Search

The outcome of patients with diffuse large B-cell lymphoma (DLBCL) with central nervous system (CNS) recurrence is poor. However, there is currently no consensus regarding diagnostic techniques. The aim of the present study was to investigate the cerebrospinal fluid (CSF) protein profile of DLBCL and identify a potential novel method for the early diagnosis of patients with DLBCL at high risk for subsequent CNS involvement. The CSF proteomic profiling of patients with DLBCL and a control group were compared using label-free liquid chromatography-tandem mass spectrometry. Gene Ontology and pathway analyses were conducted using the Database for Annotation, Visualization and Integrated Discovery. The protein interactions were analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. In the present study, a total of 53 differentially expressed proteins with >1 log2 fold change (false discovery rate

Genetic biomarkers for the diagnosis of ankylosing spondylitis (AS) remain unreported except for human leukocyte antigen B27 (HLA‑B27). Therefore, the aim of the present study was to screen the differentially expressed genes (DEGs), and those that also possess differential single nucleotide polymorphism (SNP) loci in the whole blood of AS patients compared with healthy controls by integrating two mRNA expression profiles (GSE73754 and GSE25101) and SNP microarray data (GSE39428) collected from the Gene Expression Omnibus (GEO). Using the t‑test, 1,056 and 1,073 DEGs were identified in the GSE73754 and GSE25101 datasets, respectively. Among them, 234 DEGs were found to be shared in both datasets, which were subsequently overlapped with 122 differential SNPs of genes in the GSE39428 dataset, resulting in identification of two common genes [eukaryotic translation elongation factor 1 epsilon 1 (EEF1E1) and serpin family A member 1 (SERPINA1)]. Their expression levels were significantly upregulated and the average expression log R ratios of SNP sites in these genes were significantly higher in AS patients than those in controls. Function enrichment analysis revealed that EEF1E1 was involved in AS by influencing the aminoacyl‑tRNA biosynthesis, while SERPINA1 may be associated with AS by participating in platelet degranulation. However, only the genotype and allele frequencies of SNPs (rs7763907 and rs7751386) in EEF1E1 between AS and controls were significantly different between AS and the controls, but not SERPINA1. These findings suggest that EEF1E1 may be an underlying genetic biomarker for the diagnosis of AS.

Cardiomyopathy is a disease of the heart muscle with varying etiologies and leads to heart failure. The pathways altered in the three forms of cardiomyopathy are not very clearly understood and hence in this study we attempted to identify differentially expressed proteins and pathways that are altered in the plasma of dilated, hypertrophic, and restrictive cardiomyopathy patients. For relative quantitation of the proteins we used both serial window acquisition of all theoretical mass spectra (SWATH-MS) and isobaric tags for relative and absolute quantitation techniques (iTRAQ). A total of 20 samples of DCM, HCM, RCM, and controls (5 each) were analyzed using SWATH while 3 samples in each group were analyzed using iTRAQ technique. Using SWATH, we could identify approximately 300 proteins in each of the four groups of which 205 proteins were found to be common. Of these 205 common proteins, 52, 58, and 52 proteins were found to be significantly differentially expressed in DCM, HCM, and RCM groups, respectively. Using iTRAQ, we could identify only about 150–180 proteins in the three experiments of which 96 were common. Our results indicated that most of the pathways that were enriched with the differentially expressed proteins, such as complement activation, platelet degranulation, immune response, etc., were common for DCM, RCM, and HCM. However, some of the pathways were unique as well to these groups. This study suggested that label-free SWATH in conjunction with iTRAQ-based quantitative proteomics approach could identify larger number of proteins and also highlights the importance of integrating two methods to dissect the molecular pathways involved in the progression of cardiomyopathies.

Background/Aims: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic form of kidney disease. High-throughput microarray analysis has been applied for elucidating key genes and pathways associated with ADPKD. Most genetic profiling data from ADPKD patients have been uploaded to public databases but not thoroughly analyzed. This study integrated 2 human microarray profile datasets to elucidate the potential pathways and protein-protein interactions (PPIs) involved in ADPKD via bioinformatics analysis in order to identify possible therapeutic targets. Methods: The kidney tissue microarray data of ADPKD patients and normal individuals were searched and obtained from NCBI Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified, and enriched pathways and central node genes were elucidated using related websites and software according to bioinformatics analysis protocols. Seven DEGs were validated between polycystic kidney disease and control kidney samples by quantitative real-time polymerase chain reaction. Results: Two original human microarray datasets, GSE7869 and GSE35831, were integrated and thoroughly analyzed. In total, 6,422 and 1,152 DEGs were extracted from GSE7869 and GSE35831, respectively, and of these, 561 DEGs were consistent between the databases (291 upregulated genes and 270 downregulated genes). From 421 nodes, 34 central node genes were obtained from a PPI network complex of DEGs. Two significant modules were selected from the PPI network complex by using Cytotype MCODE. Most of the identified genes are involved in protein binding, extracellular region or space, platelet degranulation, mitochondrion, and metabolic pathways. Conclusions: The DEGs and related enriched pathways in ADPKD identified through this integrated bioinformatics analysis provide insights into the molecular mechanisms of ADPKD and potential therapeutic strategies. Specifically, abnormal decorin expression in different stages of ADPKD may represent a new therapeutic target in ADPKD, and regulation of metabolism and mitochondrial function in ADPKD may become a focus of future research.

Background Inflammation and coagulation are key processes in cardiovascular diseases (CVDs). The Canakinumab Anti-inflammatory Thrombosis Outcome Study trial affirmed the importance of inflammation in CVD by showing that inhibition of the interleukin (IL)-1β pathway prevents recurrent CVD. A bi-directional relationship exists between inflammation and coagulation, but the precise interaction of platelets and IL-1β-mediated inflammation is incompletely understood. We aimed to determine the inter-relationship between platelets and inflammation—and especially IL-1β—in a cohort of healthy volunteers. Methods We used data from the 500-Human Functional Genomics cohort, which consists of approximately 500 Caucasian, healthy individuals. We determined associations of plasma levels of IL-1β and other inflammatory proteins with platelet number and reactivity, the association of platelet reactivity with ex vivo cytokine production as well as the impact of genetic variations through a genome-wide association study (GWAS). Results Platelets were associated with IL-1β on different levels. First, platelet number was positively associated with plasma IL-1β concentrations (p = 8.9 × 10−9) and inversely with concentrations of α-1-anti-trypsin (p = 1.04 × 10−18), which is a known antagonist of IL-1β. Second, platelet degranulation capacity, as determined by agonist-induced P-selectin expression, was associated with ex vivo IL-1β and IL-6 production. Third, several platelet single-nucleotide polymorphisms (SNPs) were associated with cytokine production and there was a significant platelet SNP enrichment in specific biological important pathways. Finally, platelet SNPs were enriched among SNPs earlier identified in GWAS studies in blood-related diseases and immune-mediated diseases. Conclusion This comprehensive assessment of factors associated with platelet number and reactivity reinforces the important inter-relationship of platelets and IL-1β-mediated inflammation.

International audience; Purpose : Objective markers of usual diet are of interest as alternative or validating tools in nutritional epidemiology research. The main purpose of the work was to assess whether saliva protein composition can reflect dietary habits in older adults, and how type 2 diabetes impacted on the saliva-diet correlates. Methods : 214 participants were selected from two European cohorts of community-dwelling older adults (3C-Bordeaux and Seniors-ENRICA-2), using a case-control design nested in each cohort. Cases were individuals with type 2 diabetes. Dietary information was obtained using the Mediterranean Diet Adherence Screener (MEDAS). Saliva was successfully obtained from 211 subjects, and its proteome analyzed by liquid chromatography-tandem mass spectrometry. Results : The relative abundance of 246 saliva proteins was obtained across all participants. The salivary proteome differed depending on the intake level of some food groups (especially vegetables, fruits, sweet snacks and red meat), in a diabetic status-and cohort-specific manner. Gene Set Enrichment Analysis suggested that some biological processes were consistently affected by diet across cohorts, for example enhanced platelet degranulation in high consumers of sweet snacks. Minimal models were then fitted to predict dietary variables by sociodemographic, clinical and salivary proteome variables. For the food group « sweet snacks », selected salivary proteins contributed to the predictive model and improved its performance in the Seniors-ENRICA-2 cohort and when both cohorts were combined. Conclusion : Saliva proteome composition of elderly individuals can reflect some aspects of dietary patterns.

Exosomes and other small extracellular vesicles (EVs) are potential sources of cancer biomarkers. Plasma-derived EVs have not yet been studied in pediatric Hodgkin lymphoma (HL), for which predictive biomarkers of relapse are greatly needed. In this two-part proteomic study, we used two-dimensional difference gel electrophoresis (2D-DIGE) followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) to analyze EV proteins of plasma collected at diagnosis from children with nodular sclerosis HL, relapsed or not. EVs isolated using membrane affinity had radii ranging from 20 to 130 nm and contained the programmed cell death 6-interacting (ALIX) and the tumor susceptibility gene 101 (TSG101) proteins, whereas calnexin (CANX) was not detected. 2D-DIGE identified 16 spots as differentially abundant between non-relapsed and relapsed HL (|fold change| ≥ 1.5, p &lt, 0.05). LC–MS/MS identified these spots as 11 unique proteins, including five more abundant in non-relapsed HL (e.g., complement C4b, C4B, fibrinogen γ chain, FGG) and six more abundant in relapsed HL (e.g., transthyretin, TTR). Shotgun LC–MS/MS on pooled EV proteins from non-relapsed HL identified 161 proteins, including 127 already identified in human exosomes (ExoCarta data). This EV cargo included 89 proteins not yet identified in exosomes from healthy plasma. Functional interrogation by the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that the EV proteins participate in platelet degranulation and serine-type endopeptidase activity as the most significant Gene Ontology (GO) biological process and molecular function (p &lt, 0.01).

Background: Bladder cancer is one of the most prevalent cancers, accounting for 2.1% of cancer mortalities worldwide. Bladder cancer is categorized into non-muscle invasive and muscle-invasive bladder cancers. Non-muscle invasive bladder cancer (NMIBC) is the most common and widely heterogeneous type with different outcomes. Objectives: This study was designed to categorize NMIBC tumor grade based on microarray data analysis. Methods: We performed microarray data analysis using GSE7476, GSE13507, and GSE37815 in patients diagnosed with NMIBC. Differentially expressed genes (DEGs) were identified based on low-grade and high-grade NMIBC. Protein-protein interaction (PPI) network analysis was carried out, and hub genes and underlying molecular pathways were identified. Results: We observed low-grade Hub genes, including GAS6, TGFB3, TPM1, COL5A1, COL1A2, SERPING1, ACTA2, TPM2, SDC1, and A2M involved in a variety of gene ontology (GO) biological processes, while high-grade genes were involved in cell cycle and cell division. The most relevant pathways suggested for low-grade NMIBC were extracellular matrix organization, platelet degranulation, and muscle contraction. Conclusions: The identification of gene hubs and underlying pathways in several low and high-grade NMIBC samples may offer better treatment management and prognostication based on molecular profiling.

Serum lactate dehydrogenase (LDH) has been established as a prognostic indicator given its differential expression in COVID‐19 patients. However, the molecular mechanisms underneath remain poorly understood. In this study, 144 COVID‐19 patients were enrolled to monitor the clinical and laboratory parameters over 3 weeks. Serum LDH was shown elevated in the COVID‐19 patients on admission and declined throughout disease course, and its ability to classify patient severity outperformed other biochemical indicators. A threshold of 247 U/L serum LDH on admission was determined for severity prognosis. Next, we classified a subset of 14 patients into high‐ and low‐risk groups based on serum LDH expression and compared their quantitative serum proteomic and metabolomic differences. The results showed that COVID‐19 patients with high serum LDH exhibited differentially expressed blood coagulation and immune responses including acute inflammatory responses, platelet degranulation, complement cascade, as well as multiple different metabolic responses including lipid metabolism, protein ubiquitination and pyruvate fermentation. Specifically, activation of hypoxia responses was highlighted in patients with high LDH expressions. Taken together, our data showed that serum LDH levels are associated with COVID‐19 severity, and that elevated serum LDH might be consequences of hypoxia and tissue injuries induced by inflammation.

Shear-mediated platelet activation (SMPA) in the "free flow" is the net result of a range of cell mechanobiological mechanisms. Previously, we outlined three main groups of mechanisms including: 1) mechano-destruction - i.e. additive platelet (membrane) damage; 2) mechano-activation - i.e. activation of shear-sensitive ion channels and pores; and 3) mechano-transduction - i.e. "outside-in" signaling via a range of transducers. Here, we report on recent advances since our original report which describes additional features of SMPA. A clear "signature" of SMPA has been defined, allowing differentiation from biochemically-mediated activation. Notably, SMPA is characterized by mitochondrial dysfunction, platelet membrane eversion, externalization of anionic phospholipids, and increased thrombin generation on the platelet surface. However, SMPA does not lead to integrin αIIbβ3 activation or P-selectin exposure due to platelet degranulation, as is commonly observed in biochemical activation. Rather, downregulation of GPIb, αIIbβ3, and P-selectin surface expression is evident. Furthermore, SMPA is accompanied by a decrease in overall platelet size coupled with a concomitant, progressive increase in microparticle generation. Shear-ejected microparticles are highly enriched in GPIb and αIIbβ3. These observations indicate the enhanced diffusion, migration, or otherwise dispersion of platelet adhesion receptors to membrane zones, which are ultimately shed as receptor-rich PDMPs. The pathophysiological consequence of this progressive shear accumulation phenomenon is an associated dyscrasia of remaining platelets - being both reduced in size and less activatable via biochemical means - a tendency to favor bleeding, while concomitantly shed microparticles are highly prothrombotic and increase the tendency for thrombosis in both local and systemic milieu. These mechanisms and observations offer direct clinical utility in allowing measurement and guidance of the net balance of platelet driven events in patients with implanted cardiovascular therapeutic devices.

Cystinuria is one of various disorders that cause biomineralization in the urinary system, including bladder stone formation in humans. It is most prevalent in children and adolescents and more aggressive in males. There is no cure, and only limited disease management techniques help to solubilize the stones. Recurrence, even after treatment, occurs frequently. Other than a buildup of cystine, little is known about factors involved in the formation, expansion, and recurrence of these stones. This study sought to define the growth of bladder stones, guided by micro-computed tomography imaging, and to profile dynamic stone proteome changes in a cystinuria mouse model. After bladder stones developed in vivo, they were harvested and separated into four developmental stages (sand, small, medium and large stone), based on their size. Data-dependent and data-independent acquisitions allowed deep profiling of stone proteomics. The proteomic signatures and pathways illustrated major changes as the stones grew. Stones initiate from a small nidus, grow outward, and show major enrichment in ribosomal proteins and factors related to coagulation and platelet degranulation, suggesting a major dysregulation in specific pathways that can be targeted for new therapeutic options.

In managing patients with coronavirus disease 2019 (COVID‐19), early identification of those at high risk and real‐time monitoring of disease progression to severe COVID‐19 is a major challenge. We aimed to identify potential early prognostic protein markers and to expand understanding of proteome dynamics during clinical progression of the disease. We performed in‐depth proteome profiling on 137 sera, longitudinally collected from 25 patients with COVID‐19 (non‐severe patients, n = 13; patients who progressed to severe COVID‐19, n = 12). We identified 11 potential biomarkers, including the novel markers IGLV3‐19 and BNC2, as early potential prognostic indicators of severe COVID‐19. These potential biomarkers are mainly involved in biological processes associated with humoral immune response, interferon signalling, acute phase response, lipid metabolism, and platelet degranulation. We further revealed that the longitudinal changes of 40 proteins persistently increased or decreased as the disease progressed to severe COVID‐19. These 40 potential biomarkers could effectively reflect the clinical progression of the disease. Our findings provide some new insights into host response to SARS‐CoV‐2 infection, which are valuable for understanding of COVID‐19 disease progression. This study also identified potential biomarkers that could be further validated, which may support better predicting and monitoring progression to severe COVID‐19.

We aim to establish a comprehensive COVID-19 autoantigen atlas in order to understand autoimmune diseases caused by SARS-CoV-2 infection. Based on the unique affinity between dermatan sulfate and autoantigens, we identified 348 proteins from human lung A549 cells, of which 198 are known targets of autoantibodies. Comparison with current COVID data identified 291 proteins that are altered at protein or transcript level in SARS-CoV-2 infection, with 191 being known autoantigens. These known and putative autoantigens are significantly associated with viral replication and trafficking processes, including gene expression, ribonucleoprotein biogenesis, mRNA metabolism, translation, vesicle and vesicle-mediated transport, and apoptosis. They are also associated with cytoskeleton, platelet degranulation, IL-12 signaling, and smooth muscle contraction. Host proteins that interact with and that are perturbed by viral proteins are a major source of autoantigens. Orf3 induces the largest number of protein alterations, Orf9 affects the mitochondrial ribosome, and they and E, M, N, and Nsp proteins affect protein localization to membrane, immune responses, and apoptosis. Phosphorylation and ubiquitination alterations by viral infection define major molecular changes in autoantigen origination. This study provides a large list of autoantigens as well as new targets for future investigation, e.g., UBA1, UCHL1, USP7, CDK11A, PRKDC, PLD3, PSAT1, RAB1A, SLC2A1, platelet activating factor acetylhydrolase, and mitochondrial ribosomal proteins. This study illustrates how viral infection can modify host cellular proteins extensively, yield diverse autoantigens, and trigger a myriad of autoimmune sequelae. Our work provides a rich resource for studies into “long COVID” and related autoimmune sequelae.

BACKGROUND CONTEXT: Patients with modic changes (MC) form a distinct clinical subset with reports of higher intensity of pain, poor clinical and surgical outcomes and higher incidence of recurrence. MC also is an independent risk factor for increased post-operative surgical site infection.PURPOSE: This study aimed to investigate the biological changes at molecular level, in discs with MCs. We also aim to identify biological biomarkers and potential targets for molecular therapy.STUDY DESIGN: Experimental analysisMATERIALS AND METHODS: Nucleus pulposus (NP) from 24 patients undergoing microdiscectomy for disc herniation [14 discs with MC and 10 without modic changes (NMC)] were procured. The overall expression of proteins, biological processes, protein-protein and metabolite interactions were analysed and compared. Host defense response proteins (HDRPs) and immunological pathways activated in patients with MC were documented and analysed.RESULTS: Label-free proteomic approach with stringent filters revealed a total of 208 proteins in MC and 193 in NMC groups. 45 proteins were specific to MC; 30 to NMC and 163 common to both. Downregulated proteins in MC belonged to components of extracellular matrix such as collagens (COL-6A1, 6A2, 6A3, 11A1, 12A1, and 20A1), and proteoglycans (versican (VCAN), and biglycan (BGN)). Inflammatory molecules [plasminogen (PLG), angiogenin (ANG), fibroblast growth factor-binding protein 2 (FGFBP2), tetranectin (CLEC3B), cartilage acidic protein 1 (CRTAC1), kininogen (KNG-1), chitinase-3-like protein 2 (CHI3L2), and ferritin (FTL) were expressed only in the MC group. The significantly altered pathways in MC included Fc Fragment of IgG Receptor IIIa (FCGR3A)-mediated phagocytosis, regulation of Toll-like receptors (TLR) by endogenous ligand, neutrophil and platelet degranulation.50 HDRPs were identified in the study, 14 of which were specific to MC and included acute phase reactants, antimicrobial peptides, complement cascade proteins, inflammatory molecule and stress response proteins. Metabolite-protein interaction analysis revealed a significant interaction between 19 proteins, specifically involving ubiquitin mediating proteasome degradative pathway and an association with the metabolite-glutamic acid in the MC group. Accumulation of glutamic acid in MC discs was confirmed by quantitative amino acid analysis using High-performance liquid chromatography.CONCLUSION: Our study confirms that MC represents an intense inflammatory status and activation of host defense response and immunological pathways. Downstream effects leading to ubiquitin mediated proteasomal degradation of ECM proteins and the resulting metabolites such as glutamic acid could cause excessive pain and needs further investigation.CLINICAL SIGNIFICANCE: We have documented the expression of inflammatory molecules, immune mechanisms and host defense response proteins which throw molecular insights into the pathological mechanisms of MC. Further, ubiquitin mediated proteasomal degradation and accumulation of glutamate in discs with MC might serve as targets for molecular therapy. (C) 2021 Elsevier Inc. All rights reserved.

Aberrant expression of CD43 in malignant tumors of nonhematopoietic origin such as those from lung, cervix, colon, and breast has been shown to correlate with poor prognosis, providing tumor cells with enhanced motility, anchorage-independent growth, and in vivo tumor size, while protecting the cells of NK lysis and apoptosis. To further characterize the role of CD43 in cell transformation, we tested whether interfering its expression modified the capacity of the A549 non-small cell lung cancer cells to secrete molecules contributing to malignancy. The proteomic analysis of the secretome of serum-starved A549 cells revealed that cells expressing normal levels of CD43 released significantly high levels of molecules involved in extracellular matrix organization, angiogenesis, platelet degranulation, collagen degradation, and inflammation, as compared to CD43 RNAi cells. This data reveals a novel and unexpected role for CD43 in lung cancer development, mainly in remodeling the tumor microenvironment.

Serum lactate dehydrogenase (LDH) has been established as a prognostic indicator given its differential expression in COVID-19 patients. However, the molecular mechanisms underneath remain poorly understood. In this study, 144 COVID-19 patients were enrolled to monitor the clinical and laboratory parameters over three weeks. Serum lactate dehydrogenase (LDH) was shown elevated in the COVID-19 patients on admission and declined throughout disease course, and its ability to classify patient severity outperformed other biochemical indicators. A threshold of 247 U/L serum LDH on admission was determined for severity prognosis. Next, we classified a subset of 14 patients into high- and low-risk groups based on serum LDH expression and compared their quantitative serum proteomic and metabolomic differences. The results found COVID-19 patients with high serum LDH exhibited differentially expressed blood coagulation and immune responses including acute inflammatory responses, platelet degranulation, complement cascade, as well as multiple different metabolic responses including lipid metabolism, protein ubiquitination and pyruvate fermentation. Specifically, activation of hypoxia responses was highlighted in patients with high LDH expressions. Taken together, our data showed that serum LDH levels are associated COVID-19 severity, and that elevated serum LDH might be consequences of hypoxia and tissue injuries induced by inflammation.

Platelet degranulation allows the release of large secretable pools of biologically active proteins which are critical in wound healing initiation and angiogenesis. Exosomes, which can transport a diverse suite of macromolecules, derived from amniotic epithelial cells (AEC-Exo) improve wound healing and angiogenesis. However, the underlying mechanisms are still unclear. In this investigation, we performed a user-friendly bioinformatics analysis system to identify association among the angiogenic and wound healing effects of AEC-Exo treatments. To this end, FunRich software was used, and linked to the Universal Protein Resource (UniProt) as a background database. Several enrichment analyses, including biological process, cellular component, molecular function, and protein domains were conducted on AEC-Exo proteome. Furthermore, to identify the proteins involved in platelet degranulation and evaluate protein–protein association information, comparative analyses and interaction network analyses were illustrated using the NCBI BioSystems, ExoCarta, and STRING databases. Our results indicated the statistically significant association between the proteome in AEC-Exo, platelet degranulation, and their corresponding processes. Therefore, the involvement of platelet degranulation in AEC-Exo proteins may elucidate the angiogenic and wound-healing effects of AEC-Exo treatments.

The accumulation of DNA and nuclear components in blood and their recognition by autoantibodies play a central role in the pathophysiology of systemic lupus erythematosus (SLE). Despite the efforts, the sources of circulating autoantigens in SLE are still unclear. Here, we show that in SLE, platelets release mitochondrial DNA, the majority of which is associated with the extracellular mitochondrial organelle. Mitochondrial release in patients with SLE correlates with platelet degranulation. This process requires the stimulation of platelet Fc gamma RIIA, a receptor for immune complexes. Because mice lack Fc gamma RIIA and murine platelets are completely devoid of receptor capable of binding IgG-containing immune complexes, we used transgenic mice expressing Fc gamma RIIA for our in vivo investigations. Fc gamma RIIA expression in lupus-prone mice led to the recruitment of platelets in kidneys and to the release of mitochondria in vivo. Using a reporter mouse with red fluorescent protein targeted to the mitochondrion, we confirmed platelets as a source of extracellular mitochondria driven by Fc gamma RIIA and its cosignaling by the fibrinogen receptor alpha 2b beta 3 in vivo. These findings suggest that platelets might be a key source of mitochondrial antigens in SLE and might be a therapeutic target for treating SLE.

This study used established biomarkers of death from ischemic stroke (IS) versus stroke survival to perform network, enrichment, and annotation analyses. Protein-protein interaction (PPI) network analysis revealed that the backbone of the highly connective network of IS death consisted of IL6, ALB, TNF, SERPINE1, VWF, VCAM1, TGFB1, and SELE. Cluster analysis revealed immune and hemostasis subnetworks, which were strongly interconnected through the major switches ALB and VWF. Enrichment analysis revealed that the PPI immune subnetwork of death due to IS was highly associated with TLR2/4, TNF, JAK-STAT, NOD, IL10, IL13, IL4, and TGF-β1/SMAD pathways. The top biological and molecular functions and pathways enriched in the hemostasis network of death due to IS were platelet degranulation and activation, the intrinsic pathway of fibrin clot formation, the urokinase-type plasminogen activator pathway, post-translational protein phosphorylation, integrin cell-surface interactions, and the proteoglycan-integrin extracellular matrix complex (ECM). Regulation Explorer analysis of transcriptional factors shows: (a) that NFKB1, RELA and SP1 were the major regulating actors of the PPI network; and (b) hsa-mir-26-5p and hsa-16-5p were the major regulating microRNA actors. In conclusion, prevention of death due to IS should consider that current IS treatments may be improved by targeting VWF, the proteoglycan-integrin-ECM complex, TGF-β1/SMAD, NF-κB/RELA and SP1.

The atypical pneumonia (COVID-19) caused by SARS-CoV-2 is a serious threat to global public health. However, early detection and effective prediction of patients with mild to severe symptoms remain challenging. The proteomic profiling of urine samples from healthy individuals, mild and severe COVID-19 positive patients with comorbidities can be clearly differentiated. Multiple pathways have been compromised after the COVID-19 infection, including the dysregulation of complement activation, platelet degranulation, lipoprotein metabolic process and response to hypoxia. This study demonstrates the COVID-19 pathophysiology related molecular alterations could be detected in the urine and the potential application in auxiliary diagnosis of COVID-19.

Background Immune checkpoint inhibitors have improved clinical responses and overall survival for patients with non-small cell lung cancer (NSCLC). However, the response is not equal and known NSCLC biomarkers are not sufficient in predicting therapy outcome. Deep proteomic analysis of NSCLC patient‘s plasma treated with anti-PD-1-blockade using a state-of-the-art data independent acquisition mass spectrometry (DIA-MS) is a powerful and unbiased way of identifying protein signatures associated with disease stage or response to treatment. However, to unravel these associations large-scale omics data should be analyzed with respect to available clinical information. To achieve this goal, we have used an approach previously applied by Uhlen et al., 20171 for transcriptomic datasets. In this approach survival data is used to set the most optimal thresholds for candidate biomarkers. Methods 125 plasma samples were analyzed by capillary flow liquid chromatography coupled to DIA-MS. Data were extracted with latest SpectronautTM and proteins were quantified. Each recorded protein intensity was used as a threshold for two groups of samples for which Kaplan-Meier estimates were generated using ‘survival’2 package in R. Benjamini-Hochberg correction was applied and p-values with corresponding intensity cut-offs were extracted to generate panels of potential biomarkers. Results 125 plasma samples (in total 75 baseline and 50 after 8-weeks treatment) from advanced NSCLC patients treated with an anti-PD-1 inhibitor following at least 1 prior line of treatment were analyzed. 727 unique proteins were quantified across all samples. Data analysis was performed separately for each line of treatment and treatment status resulting in more than 100’000 p-values. For each group, panels of proteins with best performance in separating progression free survivals were defined at FDR of 0.10, giving 64 unique proteins which were mapped to acute phase response, platelet degranulation and complement activation. Several of these proteins were listed in the Early Detection Research Network database of the National Cancer Institute, and one of them – LYPD3, was a potential therapeutic target in a preclinical study for NSCL treatment.3 Selected proteins were then used to cluster patients into cohorts that showed association with the response to therapy. Conclusions Deep proteomic profiling of plasma samples using DIA-MS in conjunction with clinical outcome enables a holistic and stringent analysis of potential circulating biomarkers. Such analysis generates functional insights into the plasma proteome that enable deeper understanding and comprehensive integration of clinical data with proteomics markers at different disease stages and treatment phases. References Uhlen M, Zhang C, Lee S, Sjostedt E, Fagerberg L, Bidkhori G, Benfeitas R, Arif M, Liu Z, Edfors F, Sanli K, von Feilitzen K, Oksvold P, Lundberg E, Hober S, Nilsson P, Mattsson J, Schwenk J. Therneau TM, Grambsch PM. Modeling Survival Data: Extending the Cox Model. Springer. 2000, New York, ISBN 0-387-98784-3. Willuda J, Linden L, Lerchen H, Kopitz C, Stelte-Ludwig B, Pena C, Lange C, Golfier S, Kneip C, Carrigan P E, Mclean K, Schuhmacher J, von Ahsen O, Muller J, Dittmer F, Beier R, El Sheikh S, Tebbe J, Leder G, Apeler H, Jautelat R, Ziegelbauer K, Kreft B, Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer. Mol Caner Ther 2017;16(5):893–904

Envenomation by viperid snakes may lead to severe bleeding, consumption coagulopathy, and thrombotic microangiopathy symptoms. The exact etiology or toxins responsible for thrombotic microangiopathy symptoms after snake envenomation remain obscure. Snake C-type lectin-like proteins (snaclecs) are one of the main non-enzymatic protein constituents in viper venoms, of which a majority are considered as modulators of thrombosis and hemostasis. In this study, we demonstrated that two snaclecs (mucetin and stejnulxin), isolated and identified from Protobothrops mucrosquamatus and Trimeresurus stejnegeri venoms, directly induced platelet degranulation and clot-retraction in vitro, and microvascular thrombosis has been confirmed in various organs in vivo. These snaclecs reduced cerebral blood flow and impaired motor balance and spatial memories in mice, which partially represent the thrombotic microangiopathy symptoms in some snakebite patients. The functional blocking of these snaclecs with antibodies alleviated the viper venom induced platelet activation and thrombotic microangiopathy-like symptoms. Understanding the pathophysiology of thrombotic microangiopathy associated with snake envenoming may lead to emerging therapeutic strategies.

Background aims Platelet-rich plasma (PRP) and bone marrow aspirate are commonly used in orthobiologics for their anti-inflammatory, anabolic/regenerative and immunomodulatory characteristics via platelet degranulation and cell secretions. Although platelets are derived from megakaryocytes in the bone marrow, no attention has been paid to the potential benefits of bone marrow platelets and whether their contents differ from aging platelets in peripheral blood. Methods In the present study, leukocyte-poor peripheral blood-derived platelets in plasma (LPP) and leukocyte-poor bone marrow platelets in plasma (BMP) were prepared from six donors, activated with calcium chloride, incubated and sampled at day 0, day 3 and day 6. LPP and BMP are platelet preparations intended to evaluate the respective platelet secretomes but are not classified as conventional PRPs, as they are not concentrated to the extent necessary to meet the qualifying criteria. At each time point, 15 growth and immunomodulatory factors were quantitated in LPP and BMP: platelet-derived growth factor AA, basic fibroblast growth factor/fibroblast growth factor 2, granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, macrophage colony-stimulating factor, stem cell factor, vascular endothelial growth factor, tumor necrosis factor alpha, IL-1β, interferon gamma, IL-4, IL-10, IL-1 receptor antagonist protein, IL-12p40 and arginase-1. Results The results illustrate that platelets derived from bone marrow have a unique secretome profile compared with those derived from peripheral blood, with significant differences in anti-inflammatory cytokines, which are associated with monocyte polarization. Conclusions Ultimately, bone marrow-derived platelets may be useful as a stand-alone orthobiologic or as an effective adjuvant to autologous cell therapies where anti-inflammatory and anabolic processes are desired, especially with respect to monocyte function.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the developed world, yet no pharmacological therapy exists. Here, we hypothesize that the integration of multiple omic data represents an approach towards unveiling novel molecular networks in CAVD. Databases were searched for CAVD omic studies. Differentially expressed molecules from calcified and control samples were retrieved, identifying 32 micro RNAs (miRNA), 596 mRNAs and 80 proteins. Over-representation pathway analysis revealed platelet degranulation and complement/coagulation cascade as dysregulated pathways. Multi-omics integration of overlapping proteome/transcriptome molecules, with the miRNAs, identified a CAVD protein&ndash, protein interaction network containing seven seed genes (apolipoprotein A1 (APOA1), hemoglobin subunit &beta, (HBB), transferrin (TF), &alpha, 2-macroglobulin (A2M), transforming growth factor &beta, induced protein (TGFBI), serpin family A member 1 (SERPINA1), lipopolysaccharide binding protein (LBP), inter-&alpha, trypsin inhibitor heavy chain 3 (ITIH3) and immunoglobulin &kappa, constant (IGKC)), four input miRNAs (miR-335-5p, miR-3663-3p, miR-21-5p, miR-93-5p) and two connector genes (amyloid beta precursor protein (APP) and transthyretin (TTR)). In a metabolite&ndash, gene&ndash, disease network, Alzheimer&rsquo, s disease exhibited the highest degree of betweenness. To further strengthen the associations based on the multi-omics approach, we validated the presence of APP and TTR in calcified valves from CAVD patients by immunohistochemistry. Our study suggests a novel molecular CAVD network potentially linked to the formation of amyloid-like structures. Further investigations on the associated mechanisms and therapeutic potential of targeting amyloid-like deposits in CAVD may offer significant health benefits.

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [18F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [18F]FDG activity as a surrogate for lung inflammation. Our linear mixed-effects models required that for each individual the slope of the line of [18F]FDG data in the outcome and the slope of the peripheral blood transcript expression data correlate, i.e., the slopes of the outcome and explanatory variables had to be similar. Of 10,295 genes that changed as a function of time, we identified 639 genes whose expression profiles correlated with decreasing [18F]FDG uptake levels in the lungs. Gene enrichment over-representation analysis revealed that numerous biological processes were significantly enriched in the 639 genes, including several well known in TB transcriptomics such as platelet degranulation and response to interferon gamma, thus validating our novel approach. Others not previously associated with TB pathobiology included smooth muscle contraction, a set of pathways related to mitochondrial function and cell death, as well as a set of pathways connecting transcription, translation and vesicle formation. We observed up-regulation in genes associated with B cells, and down-regulation in genes associated with platelet activation. We found 254 transcription factor binding sites to be enriched among the 639 gene promoters. In conclusion, we demonstrated that of the 10,295 gene expression changes in peripheral blood, only a subset of 639 genes correlated with inflammation in the lungs, and the enriched pathways provide a description of the biology of resolution of lung inflammation as detectable in peripheral blood. Surprisingly, resolution of PTB inflammation is positively correlated with smooth muscle contraction and, extending our previous observation on mitochondrial genes, shows the presence of mitochondrial stress. We focused on pathway analysis which can enable therapeutic target discovery and potential modulation of the host response to TB.

Background: Hepatocellular carcinoma (HCC) is the cause of an overwhelming number of cancer-related deaths across the world. Developing precise and noninvasive biomarkers is critical for diagnosing HCC. Our research was designed to explore potentially useful biomarkers of host peripheral blood mononuclear cell (PBMC) in HCC by integrating comprehensive bioinformatic analysis. Methods: Gene expression data of PBMC in both healthy individuals and patients with HCC were extracted from the Gene Expression Omnibus (GEO) to identify differentially expressed genes (DEGs). The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to annotate the function of DEGs. Protein-protein interaction analysis was performed to screen the hub genes from DEGs. cBioportal database analysis was performed to assess the prognostic significance of hub genes. The Cancer Cell Line Encyclopedia (CCLE) and The Human Protein Atlas (HPA) database analyses were performed to confirm the expression levels of the hub genes in HCC cells and tissue. Results: A total of 95 DEGs were screened. Results of the GO analysis revealed that DEGs were primarily involved in platelet degranulation, cytoplasm, and protein binding. Results of the KEGG analysis indicated that DEGs were primarily enriched in focal adhesion. Five genes, namely, myosin light chain kinase (MYLK), interleukin 1 beta (IL1B), phospholipase D1 (PLD1), cortactin (CTTN), and moesin (MSN), were identified as hub genes. A search in the CCLE and HPA database showed that the expression levels of these hub genes were remarkably increased in the HCC samples. Survival analysis revealed that the overexpression of MYLK, IL1B, and PLD1 may have a significant effect on HCC survival. The aberrant high expression levels of MYLK, IL1B, and PLD1 strongly indicated worse prognosis in patients with HCC. Conclusions: The identified hub genes may be closely linked with HCC tumorigenicity and may act as potentially useful biomarkers for the prognostic prediction of HCC in PBMC samples.

Summary Early detection and effective treatment of severe COVID-19 patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model was validated using 10 independent patients, 7 of which were correctly classified. Targeted proteomics and metabolomics assays were employed to further validate this molecular classifier in a second test cohort of 19 COVID-19 patients, leading to 16 correct assignments. We identified molecular changes in the sera of COVID-19 patients compared to other groups implicating dysregulation of macrophage, platelet degranulation, complement system pathways, and massive metabolic suppression. This study revealed characteristic protein and metabolite changes in the sera of severe COVID-19 patients, which might be used in selection of potential blood biomarkers for severity evaluation., Graphical Abstract, Highlights • 93 proteins show differential expression in severe COVID-19 patient sera • 204 metabolites in COVID-19 patient sera correlate with disease severity • A model composed of 29 serum factors shows patient stratification potential • Pathway analysis highlights metabolic and immune dysregulation in COVID-19 patients, Proteomic and metabolomic analysis of COVID-19 sera identifies differentially expressed factors that correlate with disease severity and highlights dysregulation of multiple immune and metabolic components in clinically severe patients.

Type 1 diabetes (T1D) is one of the most common autoimmune diseases in children. Previous studies have suggested that endothelial progenitor cells (EPCs) might be engaged in the regulating of the biological processes in T1D and folic acid (FA) might be engaged in regulating EPC function. The present study has identified 716 downregulated genes and 617 upregulated genes in T1D EPC cases after treated with FA. Bioinformatics analysis has shown that these DEGs were engaged in regulating metabolic processes, cell proliferation-related processes, bone marrow development, cell adhesion, platelet degranulation, and cellular response to growth factor stimulus. Furthermore, we have conducted and identified hub PPI networks. Importantly, we have identified 6 upregulated genes (POLR2A, BDNF, CDC27, LTN1, RAB1A, and CUL2) and 8 downregulated genes (SHC1, GRIN2B, TTN, GNAL, GNB2, PTK2, TF, and TLR9) as key regulators involved in the effect of FA on endothelial progenitor cell transcriptome of patients with T1D. We think that this study could provide novel information to understand the roles of FA in regulating EPCs of T1D patients.

Macrolides and corticosteroid resistant have been reported in mycoplasma pneumoniae (MP) pneumonia (MPP). MP clearance is difficult even by sensitive antibiotics in severe MPP (SMPP). SMPP children might develop into airway remodeling even bronchiolitis/bronchitis obliterans. There is an urgent need to identify serum biomarkers indicating the progress of MPP and to discover new target drugs for the treatment of SMPP. In this study, we collected serum samples from general MPP (GMPP) and SMPP patients to perform proteomics profiling. Total 130 differentially expressed proteins with 61 up-regulated in GMPP and 69 up-regulated in SMPP were identified. Among these, FCGBP was one of the most altered protein with highest fold change. Biological enrichment analysis indicated an uncontrolled inflammation catastrophe in SMPP. In addition, complement, coagulation cascades, collagen-containing extracellular matrix and platelet degranulation pathway were enriched in both groups. KEGG analysis indicated an enriched platelet activation in SMPP. ELISA was then performed to verify the dynamic serum FCGBP expression level between other GMPP and SMPP patients. FCGBP level in SMPP was significantly higher than that in GMPP. FCGBP level in GMPP exhibited a decreased trend while SMPP showed the opposite trend during the disease course. Our study demonstrates the first proteomics characteristic of GMPP and SMPP and provides FCGBP as a new serum biomarker indicating the progress of SMPP. Further CMap analysis identified 25 drugs target for the treatment of SMPP. Among them, MTOR inhibitor, a macrolide compound and cell proliferation inhibitor, is the most promising drug targeting for the treatment of SMPP.

SUMMARYThe atypical pneumonia (COVID-19) caused by SARS-CoV-2 is an ongoing pandemic and a serious threat to global public health. The COVID-19 patients with severe symptoms account for a majority of mortality of this disease. However, early detection and effective prediction of patients with mild to severe symptoms remains challenging. In this study, we performed proteomic profiling of urine samples from 32 healthy control individuals and 6 COVID-19 positive patients (3 mild and 3 severe). We found that urine proteome samples from the mild and severe COVID-19 patients with comorbidities can be clearly differentiated from healthy proteome samples based on the clustering analysis. Multiple pathways have been compromised after the COVID-19 infection, including the dysregulation of immune response, complement activation, platelet degranulation, lipoprotein metabolic process and response to hypoxia. We further validated our finding by directly comparing the same patients’ urine proteome after recovery. This study demonstrates the COVID-19 pathophysiology related molecular alterations could be detected in the urine and the potential application of urinary proteome in auxiliary diagnosis, severity determination and therapy development of COVID-19.

Sepsis is a major threat with high mortality rate for critically ill patients. Response to pathogen infection by the host immune system is a key biological process involved in the onset and development of sepsis. Heterogeneous host genome variation, especially single nucleotide polymorphisms (SNPs), has long been suggested to contribute to differences in disease progression. However, the function of SNPs located in non-coding regions remains to be elucidated. Recently, m6A mRNA modification levels were revealed to differ at SNPs. As m6A is a crucial regulator of gene expression, these SNPs might control genes by changing the m6A level on mRNA. To investigate the potential role of m6A SNPs in sepsis, we integrated m6A-SNP and expression quantitative trait loci (eQTLs) data. Analysis revealed 15,720 m6A-cis-eQTLs and 381 m6A-trans-eQTLs associated with sepsis. We identified 1321 genes as locations of m6A-cis-eQTLs. These were enriched in platelet degranulation and Staphylococcus aureus infection pathways, which are vital for the pathophysiological process of sepsis. We conclude that m6A modification of mRNA plays a very important role in sepsis, with m6A-cis-eQTLs potentially having the most effect on individual variation in sepsis progression.

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive pulmonary and systemic inflammation. Acute exacerbations of COPD (AECOPD) are associated with acute inflammation and infection, increase in the rates of morbidity and mortality. Previous proteomic studies have focussed on identifying proteins involved in COPD pathogenesis in samples collected from the lung (e.g. lung tissue biopsies, bronchoalveolar lavage and sputum) but not from blood of patients who experienced AECOPD. In this study, plasma was analysed by two independent quantitative proteomics techniques; isobaric tag for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM) to identify differential expression of circulating proteins in patients with stable COPD (sCOPD) and AECOPD. Firstly, iTRAQ performed on pooled plasma samples from AECOPD, sCOPD, and healthy non-smoking controls (HC) revealed 15 differentially expressed proteins between the 3 groups. MRM subsequently performed on a separate cohort of AECOPD, sCOPD, and HC patients confirmed 9 proteins to be differentially expressed by AECOPD compared to HC (Afamin, alpha-1-antichymotrypsin, Apolipoprotein E, Beta-2-glycoprotein 1, Complement component C9, Fibronectin, Immunoglobulin lambda like polypeptide 5, Inter-alpha-trypsin inhibitor heavy chain H3, Leucine rich alpha-2-glycoprotein 1). Network analysis demonstrates that most of these proteins are involved in proteolysis regulation, platelet degranulation and cholesterol metabolism. In conclusion, several potential plasma biomarkers for AECOPD were identified in this study. Further validation studies of these proteins may elucidate their roles in the development of AECOPD.

Background: Hypercoagulative conditions play a key role in venous thromboembolism (VTE). Inflammation is currently linked to VTE, but the potential role of circulating microparticles and oxidative stress (OxS) must be elucidated. The aim of this study was to evaluate platelet-derived microparticles and surrogate OxS biomarkers in patients diagnosed with VTE through a case&ndash, control study. Methods: Platelet-derived microparticles (MPs), pro-thrombinase-induced clotting time assay (PiCT), phospholipids (PLPs), malondialdehyde (MDA), 4-hydroxynonenale (4-HNE), thiobarbituric acid reactive substances (TBARs), superoxide dismutase (SOD), and galectin-3 (Gal-3) were measured in VTE patients and in healthy controls. Results: PLPs, 4-HNE, TBARs, and Gal-3 were higher in VTE patients compared to controls, conversely, SOD was lower. A significant non-linear regression between OxS biomarkers and the markers of platelet degranulation was found. Conclusion: Our results suggest that OxS and platelet degranulation are concomitant pathophysiological mechanisms in VTE.

Soybean toxin (SBTX) is a protein isolated from soybean seeds and composed of two polypeptide subunits (17 and 27 kDa). SBTX has in vitro activity against phytopathogenic fungi such as Cercospora sojina, Aspergillus niger, and Penicillium herguei, and yeasts like Candida albicans, C. parapsilosis, Kluyveromyces marxiannus, and Pichia membranifaciens. The present study aimed to analyze in vitro whether SBTX causes any side effects on non-target bacterial and mammalian cells that could impede its potential use as a novel antifungal agent. SBTX at 100 mu g/mL and 200 mu g/mL did not hinder the growth of the bacteria Salmonella enterica (subspecies enterica serovar choleraesuis), Bacillus subtilis (subspecies spizizenii) and Staphylococcus aureus. Moreover, SBTX at concentrations up to 500 mu g/mL did not significantly affect the viability of erythrocytes, neutrophils, and human intestinal Caco-2 cells. To study whether SBTX could induce relevant alterations in gene expression, in vitro DNA microarray experiments were conducted in which differentiated Caco-2 cells were exposed for 24 h to 100 mu g/mL or 200 mu g/mL SBTX. SBTX up-regulated genes involved in cell cycle and immune response pathways, but downregulated genes that play a role in cholesterol biosynthesis and platelet degranulation pathways. Thus, although SBTX did not affect bacteria, nor induced cytotoxity in mammalian cells, it affected some biological pathways in the human Caco-2 cell line that warrants further investigation.

Platelet accumulation at the site of a vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood. Here we describe a novel computational model of microvascular clot formation for the detailed analysis of thrombus mechanics. We adopt a previously developed two-dimensional particle-based model focused on the thrombus shell formation and revise it to introduce the platelet agonists. Blood flow is simulated via a computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process. The new model qualitatively reproduces the enhanced thrombus formation due to dense granule secretion, in line with in vivo findings, and provides a mechanism for the thrombin confinement at the early stages of clot formation. Our calculations also predict that the release of platelet dense granules results in the additional mechanical stabilization of the inner layers of thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of a thrombus, which are expected to result in the mechanical disruptions at the later stages of clot formation.

It is generally accepted that inflammation within the CNS contributes to neurodegeneration after traumatic brain injury (TBI), but it is not clear how inflammation is initiated in the absence of infection and whether this neuroinflammation is predominantly beneficial or detrimental. We have previously found that brain-enriched glycosphingolipids within neuronal lipid rafts (NLR) induced platelet degranulation and secretion of neuro-transmitters and pro-inflammatory factors. In the present study, we compared TBI-induced inflammation and neurodegeneration in wild-type vs. St3gal5 deficient (ST3(-/-)) mice that lack major CNS-specific glycosphingolipids. After TBI, microglial activation and CNS macrophage infiltration were substantially reduced in ST3(-/-) animals. However, ST3(-/-) mice had a larger area of CNS damage with marked neuronal/axonal loss. The interaction of platelets with NLR stimulated neurite growth, increased the number of PSD95-positive dendritic spines, and intensified neuronal activity. Adoptive transfer and blocking experiments provide further that platelet-derived serotonin and platelet activating factor plays a key role in the regulation of sterile neuroinflammation,hemorrhage and neuronal plasticity after TBI.

Coronary artery disease (CAD) is a major cause of end-stage cardiac disease. Although profound efforts have been made to illuminate the pathogenesis, the molecular mechanisms of CAD remain to be analyzed. To identify the candidate genes in the advancement of CAD, microarray dataset GSE23766 was downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified, and pathway and gene ontology (GO) enrichment analyses were performed. The protein-protein interaction network was constructed and the module analysis was performed using the Biological General Repository for Interaction Datasets (BioGRID) and Cytoscape. Additionally, target genes-miRNA regulatory network and target genes-TF regulatory network were constructed and analyzed. There were 894 DEGs between male human CAD samples and female human CAD samples, including 456 up regulated genes and 438 down regulated genes. Pathway enrichment analyses revealed that DEGs (up and down regulated) were mostly enriched in the superpathway of steroid hormone biosynthesis, ABC transporters, oxidative ethanol degradation III and Complement and coagulation cascades. Similarly, geneontology enrichment analyses revealed that DEGs (up and down regulated) were mostly enriched in the forebrain neuron differentiation, filopodium membrane, platelet degranulation and blood microparticle. In the PPI network and modules (up and down regulated), MYC, NPM1, TRPC7, UBC, FN1, HEMK1, IFT74 and VHL were hub genes. In the target genes-miRNA regulatory network and target genes&mdash, TF regulatory network (up and down regulated), TAOK1, KHSRP, HSD17B11 and PAH were target genes. In conclusion, the pathway and GO ontology enriched by DEGs may reveal the molecular mechanism of CAD. Its hub and target genes, MYC, NPM1, TRPC7, UBC, FN1, HEMK1, IFT74, VHL, TAOK1, KHSRP, HSD17B11 and PAH were expected to be new targets for CAD. Our finding provided clues for exploring molecular mechanism and developing new prognostics, diagnostic and therapeutic strategies for CAD.

Purpose: Primary angle-closure glaucoma (PACG) is associated with increased intraocular pressure, optic nerve damage, and progressive vision loss, but the molecular mechanism that underpins retinal ganglion neuropathy in PACG remains poorly understood. To better understand the pathogenesis of human PACG, we performed the first comprehensive proteomic analysis of aqueous humor (AH) samples from PACG patients and matched control donors to study pathogenic alteration in AH composition in disease. Methods: High-resolution, label-free, liquid chromatography–tandem mass spectrometry-based quantitative proteomic analyses were performed in AH samples collected from PACG patients and a matched control cohort of patients with cataracts. Results: The AH proteome comprised of 1363 distinct proteins, of which more than 50% were differentially expressed in PACG (773 total; 501 up-regulated, 272 down-regulated). AH from PACG patients was enriched in atypical collagens and fibronectins, suggesting that the composition of the trabecular matrix is significantly altered in disease. Pathway and cluster analyses revealed that AH protein modulation in PACG is closely associated with biological processes including platelet degranulation, cellular import/export mechanisms, and control of protease activity. In addition, critical mediators of oxygen homeostasis and neuronal function in AH were significantly dysregulated in disease, strongly implicating oxidative stress responses in PACG-associated nerve damage. Conclusions: Altered AH proteome in human PACG indicated oxidative stress in the neuronal damage that preceded vision loss. Identifying key mediators of PACG pathology will yield new prognostic biomarkers and novel targets for future therapeutic interventions. Ministry of Education (MOE) National Medical Research Council (NMRC) This work is in part supported by grants from the Singapore Ministry of Education (MOE2014-T2-2-043 and MOE2016-T2-2-018), the National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016), and National Healthcare Group Small Innovative Grant (Grant # 13018).

Purpose Age-related macular degeneration (AMD) is associated with altered gene and protein expression in the retina. We characterize the aqueous humor (AH) proteome in AMD to gain insight into the pathogenesis of the disease and identify potential biomarkers. Methods AH was collected from age and gender matched neovascular AMD (nvAMD; n = 10) patients and controls (n = 10). AH was pooled to create two samples (nvAMD and control), followed by intensity-based label-free quantification (MS1). Functional and bioinformatic analysis were then performed. A validation set (20 controls, 15 atrophic AMD and 15 nvAMD) was tested via multiplex ELISA for nine differentially expressed proteins according to the MS1 findings. Results MS1 identified 674 proteins in the AH. 239 proteins were upregulated in nvAMD (nvAMD/control > 2, peptide tags (PT) > 2), and 86 proteins were downregulated (nvAMD/control < 0.5, PT > 2). Functional analysis of proteins upregulated in AMD demonstrated enrichment for platelet degranulation (enrichment score (ES):28.1), negative regulation of endopeptidase activity (ES:18.8), cellular protein metabolic process (ES:11.8), epidermal growth factor-like domain (ES:10.3), sushi/SCR/CCP (ES:10.1), and complement/coagulation cascades (ES:9.2). AMD protein clusters were upregulated for 3/6 (χ2 < 0.05 compared to randomization). Validation via ELISA confirmed MS1 in 2/9 proteins (Clusterin and Serpin A4, P < 0.05), while 3/9 showed differential expression between aAMD and nvAMD (Clusterin, Serpin A4, and TF P < 0.05). Receiver operating characteristic curve calculation identified the area under the curve of 0.82 for clusterin as a biomarker for distinction of AMD. Conclusions AH proteomics in AMD patients identified several proteins and functional clusters with altered expression. Further research should confirm if these proteins may serve as biomarkers or therapeutic target for the disease.

Background Papillary renal cell carcinoma (PRCC) is the most common type of renal cell carcinoma after clear cell renal cell carcinoma (ccRCC). Its pathological classification is controversial, and its molecular mechanism is poorly understood. Therefore, the identification of key genes and their biological pathways is of great significance to elucidate the molecular mechanisms of PRCC occurrence and progression. Methods The PRCC-related datasets GSE7023, GSE48352 and GSE15641 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, and gene ontology (GO) term enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. Cytoscape and STRING were used to construct the protein-protein interaction network (PPI) and perform module analysis to identify hub genes and key pathways. A heatmap of hub genes was constructed using the UCSC cancer genomics browser. Overall survival and recurrence-free survival of patients stratified by the expression levels of hub genes were analysed using Kaplan-Meier Plotter. The online database UALCAN was applied to analyse gene expression based on tissue type, stage, subtype and race. Results A total of 214 DEGs, specifically, 205 downregulated genes and 9 upregulated genes, were identified. The DEGs were mainly enriched in angiogenesis, kidney development, oxidation-reduction process, metabolic pathways, etc. The 17 hub genes identified were mainly enriched in the biological processes of angiogenesis, cell adhesion, platelet degranulation, and leukocyte transendothelial migration. Survival analysis showed that EGF, KDR, CXCL12, REN, PECAM1, CDH5, THY1, WT1, PLAU and DCN might be related to the carcinogenesis, metastasis or recurrence of PRCC. UALCAN analysis showed that low expression of PECAM1 and PLAU in PRCC tissues was related to stage, subtype and race. Conclusions The DEGs and hub genes identified in the present study provide insight into the specific molecular mechanisms of PRCC occurrence and development and may be potential molecular markers and therapeutic targets for the accurate classification and efficient diagnosis and treatment of PRCC.

When blood passes through the extracorporeal circuit during haemodialysis (HD) undesirable effects including platelet degranulation and coagulation activation take place. β-thromboglobulin (β-TG) is a sensitive marker of platelet activation. The aim of this study was to investigate platelet degranulation and coagulation activation during HD with the heparin-coated dialysis membrane HeprAN.Four HD sessions were evaluated in each of 12 chronic HD patients. None of the patients used oral warfarin, other anticoagulants or antiplatelet drugs. In the first session the HeprAN membrane or a conventional polyflux membrane was used in a randomized manner and thereafter alternately in a cross-over design, and 50% of the conventional dalteparin dose was given at start of HD. Prothrombin fragment 1 + 2 (PF1 + 2), β-TG and anti-factor Xa activity were measured repeatedly.No dialysis sessions were terminated early due to clotting of the extracorporeal system. Activation of intravascular coagulation as assessed by change in PF1 + 2 during 4 hours of HD was the same with the two membranes. β-TG concentration decreased significantly during 4 hours of HD with the HeprAN membrane but remained stable with the polyflux membrane.There were no differences in clotting scores or coagulation activation with the two membranes. The decrease in β-TG during HD with the HeprAN membrane suggests β-TG to be an inferior marker of platelet degranulation when using a heparin-coated dialysis membrane. A possible mechanism for the decline in β-TG concentration may be adherence of this heparin-binding protein to the heparin-coated dialysis membrane.

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbβ3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

Platelet-rich plasma (PRP) is blood plasma with a high concentration of autologous platelets which constitute an immense reservoir of growth factors. The clinical use of PRP is widespread in various medical applications. Although highly popular with athletes, the use of PRP for the treatment of tendinopathies remains scientifically controversial, particularly due to the diversity of products that go by the name of "PRP." To optimize its use, it is important to look at the various stages of obtaining PRP. In this literature review, we take a closer look at eight parameters which may influence the quality of PRP: 1) anticoagulants used to preserve the best platelet function, 2) the speed of centrifugation used to extract the platelets, 3) the platelet concentrations obtained, 4) the impact of the concentration of red and while blood cells on PRP actions, 5) platelet activators encouraging platelet degranulation and, hence, the release of growth factors, and 6) the use or nonuse of local anesthetics when carrying out infiltration. In addition to these parameters, it may be interesting to analyze other variables such as 7) the use of ultrasound guidance during the injection with a view to determining the influence they have on potential recovery.

A 71 year-old woman is admitted to Le Mans hospital center for management of a chronic skin lesion. She has no personal nor familial bleeding history and does not take any medication. In peripheral blood collected with EDTA (ethylene diamine tetra-acetate), the platelet count is elevated and the blood film shows uniformly grey platelets. In sodium citrate-collected blood, platelets show no abnormality. We describe an EDTA-related artifact that is not to be mistaken for grey platelet syndrome.

Familial hemophagocytic lymphohistiocytosis (FHL) is caused by biallelic variants in genes regulating granule secretion in cytotoxic lymphocytes. In FHL3-5, the affected genes UNC13D, STX11 and STXBP2 have further been shown to regulate the secretion of platelet granules, giving rise to compromised platelet function. Therefore, we aimed to investigate platelet degranulation in patients heterozygous for variants in UNC13D, STX11 and STXBP2. During the work-up of patients referred to the Coagulation Unit, Skåne University Hospital, Malmö, Sweden and the Department of Hematology, Rigshospitalet, Copenhagen, Denmark due to bleeding tendencies, 12 patients harboring heterozygous variants in UNC13D, STX11 or STXBP2 were identified using targeted whole exome sequencing. Transmission electron microscopy (TEM) was used to assess the secretion of platelet dense granules following thrombin stimulation. Platelet degranulation, activation and aggregation were further assessed by flow cytometry (FC) and light transmission aggregometry (LTA) with lumi-aggregometry. In total, eight out of twelve (67%) patients showed impaired degranulation by at least one of the assays (TEM, FC and LTA). In the 12 patients, eight different heterozygous variants were identified. One variant was strongly associated with impaired degranulation, while four of the variants were associated with impaired granule secretion to a slightly lesser extent. One additional variant was found in six out of the twelve patients, and was associated with varying degrees of degranulation impairment. Accordingly, six out of the eight (75%) identified variants were associated with impaired platelet degranulation. Our results suggest that heterozygous variants in UNC13D, STX11 and STXBP2 are sufficient to cause platelet secretion defects resulting in increased bleeding.

In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.Amaç: Son on yıl içerisinde, dondurulup saklanan trombositlerin özellikle travma hastalarında kullanımı ile ilgili önemli bir bilgi birikimi oluşmuştur. Bununla birlikte bu çalışmalarda trombositlerin morfolojik ve fonksiyonel değişikliklerinden çok az bahsedilmektedir. Son zamanlarda dondurulup saklanan trombositlerin aktive olarak trombosit kaynaklı mikropartikül (TKM) oluşumu ve trombosit kaynaklı büyüme faktörü (TKBF) salınımı ile sonuçlanan prokoagulan membran değişiklikleri olduğu saptanmıştır. Çalışmamızda dondurulup saklanan trombositlerin canlılıkları, TKM ve TKBF düzeyleri incelenerek trombin olumuyla ilişkileri değerlendirildi. Gereç ve Yöntemler: Yirmi bağışçıdan alınan aferez trombosit süspansiyonları (ATS) bir gün bekletildikten sonra %6 dimetil sülfoksid ile dondurularak -80 °C’de bir gün saklandı. Trombositler eritildikten sonra 20 mL. otolog, plazma ile seyreltildi ve alınan örneklerden incelemeler yapıldı. Bulgular: Dondurulup çözülmüş ATS’lerdeki TKM seviyeleri tazeATS’lerdekinden anlamlı düzeyde daha yüksekti (2763±399,4/µL ve 319,9±80,5/µL; p0,001). Dondurulup çözülmüş ATS’lerdeki PDGF seviyeleri de taze ATS’lerdekinden anlamlı düzeyde daha yüksekti (550,9±73,6 pg/mL ve 96,5±49 pg/mL; p0,001). Bununla birlikte dondurulup çözülmüş ATS’lerin canlılıkları taze ATS’lere göre anlamlı düzeyde düşüktü (68,2±13,7% ve 94±7,5%, p0,001). Dondurulup çözülmüş ATS’lerin ortalama endojen thrombin potensiyelleri (ETP) taze ATS’lerinkinden istatiksel olarak anlamlı düzeyde yüksekti (3406,1±430,4 nM.min ve 2757,6±485,7 nM.min, p0,001). Ayrıca ETP ile TKM düzeyleri arasında istatiksel olarak anlamlı zayıf pozitif korelasyon mevcuttu (r=0,192, p=0,014). Sonuç: Çalışmamızdaki sonuçlar dondurulup çözüldükten sonra ATS’lerdeki trombositlerin canlılıklarında önemli düzeyde azalma olsa da, daha erken ve daha yüksek trombin oluşumunun gerçekleştiğini ve bunun da dondurma işlemi sonrası anlamlı düzeyde artan TKM’ler ile ilişkili olarak meydana geldiğini göstermektedir. Dondurma işlemi ile trombositlerde hasarlanmalar meydana gelmektedir ve dondurulmuş trombositlerin in vivo kullanımlarının taze trombositlere göre üstünlüğü ile ilgili bilimsel kanıtlar yetersizdir. Bu sebeple dondurulmuş trombosit süspansiyonlarının travma şartlarında kullanılmalarının ve taze trombosit süspansiyonlarının ise kan bankalarında, profilaktik kullanım amacıyla bulundurulmalarının daha uygun olacağı değerlendirilmiştir.

Background/Aims: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+]i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+]i, P-selectin abundance, and active αIIbβ3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Conclusions: Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation.

SummaryPlatelet degranulation at the site of vascular injury prevents bleeding and may affect the chronic vascular wound healing response. Transforming Growth Factor (TGF)-β1 is a major component of platelet α-granules known to accumulating in thrombi. It was our aim to determine the role of TGFβ1 released from activated platelets for neointima formation following arterial injury and thrombosis. Mice with platelet-specific deletion of TGFβ1 (Plt.TGFβ-KO) underwent carotid artery injury. Immunoassays confirmed the absence of active TGFβ1 in platelet releasates and plasma of Plt.TGFβ-KO mice. Whole blood analyses revealed similar haematological parameters, and tail cut assays excluded major bleeding defects. Platelet aggregation and the acute thrombotic response to injury in vivo did not differ between Plt.TGFβ-KO and Plt.TGFβ-WT mice. Morphometric analysis revealed that absence of TGFβ1 in platelets resulted in a significant reduction of neointima formation with lower neointima area, intima-to-media ratio, and lumen stenosis. On the other hand, the media area was enlarged in mice lacking TGFβ1 in platelets and contained increased amounts of proteases involved in latent TGFβ activation, including MMP2, MMP9 and thrombin. Significantly increased numbers of proliferating cells and cells expressing the mesenchymal markers platelet-derived growth factor receptor-β or fibroblast-specific protein-1, and the macrophage antigen F4/80, were observed in the media of Plt.TGFβ-KO mice, whereas the medial smooth muscle-actin-immuno-positive area and collagen content did not differ between genotypes. Our findings support an essential role for platelet-derived TGFβ1 for the vascular remodelling response to arterial injury, apparently independent from the role of platelets in thrombosis or haemostasis.Supplementary Material to this article is available online at www.thrombosis-online.com.