clinical utility for EAC. Citation Format: Joe Abdo, Chris Wichman, Nick E. Dietz, John Fleegel, Pawel Ciborowski, Sumeet K. Mittal, Devendra K. Agrawal. Discovery of novel markers in Barrett's-related esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2691.

Current and Emerging Biomarkers for Pulmonary Hypertension

“A biological molecule found in blood, other body fluids, or tissues that is a sign of a normal or abnormal process, or of a condition or disease. A biomarker may be used to see how well the body responds to a treatment for a disease or condition. Also called molecular marker or signature molecule” – Biomarker definition, National Institutes of Health, National Cancer Institute, Dictionary of Cancer Terms Although this definition pertains to the field of cancer and cancer biology, it applies to many disciplines, including the field of pulmonary vascular disease. In the world of pulmonary hypertension (PH), biomarkers hold a special place. In the diagnosis of PH subtype, we are limited by the risk of lung biopsy1 and, further, in patients with pulmonary arterial hypertension (PAH) we are limited by the inability to frequently obtain our strongest predictors of mortality: invasive pulmonary hemodynamic assessment. Thus, biomarkers hold much promise for the field of PH. We are searching for markers of PH subtypes and endophenotypes. We are looking for predictors of mortality in all forms of PH and, critically, we are hoping to find peripheral blood markers that will help us discover which drugs are likely to benefit a particular patient. Although we have made inroads in all 3 areas, there are substantial opportunities for refinement of our current biomarkers and discovery of novel markers to improve the care of PH patients. This review will cover the state of current biomarkers in PH and discuss challenges and future directions.

Plasma proteomics reveals coagulation, inflammation, and metabolic shifts in H-type hypertension patients with and without acute ischemic stroke.

Systematic profiling of a larger portion of circulating plasma proteome provide opportunities for unbiased discovery of novel markers to improve diagnostic, therapeutic, or predictive accuracy. This study aimed to identify differentially expressed proteins (DEPs) in plasma that could provide overall insight into the molecular changes of both H- type hypertension (HH) and HH-related acute ischemic stroke (AIS). This study used an iTRAQ-based LC-MS/MS proteomics approach to screen for plasma DEPs in HH patients with and without AIS, and controls. After excluding highly abundant plasma proteins, more than 600 proteins, and their relative levels, were identified. Of these, 26 DEPs, each showing > 1.2-fold change, were identified in HH and HH-related AIS patients compared with controls. Bioinformatics analysis revealed that these DEPs were enriched in 21 functional gene ontology items; “blood coagulation” was the most predominant pathway showing enrichment. Of these, eight DEPs were located in the hub position of networks involved with protein-protein interactions. AT-3, CRP, ApoB, and AHSG were further validated in each group by enzyme-linked immune sorbent assays. Comparing HH-related AIS with HH, the areas under the curve for AT-3, CRP, ApoB, and AHSG were 0.698, 0.892, 0.626, and 0.847, respectively. This proteomic profiling study provided enhanced pathophysiological understanding of the regulatory processes involved in coagulation, inflammation, and metabolism, and identified a panel of novel biomarkers for detecting HH-related AIS during its pre-stroke stage.

Abstract 5545: Integrated system-level analyses of androgen receptor variant networks to identify novel prostate cancer-relevant genes

Abstract Unbiased high-throughput approaches provide a global view of the massive genetic and transcriptomic changes that occur in and potentially drive cancer. Such approaches permit the discovery of novel markers and therapeutic targets. Current treatments for advanced prostate cancer (PC) focus on inhibiting the androgen receptor (AR). However, PC inevitably progresses to a stage termed castration-resistant prostate cancer (CRPC), which is incurable. In CRPC, C-terminal truncated, constitutively active AR splice variants (such as AR-V7) play key transcription-regulatory roles resulting in treatment resistance and disease progression. However, designing high-affinity drugs to target the amino terminus of AR and AR variants is a major challenge due to the intrinsic disorganized structure of this region. Thus there is an imperative need to identify novel AR-V7 hub genes in PC that may serve as therapeutic targets. We performed an extensive and highly robust gene expression meta-analysis on PC patient samples. We defined gene modules correlated with disease progression using a powerful systems biology approach termed Weighted Gene-Co-expression Network Analysis (WGCNA). Further, we mapped the AR-V7 interactome for the first time using a novel high-throughput synthetic genetic array screening in yeast, known as Yeast Augmented Network Analysis (YANA). YANA was performed by crossing yeast expressing AR-V7 with a large collection of yeast strains lacking non-essential genes, and identifying those genes that caused a change in yeast growth (fitness). Human orthologs of the identified yeast genes were used to build an AR-V7 functional gene network. Finally, we combined the results from our independent system-level analyses with experimental data to identify hub genes that are upregulated in PC patients, regulated by AR-V7, and also functionally interact with AR-V7. The identified genes not only include select genes previously linked to PC, such as members of the cyclin and topoisomerase families, but also genes that have not been previously linked to AR-V7 activity or PC progression. Moreover, our gene expression signature predicts a higher risk of PC recurrence after primary treatment in patients. In sum, we show here an unbiased and novel gene discovery strategy using bioinformatics in concert with experimental approaches to identify new candidate genes in CRPC that may lead to prognostic markers and future targeted therapies. Citation Format: Fiorella Magani, Eric R. Bray, Ning Zhao, Stephanie Peacock, Kerry L. Burnstein. Integrated system-level analyses of androgen receptor variant networks to identify novel prostate cancer-relevant genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5545. doi:10.1158/1538-7445.AM2017-5545

Emerging Affinity-Based Proteomic Technologies for Large-Scale Plasma Profiling in Cardiovascular Disease.

Plasma biomarkers that reflect molecular states of the cardiovascular system are central for clinical decision making. Routinely used plasma biomarkers include troponins, natriuretic peptides, and lipoprotein particles, yet interrogate only a modest subset of pathways relevant to cardiovascular disease. Systematic profiling of a larger portion of circulating plasma proteins (the plasma proteome) will provide opportunities for unbiased discovery of novel markers to improve diagnostic or predictive accuracy. In addition, proteomic profiling may inform pathophysiological understanding and point to novel therapeutic targets. Obstacles for comprehensive proteomic profiling include the immense size and structural heterogeneity of the proteome, and the broad range of abundance levels, as well. Proteome-wide, untargeted profiling can be performed in tissues and cells with tandem mass spectrometry. However, applications to plasma are limited by the need for complex preanalytical sample preparation stages limiting sample throughput. Multiplexing of targeted methods based on capture and detection of specific proteins are therefore receiving increasing attention in plasma proteomics. Immunoaffinity assays are the workhorse for measuring individual proteins but have been limited for proteomic applications by long development times, cross-reactivity preventing multiplexing, specificity issues, and incomplete sensitivity to detect proteins in the lower range of the abundance spectrum (below picograms per milliliter). Emerging technologies to address these issues include nucleotide-labeled immunoassays and aptamer reagents that can be automated for efficient multiplexing of thousands of proteins at high sample throughput, coupling of affinity capture methods to mass spectrometry for improved specificity, and ultrasensitive detection systems to measure low-abundance proteins. In addition, proteomics can now be integrated with modern genomics tools to comprehensively relate proteomic profiles to genetic variants, which may both influence binding of affinity reagents and serve to validate the target specificity of affinity assays. The application of deep quantitative proteomic profiling to large cohorts has thus become increasingly feasible with emerging affinity methods. The aims of this article are to provide the broad readership of Circulation with a timely overview of emerging methods for affinity proteomics and recent progress in cardiovascular medicine based on such methods.

Proteomic Approaches to Biomarker Discovery in Cutaneous T-Cell Lymphoma.

Cutaneous T-cell lymphoma (CTCL) is the most frequently encountered type of skin lymphoma in humans. CTCL encompasses multiple variants, but the most common types are mycosis fungoides (MF) and Sezary syndrome (SS). While most cases of MF run a mild course over a period of many years, other subtypes of CTCL are very aggressive. The rapidly expanding fields of proteomics and genomics have not only helped increase knowledge concerning the carcinogenesis and tumor biology of CTCL but also led to the discovery of novel markers for targeted therapy. Although multiple biomarkers linked to CTCL have been known for a relatively long time (e.g., CD25, CD45, CD45RA, and CD45R0), compared to other cancers (lymphoma, melanoma, colon carcinoma, head and neck cancer, renal cancer, and cutaneous B-cell lymphoma), information about the antigenicity of CTCL remains relatively limited and no dependable protein marker for CTCL has been discovered. Considering the aggressive nature of some types of CTCL, it is necessary to identify circulating molecules that can help in the early diagnosis, differentiation from inflammatory skin diseases (psoriasis, nummular eczema), and aid in predicting the prognosis and evolution of this pathology. This review aims to bring together some of the information concerning protein markers linked to CTCL, in an effort to further the understanding of the convolute processes involved in this complex pathology.

Kidney tissue proteomics reveals regucalcin downregulation in response to diabetic nephropathy with reflection in urinary exosomes

Diabetic nephropathy (DN) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease. DN progresses silently and without clinical symptoms at early stages. Current noninvasive available markers as albuminuria account with severe limitations (late response, unpredictable prognosis, and limited sensitivity). Thus, it urges the discovery of novel markers to help in diagnosis and outcome prediction. Tissue proteomics allows zooming-in where pathophysiological changes are taking place. We performed a differential analysis of renal tissue proteome in a rat model of early DN by 2-dimensional differential gel electrophoresis and mass spectrometry. Confirmation was performed by Western blot, immunohistochemistry (IHC), and selected reaction monitoring (SRM). Rat urine samples were collected and exosomes were isolated from urine to evaluate if these microvesicles reflect changes directly occurring at tissue level. The protein showing maximum altered expression in rat tissue in response to DN was further analyzed in human kidney tissue and urinary exosomes. Regucalcin protein or senescence marker protein-30 (SMP30) (Swiss-Prot Q03336) was found to be strongly downregulated in DN kidney tissue compared with healthy controls. The same trend was observed in exosomes isolated from urine of control and DN rats. These data were further confirmed in a pilot study with human samples. IHC revealed a significant decrease of regucalcin in human kidney disease tissue vs control kidney tissue, and regucalcin was detected in exosomes isolated from healthy donors' urine but not from kidney disease patients. In conclusion, regucalcin protein expression is reduced in DN kidney tissue and this significant change is reflected in exosomes isolated from urine. Urinary exosomal regucalcin represents a novel tool, which should be explored for early diagnosis and progression monitoring of diabetic kidney disease.