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Background. Hepatic fibrosis is a severe liver disease that has threatened human health for a long time. In order to undergo timely and adequate therapy, it is important for patients to obtain an accurate diagnosis of fibrosis. Laboratory inspection methods have been efficient in distinguishing between advanced hepatic fibrosis stages (F3, F4), but the identification of early stages of fibrosis has not been achieved. The development of proteomics may provide us with a new direction to identify the stages of fibrosis. Methods. We established serum proteomic maps for patients with hepatic fibrosis at different stages and identified differential expression of proteins between fibrosis stages through ultra-high-performance liquid chromatography tandem mass spectrometry proteomic analysis. Results. From the proteomic profiles of the serum of patients with different stages of liver fibrosis, a total of 1,338 proteins were identified. Among three early fibrosis stages (control, F1, and F2), 55 differential proteins were identified, but no proteins simultaneously exhibited differential expression between control, F1, and F2. Differential proteins were detected in the comparison between different fibrosis stages. Significant differences were found between advanced fibrosis stages (F2-vs.-F3 and F3-vs.-F4) through a series of statistical analysis, including hierarchical clustering, Gene Ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes pathway, and protein-protein interaction network analysis. The differential proteins identified by GO annotation were associated with biological processes (mainly platelet degranulation and cell adhesion), molecular functions, and cellular components. Conclusions. All potential biomarkers identified between the stages of fibrosis could be key points in determining the fibrosis staging. The differences between early stages may provide a useful reference in addressing the challenge of early fibrosis staging.

Background The aim of this study was to apply proteomic methodology for the analysis of proteome changes in women with polycystic ovary syndrome (PCOS). Material and methods All the participators including 31 PCOS patients and 31 healthy female as controls were recruited, the clinical characteristics data was recorded at the time of recruitment, the laboratory biochemical data was detected. Then, a data-independent acquisition (DIA)-based proteomics method was performed to compare the serum protein changes between PCOS patients and controls. In addition, Western blotting was used to validate the expression of identified proteomic biomarkers. Results There were 80 proteins differentially expressed between PCOS patients and controls significantly, including 54 downregulated and 26 upregulated proteins. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that downregulated proteins were enriched in platelet degranulation, cell adhesion, cell activation, blood coagulation, hemostasis, defense response and inflammatory response terms; upregulated proteins were enriched in cofactor catabolic process, hydrogen peroxide catabolic process, antioxidant activity, cellular oxidant detoxification, cellular detoxification, antibiotic catabolic process and hydrogen peroxide metabolic process. Receiver operating characteristic curves analysis showed that the area under curve of Histone H4 (H4), Histone H2A (H2A), Trem-like transcript 1 protein (TLT-1) were all over than 0.9, indicated promising diagnosis values of these proteins. Western blotting results proved that the detected significant proteins, including H4, H2A, TLT-1, Peroxiredoxin-1, Band 3 anion transport protein were all differently expressed in PCOS and control groups significantly. Conclusion These proteomic biomarkers provided the potentiality to help us understand PCOS better, but future studies comparing systemic expression and exact role of these candidate biomarkers in PCOS are essential for confirmation of this hypothesis.

Cheng Zhou,1 Chunlin Song,1 Xiang Huang,1 Shufen Chen,1 Yan Long,2 Shanshui Zeng,2 Hongling Yang,2 Min Jiang2 1Laboratory of Molecular Diagnostics, Southern Medical University Affiliated Maternal & Child Health Hospital of Foshan, Foshan, 528000, People’s Republic of China; 2Department of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, Guangzhou, 510623, People’s Republic of ChinaCorrespondence: Hongling Yang; Min JiangDepartment of Laboratory, Guangzhou Women and Children’s Medical Centre, Guangzhou Medical University, No. 9, Jinsui Road, Guangzhou, 510623, People’s Republic of ChinaTel +86-20-38857723; +86-20-38076256Email hlyang62@163.com; jiangmin2011@126.comBackground: Gestational hypertension (GH), a hypertensive disorder of pregnancy (HDP), is a leading cause of maternal and fetal mortality due to the lack of clarity on its exact etiology and clinically feasible prediction models. This study was performed to discover novel biomarkers before 20 weeks gestation and thereby construct an early GH prediction model.Methods: This study was designed based on differentially expressed protein screening followed by clinical validation. In the screening phase, a nested case-controlled study was conducted by plasma proteomic analyses using label-free LC-MS/MS and plasma samples from seven pre-GH cases before 20-week gestation and seven age- and gestational week-matched controls. In the validation phase, 10 proteins with differential expression in the screening phase were validated by ELISA or electrochemiluminescence in an independent study consisting of 29 pre-GH cases before 20-week gestation and 29 matched controls.Results: In the screening phase, 149 proteins were found to be differentially expressed between the two groups and were predominantly involved in complement and coagulation cascades, platelet degranulation and positive regulation of cell motility. Further validation showed that serpin family C member 1 (SERPINC1), serpin family A member 5 (SERPINA5), complement factor H-related protein 5 (CFHR5), clusterin, cytokeratin 18 (CK18) and histidine-rich glycoprotein (HRG) levels were significantly higher in women who later developed GH compared to women with uncomplicated pregnancies (P< 0.05). Binary logistic regression analysis was used to determine the combination efficacy of models for early prediction of GH. The model with a combination of SERPINC1, CK18 and HRG had a significantly better discriminatory power (AUC = 0.91, 95% CI 0.83– 0.98) compared to the models with those proteins alone as independent predictors of GH.Conclusion: Plasma levels of SERPINC1, SERPINA5, CFHR5, clusterin, CK18 and HRG are potential novel predictive biomarkers of GH, and a prediction model using a combination of SERPINC1, CK18 and HRG has good discriminatory performance for GH before 20 weeks gestation.Keywords: label-free LC-MS/MS, gestational hypertension, biomarkers

The aim of the study was to monitor and analyse injured and diseased loggerhead sea turtles (Caretta caretta) plasma proteome profiles and biochemistry parameters during their recovery period in rescue centre within different age and recovery period groups, and determine the potential biomarkers that can be used in diagnostics. The plasma biochemical parameters of total protein and glucose content, accompanied by aspartate aminotransferase (AST) and N-acetyl-cystein-activated creatinine kinase (CK-NAC) are highlighted as valuable and potential biomarkers of turtle's health status and condition. Using high throughput tandem mass tag (TMT)-based proteomic approach we identified 913 plasma proteins, 12 of which shown to be modulated in loggerheads age groups, and identified as a part of (i) platelet degranulation, (ii) neutrophil degranulation, and (iii) innate immune system pathways. The neurofascin (NFASC) is shown to be differentially abundant among all the age groups, and alpha-1-acid glycoprotein 2-like (ORM2) and alpha-1-antitrypsin-like (SERPINA1) proteins were recognized as members of all three above mentioned REACTOME pathways. Furthermore, 29 of plasma proteins were significantly differentially abundant in loggerheads age and recovery period groups. Out of 15 recognized pathways, those proteins were mostly included in three specific REACTOME pathways: (i) post-translational phosphorylation, (ii) regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs), and (iii) platelet degranulation. The alpha-fetoprotein (AFP) was the only protein which showed statistically significant up-regulation patterns in all loggerhead age groups before release from the rescue centre, and the complement component 3 (C3) protein was the only protein modulated in all recovery period groups. Furthermore, C3 protein takes part in 9; and followed up with apolipoprotein A-I (APOA1) in 7; complement component 4 (C4), complement component 5 (C5) and kininogen-1 (KNG1) in 6 REACTOME pathways. Thereby, those proteins are highlighted and recommended as potential biomarkers of turtle's health status. Data are available via ProteomeXchange with identifier PXD029569. Finally, based on our results, we believe that comprehensive omics approach and routine plasma biochemical analysis, accompanied by proteins of acute phase, acid-base status and immune-response indicator analysis may significantly and reliably improve assessment of captive loggerheads rehabilitation and medication. SIGNIFICANCE: Monitoring and comparison of loggerhead sea turtles (C. caretta) blood plasma biochemistry parameters and plasma proteome profiles in relation to the age, and recovery period pointed out significantly differentially abundant proteins, along with certain biochemical parameter contents as potential biomarkers of turtle's fitness, health status and physiology.

Supplemental Digital Content is available in the text., Despite growing use of mechanical circulatory support, limitations remain related to hemocompatibility. Here, we performed a head-to-head comparison of the hemocompatibility of a centrifugal cardiac assist system—the Centrimag, with that of the latest generation of an intravascular microaxial system—the Impella 5.5. Specifically, hemolysis, platelet activation, microparticle (MP) generation, and von Willebrand factor (vWF) degradation were evaluated for both devices. Freshly obtained porcine blood was recirculated within device propelled mock loops for 4 hours, and alteration of the hemocompatibility parameters was monitored over time. We found that the Impella 5.5 and Centrimag exhibited low levels of hemolysis, as indicated by minor increase in plasma free hemoglobin. Both devices did not induce platelet degranulation, as no alteration of β-thromboglobulin and P-selectin in plasma occurred, rather minor downregulation of platelet surface P-selectin was detected. Furthermore, blood exposure to shear stress via both Centrimag and Impella 5.5 resulted in a minor decrease of platelet count with associated ejection of procoagulant MPs, and a decrease of vWF functional activity (but not plasma level of vWF-antigen). Greater MP generation was observed with the Centrimag relative to the Impella 5.5. Thus, the Impella 5.5 despite having a lower profile and higher impeller rotational speed demonstrated good and equivalent hemocompatibility, in comparison with the predicate Centrimag, with the advantage of lower generation of MPs.

Intimal injury is an early stage of several cardiovascular diseases. Endothelial progenitor cells (EPCs) play a significant role in endothelial repair following vascular injury. Once the intima is damaged, EPCs are mobilized from the bone marrow to the injury site. Meanwhile, the injury to the intimal surface triggers platelet degranulation, aggregation, and adhesion to the damaged endothelium, and exposed collagen stimulates platelet to secrete platelet-derived microvesicles (PMVs). However, the role of PMVs in EPC function during this process remains unknown. In an in vivo study, EPCs and platelets were found to adhere to the injury site in Sprague-Dawley (SD) rat vascular injury model. In vitro, collagen stimulation induced the release of PMVs, and collagen-activated PMVs (ac.PMVs) significantly promoted EPC proliferation. Transforming growth factor-β1 (TGF-β1) content was increased in ac.PMVs. Activated PMVs significantly upregulated Smad3 phosphorylation in EPCs and increased Smad3 nuclear translocation from the cytoplasm. TGF-β1 knockdown ac.PMVs downregulated EPC proliferation. Recombinant TGF-β1 enhanced EPC proliferation. The TGF-β1 inhibitor SB431542 significantly repressed the intracellular signal triggered by ac.PMVs. Furthermore, the Smad3-specific phosphorylation inhibitor SIS3 effectively reversed the cell proliferation induced by ac.PMVs. Smad3 translocated to the nucleus and enhanced EPC proliferation via its downstream genes tenascin C (TNC), CDKN1A, and CDKN2A. r-TGF-β1 promoted reendothelialization and EPC proliferation in vivo. Our data demonstrate that activated PMVs deliver TGF-β1 from collagen-activated platelets to EPCs, which in turn activates Smad3 phosphorylation and regulates TNC, CDKN1A, and CDKN2A expression to promote EPC proliferation, suggesting that PMVs act as a key transporter and a potential therapeutic target for vascular injury.

Dogs infected with the cardiopulmonary nematode Angiostrongylus vasorum may suffer from respiratory distress and/or bleeding disorders. Descriptions of clinical signs in foxes are rare, despite high prevalence. To evaluate the impact of infection on coagulation and immune response, serum proteins from eight experimentally infected foxes before and after inoculation (day 0, 35, 84, 154) were subjected to differential proteomic analyses based on quantitative data and compared to available data from dogs. The number of proteins with differential abundance compared to the uninfected baseline increased with chronicity of infection. Bone marrow proteoglycan, chitinase 3-like protein 1 and pulmonary surfactant-associated protein B were among the most prominently increased proteins. The abundance of several proteins involved in coagulation was decreased. Enriched pathways obtained from both increased and decreased proteins included, among others, "platelet degranulation " and "haemostasis ", and indicated both activation and suppression of coagulation. Qualitative comparison to dog data suggests some parallel serum proteomic alterations. The comparison, however, also indicates that foxes have a more adequate immunopathological response to A. vasorum infection compared to dogs, facilitating persistent infections in foxes. Our findings imply that foxes may be more tolerant to A. vasorum infection, as compared to dogs, reflecting a longer evolutionary host-parasite adaptation in foxes, which constitute a key wildlife reservoir., Pathogens, 10 (11), ISSN:2076-0817

Accelerated healing in wild or captive South American sea lions (Otaria flavescens) is a key tool to help minimize infection and complications associated with open wounds, dental disease, and ocular pathology. Platelet-rich plasma (PRP) is an autogenous source for growth factors based on platelet concentration, which can be obtained by centrifuging whole blood collected in sodium citrate anticoagulant. Currently, there are well-defined PRP concentration protocols for humans and most domestic companion animal species. However, there is no clear centrifugation protocol for obtaining PRP in most marine mammal species. This study aimed to optimize the platelet concentration protocol based on whole blood centrifugation using speeds ranging from 500 to 5,000 rpm and times ranging from 3 to 6 min. Blood was drawn from seven adult South American sea lions, placed into 1-ml sodium citrate tubes, and centrifuged following 12 different centrifugation protocols. PRP was designated as the lower third fraction of the centrifuged plasma. Platelet counts were performed using flow cytometry and statistical analysis was carried out to establish a well-defined protocol for efficient PRP production. Transmission electron microscopy (TEM) analysis was performed to evaluate possible platelet degranulation during the different centrifugation protocols and measure platelet areas. Maximum concentration of platelets in PRP was 4.73-fold higher than the number of platelets in equal volume of whole blood, and significant differences in the concentrations obtained were found between the 12 centrifugation protocols evaluated using different speed and time combinations. The best one-step centrifugation protocol resulted from using 900-rpm speed for 3 min. The highest-fold increase was achieved using a two-step centrifugation protocol, which combined the most efficient one-step centrifugation protocol (900 rpm, 3 min) with a second centrifugation using 2,000-rpm speed for 6 min. TEM analysis confirmed that platelets were complete and maintained integrity after the proposed protocol.

Adequate endometrial stromal cell (ESC) decidualization is vital for endometrial health. Given the importance of extracellular vesicles (EVs) in intercellular communication, we investigated how their protein landscape is reprogrammed and dysregulated during decidual response. Small EVs (sEVs) from human ESC-conditioned media at Day-2 and -14 following decidual stimuli were grouped as well- (WD) or poorly decidualized (PD) based on their prolactin secretion and subjected to mass spectrometry-based quantitative proteomics. On Day 2, in PD- versus WD-ESC-sEVs, 17 sEV- proteins were down-regulated (C5, C6; complement/coagulation cascades, and SERPING1, HRG; platelet degranulation and fibrinolysis) and 39 up-regulated (FLNA, COL1A1; focal adhesion, ENO1, PKM; glycolysis/gluconeogenesis, and RAP1B, MSN; leukocyte transendothelial migration). On Day 14, in PD- versus WD-ESC-sEVs, FLNA was down-regulated while 21 proteins were up-regulated involved in complement/coagulation cascades (C3, C6), platelet degranulation (SERPINA4, ITIH4), B-cell receptor signalling and innate immune response (immunoglobulins). Changes from Days 2 to 14 suggested a subsequent response in PD-ESC-sEVs with 89 differentially expressed proteins mostly involved in complement and coagulation cascades (C3, C6, C5), but no change in WD-ESC-sEVs ESC. Poor decidualization was also associated with loss of crucial sEV-proteins for cell adhesion and invasion (ITGA5, PFN1), glycolysis (ALDOA, PGK1) and cytoskeletal reorganization (VCL, RAC1). Overall, this study indicates varied ESC response even prior to decidualization and provides insight into sEVs-proteomes as a benchmark of well-decidualized ESC. It shows distinct variation in sEV-protein composition depending on the ESC decidual response that is critical for embryo implantation, enabling and limiting trophoblast invasion during placentation and sensing a healthy embryo.

Objective. To explore the therapeutic targets, network modules, and coexpressed genes of Radix Rhei Et Rhizome intervention in cerebral infarction (CI), and to predict significant biological processes and pathways through network pharmacology. To explore the differential proteins of Radix Rhei Et Rhizome intervention in CI, conduct bioinformatics verification, and initially explain the possible therapeutic mechanism of Radix Rhei Et Rhizome intervention in CI through proteomics. Methods. The TCM database was used to predict the potential compounds of Radix Rhei Et Rhizome, and the PharmMapper was used to predict its potential targets. GeneCards and OMIM were used to search for CI-related genes. Cytoscape was used to construct a protein-protein interaction (PPI) network and to screen out core genes and detection network modules. Then, DAVID and Metascape were used for enrichment analysis. After that, in-depth analysis of the proteomics data was carried out to further explore the mechanism of Radix Rhei Et Rhizome intervention in CI. Results. (1) A total of 14 Radix Rhei Et Rhizome potential components and 425 potential targets were obtained. The core components include sennoside A, palmidin A, emodin, toralactone, and so on. The potential targets were combined with 297 CI genes to construct a PPI network. The targets shared by Radix Rhei Et Rhizome and CI include ALB, AKT1, MMP9, IGF1, CASP3, etc. The biological processes that Radix Rhei Et Rhizome may treat CI include platelet degranulation, cell migration, fibrinolysis, platelet activation, hypoxia, angiogenesis, endothelial cell apoptosis, coagulation, and neuronal apoptosis. The signaling pathways include Ras, PI3K-Akt, TNF, FoxO, HIF-1, and Rap1 signaling pathways. (2) Proteomics shows that the top 20 proteins in the differential protein PPI network were Syp, Syn1, Mbp, Gap43, Aif1, Camk2a, Syt1, Calm1, Calb1, Nsf, Nefl, Hspa5, Nefh, Ncam1, Dcx, Unc13a, Mapk1, Syt2, Dnm1, and Cltc. Differential protein enrichment results show that these proteins may be related to synaptic vesicle cycle, vesicle-mediated transport in synapse, presynaptic endocytosis, synaptic vesicle endocytosis, axon guidance, calcium signaling pathway, and so on. Conclusion. This study combined network pharmacology and proteomics to explore the main material basis of Radix Rhei Et Rhizome for the treatment of CI such as sennoside A, palmidin A, emodin, and toralactone. The mechanism may be related to the regulation of biological processes (such as synaptic vesicle cycle, vesicle-mediated transport in synapse, presynaptic endocytosis, and synaptic vesicle endocytosis) and signaling pathways (such as Ras, PI3K-Akt, TNF, FoxO, HIF-1, Rap1, and axon guidance).

Human milk (HM) is the golden standard of infant nutrition that can protect immature body function and enhance nutrition metabolism to ensure infant growth. Region specificity and lactation period could change the protein composition in HM. In this research, proteomics analysis was used to compare proteomes across eight cities, namely Harbin, Lanzhou, Guangzhou, Chengdu, Jinhua, Weihai, Zhengzhou, and Beijing, which represented the northeast, northwest, southeast, southwest, east, and north and central regions of China,. Proteins varied significantly among the cities. These different proteins were mainly involved in the process of platelet degranulation, innate immune response, and triglyceride metabolic process, which might be due to different living environments. These differences also lead to variation in protection and fat metabolism from mothers to infants in different cities. Four proteins were expressed differently during 6 months of lactation, namely Dipeptidyl peptidase 1, Lysozyme C, Carbonic anhydrase 6, and Chordin-like protein 2. The changes in these proteins might be because of the change of growth needs of the infants. The findings from our results might help to improve the understanding of HM as well as to design infant formula.

Lipoprotein(a) (Lp(a)) is one of the most important risk factors for the development of calcific aortic valve stenosis (CAVS). However, the mechanisms through which Lp(a) causes CAVS are currently unknown. Our objectives were to characterize the Lp(a) proteome and to identify proteins that may be differentially associated with Lp(a) in patients with versus without CAVS. Our second objective was to identify genes that may be differentially regulated by exposure to high versus low Lp(a) levels in explanted aortic valves from patients with CAVS. We isolated Lp(a) from the blood of 21 patients with CAVS and 22 volunteers and performed untargeted label-free analysis of the Lp(a) proteome. We also investigated the transcriptomic signature of calcified aortic valves from patients who underwent aortic valve replacement with high versus low Lp(a) levels (n = 118). Proteins involved in the protein activation cascade, platelet degranulation, leukocyte migration, and response to wounding may be associated with Lp(a) depending on CAVS status. The transcriptomic analysis identified genes involved in cardiac aging, chondrocyte development, and inflammation as potentially influenced by Lp(a). Our multi-omic analyses identified biological pathways through which Lp(a) may cause CAVS, as well as key molecular events that could be triggered by Lp(a) in CAVS development.

Platelet accumulation at the site of 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 thrombus formation for detailed analysis of thrombus mechanics. Adopting a previously developed two-dimensional particle-based model focused on the thrombus shell formation, we revise it to introduce platelet agonists. Blood flow is simulated via 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 enhanced thrombus formation due to granule secretion in line with in vivo findings and provides a mechanism for thrombin confinement at the early stages of aggregate formation. Our calculations also predict that release of dense granules results in additional mechanical stabilization of the inner layers of the thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of thrombus, which are expected to result in mechanical disruptions at the later stages of thrombus formation.

We have shown that overexpression of human alpha-1 antitrypsin (hAAT) in mesenchymal stromal cells (hAAT-MSCs) improves mobility and function of MSCs. In this study we further characterized hAAT MSCs and control MSCs and compared their therapeutic effects in the prevention of type 1 diabetes in the spontaneous non-obese diabetic (NOD) mice. Bone marrow specimens from three individual donors were purchased from StemExpress. Cells were grown to passage 3-4 and infected with hAAT-containing virus for hAAT-overexpression. Single Cell RNAseq was performed by the 10X Genomic System. Proteomic Analysis of exosomes was performed at the MUSC Proteomic Core. MSCs and hAAT-MSCs from one donor were injected into the NOD mice at 8 weeks of age. Mice blood glucose levels were measured twice per week. Cell infiltration into islets was analyzed by H&E staining of pancreatic tissues. Single cell RNAseq analysis showed that hAAT-MSCs and control MSCs have different gene expression signatures, in which hAAT-MSCs showed increased expressions of chemokine (C-X-C motif) ligands (CXCL1, 3, 8), HES5 and others. Exosome proteomic analysis indicates that exosomes from these two types of cells have distinct protein patterns. In hAAT-MSCs cells, platelet degranulation proteins were upregulated and proteins involved in cell-cell adhesion, and cell division were been downregulated. In the NOD mice, a single dose of hAAT-MSCs led to delayed onset of T1D, associated with reduced cell infiltration into the islets. hAAT-MSC therapy showed more profound protection compared to control MSCs. It is likely that overexpression of hAAT enhanced the anti-inflammatory function (via regulating chemokines) promoting the Notch signaling pathway (via regulating HES5) and lead to improved protection in the NOD mice. Disclosure H. Wei: None. W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (1R01DK105183, DK120394, DK118529); U.S. Department of Veterans Affairs (I01BX004536)

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

BackgroundPolyembryony is defined as the formation of several embryos from a single egg. This phenomenon can occur in humans, armadillo, and some endoparasitoid insects. However, the mechanism underlying polyembryogenesis in animals remains to be elucidated. The polyembryonic parasitoid waspCopidosoma floridanumoviposits its egg into an egg of the host insect; eventually, over 2000 individuals will arise from one egg. Previously, we reported that polyembryogenesis is enhanced when the juvenile hormone (JH) added to the culture medium in the embryo culture. Hence, in the present study, we performed RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanisms controlling polyembryogenesis ofC. floridanum. Functional annotation of genes is not fully available forC.floridanum; however, whole genome assembly has been archived. Hence, we constructed a pipeline for gene functional annotation inC. floridanumand performed molecular network analysis. We analyzed differentially expressed genes between control and JH-treated molura after 48 h of culture, then used the tblastx program to assign wholeC. floridanumtranscripts to human gene.ResultsWe obtained 11,117 transcripts in the JH treatment group and identified 217 differentially expressed genes compared with the control group. As a result, 76% ofC. floridanumtranscripts were assigned to human genes. Gene enrichment analysis revealed genes associated with platelet degranulation, fatty acid biosynthesis, cell morphogenesis in the differentiation and integrin signaling pathways were fluctuated following JH treatment. Furthermore, Cytoscape analysis revealed a molecular interaction that was possibly associated with polyembryogenesis .ConclusionsWe have constructed a pipeline for gene functional annotation ofC. floridanum, and identified transcripts with high similarity to human genes during early embryo developmental. Additionally, this study reveals new molecular interactions associated with polyembryogenesis; these interactions could indicate the molecular mechanisms underlying polyembryony. Our results highlight the potential utility of molecular interaction analysis in human twins.

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.

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.

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.

Garcinol, an anti-inflammatory and anti-carcinogenic polyisoprenylated benzophenone isolated from Garcinia plants, stimulates tumor cell apoptosis and suicidal erythrocyte death, but supports the survival of hepatocytes and neurons. The present study explored whether the substance influences platelet function and/or apoptosis. To this end, we exposed murine blood platelets to garcinol (33 &micro, M, 30 min) without and with activation by collagen-related peptide (CRP) (2&ndash, 5 &micro, g/mL) or thrombin (0.01 U/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 &alpha, IIb&beta, 3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, and aggregation utilizing staining with CD9-APC and CD9-PE. As a result, in the absence of CRP and thrombin, the exposure of the platelets to garcinol did not significantly modify [Ca2+]i, P-selectin abundance, activated &alpha, 3 integrin, annexin-V-binding, cell volume, caspase activity, and aggregation. Exposure of platelets to CRP or thrombin was followed by a significant increase of [Ca2+]i, P-selectin abundance, &alpha, 3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as significant cell shrinkage. All effects of CRP were strong and significant, those of thrombin were only partially and slightly blunted in the presence of garcinol. In conclusion, garcinol blunts CRP-induced platelet activity, apoptosis and aggregation.

Background Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. Methods The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer’s Z-score method. Results A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. Conclusions We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type. Electronic supplementary material The online version of this article (10.1186/s12885-019-5547-y) contains supplementary material, which is available to authorized users.

Skin blistering as a result of snakebite envenomation is characteristic of some bites, however little is known regarding the mechanism of blister formation or the composition of the blister fluid. In order to investigate if blister fluid proteomes from humans suffering snakebite envenomation could provide insights on the pathophysiology of these skin alterations, blister fluid was collected from six patients upon presentation at a clinic in India bitten by three species of snakes, Daboia russelii (3), Hypnale hypnale (2), or Naja naja (1). Standard clinical data were recorded throughout the treatment. Approximately 805 proteins were identified in blister fluids using proteomic analyses. Informatics analyses of the proteomes identified the top biological response categories as: platelet degranulation, innate immune response, receptor-mediated endocytosis, complement activation, and blood coagulation. Hierarchical clustering did not show a clear segregation of patients&rsquo, proteomes being associated with the species of snake involved, suggesting that either the proteomic profiles described reflect a general response to venom-induced tissue damage or more patient data sets will be required to observe significant differences. Finally, it is of interest that venom proteins were also identified in the blister fluids suggesting that this fluid may serve as a reservoir of venom biologically active proteins/toxins, and as such, may indicate the clinical value of removing blister fluid to attenuate further tissue damage.

Background Hypothermia, either therapeutically induced or accidental (ie, an involuntary decrease in core body temperature to

The progression of Alzheimer’s dementia is associated with neurovasculature impairment, which includes inflammation, microthromboses, and reduced cerebral blood flow. Here, we investigate the effects ofβamyloid peptides on the function of platelets, the cells driving haemostasis. Amyloid peptideβ1-42 (Aβ1-42), Aβ1-40, and Aβ25-35 were tested in static adhesion experiments, and it was found that platelets preferentially adhere to Aβ1-42 compared to other Aβpeptides. In addition, significant platelet spreading was observed over Aβ1-42, while Aβ1-40, Aβ25-35, and the scAβ1-42 control did not seem to induce any platelet spreading, which suggested that only Aβ1-42 activates platelet signalling in our experimental conditions. Aβ1-42 also induced significant platelet adhesion and thrombus formation in whole blood under venous flow condition, while other Aβpeptides did not. The molecular mechanism of Aβ1-42 was investigated by flow cytometry, which revealed that this peptide induces a significant activation of integrinαIIbβ3, but does not induce platelet degranulation (as measured by P-selectin membrane translocation). Finally, Aβ1-42 treatment of human platelets led to detectable levels of protein kinase C (PKC) activation and tyrosine phosphorylation, which are hallmarks of platelet signalling. Interestingly, the NADPH oxidase (NOX) inhibitor VAS2870 completely abolished Aβ1-42-dependent platelet adhesion in static conditions, thrombus formation in physiological flow conditions, integrinαIIbβ3 activation, and tyrosine- and PKC-dependent platelet signalling. In summary, this study highlights the importance of NOXs in the activation of platelets in response to amyloid peptideβ1-42. The molecular mechanisms described in this manuscript may play an important role in the neurovascular impairment observed in Alzheimer’s patients.

Metastatic neuroblastoma (NB) is the leading cause of cancer death in children under the age of 5. Interestingly, infants develop a special pattern of metastasis to skin, liver and bone marrow (stage 4s) and typically undergo spontaneous remission with very low mortality. There is accumulating evidence that patients with intermediate risk group, e.g. infants with bone metastasis, have the same natural history of spontaneous regression and they do not require intensive therapy [1]. This highlights the importance of characterizing this group of patients at molecular level. We searched open repositories for quantitative gene expression data and identified GSE49711 as a suitable data source. This series includes data on 498 patients with NB including extensive phenotypic data and RNA-seq counts generated by Illumina Hiseq 2000 platform. First, we applied exploratory analysis to check the quality of data and the need for normalization before further analysis. Also, patients were categorized into 3 groups: Very low risk (VLR, N= 45), very high risk (VHR, N=92) and others (N= 361). The first 2 groups were further analyzed for differential gene expression (DGE) to identify genes responsible for their contrasting outcome. The edgeR package was used to define the top 100 differentially expressed genes with lowest adjusted p-values. These genes were applied to segregate samples using principal component analysis (PC). Interestingly, PC2 identified a special subset of patients who belong the VLR but have a unique gene expression pattern. Indeed, a small subset of VLR patients (N=6) clustered away from the main group and at extreme PC2 values. When the rotation of PC2 was analyzed, a group of 20 genes were found to have contributed to its values. The selected top 100 genes were then used to build a model of hierarchal clustering and constructing a heatmap. The same subset of samples appeared on a distinct limp of the dendogram with remarkable increased expression in the same exact group of 20 genes mentioned above. Gene ontology (GO) enrichment analysis of these specific genes was performed using an online tool developed by the GO Consortium [2], [3] revealed involvement of the following processes: Acute inflammatory response, regulation of blood coagulation, regulation of hydrolase activity and platelet degranulation. These processes indicate the role of immune system in the process, possibly combined by platelet activation. Interestingly, recent literature is highlighting the role of platelets in initiating inflammatory processes in neural tissue [4]. The model (top 100 genes selected by previous analysis) was used to test the original dataset (N=498) and segregated patients into 3 groups of patients based on outcome. Differential expression of MYCN, NME2(C-Myc transcription factor) and NCAN (neuronal adhesion) were most significantly associated with variable mortality. In conclusion, when 2 extreme groups of NB patients were defined and DGE was applied, a model was developed that showed similarity between the groups with categorization of a small subset of VLR with unique pattern of gene expression. The model worked well in defining risk of mortality and identified certain genes that can serve for prognostication.

Studies of sex-dependent differences in platelet aggregation and glycoprotein (GP)IIb/IIIa activation have demonstrated that platelets from females are more sensitive to agonists than those from males. To date, there is little understanding of these differences at a molecular level. Here, sex differences in reactivity of platelets from 86 women and 86 men were investigated. Platelet degranulation (CD62P expression) and activation of GPIIb/IIIa (PAC-1 binding), with and without ADP, were assessed. Extent of shape change (ESC) in response to ADP was measured. Basal CD62P and PAC-1 expression did not differ between the sexes. In response to ADP activation, mean PAC-1 binding in platelets from female donors was 17.9±3.5% vs. 14.0±4.1% in platelets from male donors, and ESC was significantly greater in platelets from females (p0.05). Evaluation of basal expression of signaling molecules along the ADP receptor pathway leading to GPIIb/IIIa activation and subsequent RhoA/ROCK signaling via GPIIb/IIIa 'outside-in' signaling showed that platelets from females produce 3-fold greater levels of phosphorylated protein kinase C (PKC) substrates. There was a 2.5-fold greater level of activated RhoA, and platelet sub-fractionation analysis demonstrated 2.7-fold more RhoA in the membrane fraction of female vs. male platelets. Similarly, there was a 2.8-fold increase in levels of phosphorylated myosin light chain (MLC) in platelets from females vs. males. The increased signaling activity in platelets from females mirrors their greater sensitivity to agonists. These findings further our understanding of the molecular differences between platelets from males and females.

SummaryCD44 is required for signalling of macrophage migration inhibitory factor (MIF), an anti-apoptotic pro-inflammatory cytokine. MIF is expressed and released from blood platelets, key players in the orchestration of occlusive vascular disease. Nothing is known about a role of CD44 in the regulation of platelet function. The present study thus explored whether CD44 modifies degranulation (P-selectin exposure), integrin activation, caspase activity, phosphatidylserine exposure on the platelet surface, platelet volume, Orai1 protein abundance and cytosolic Ca2+-activity ([Ca2+]i). Platelets from mice lacking CD44 (cd44-/- ) were compared to platelets from corresponding wild-type mice (cd44+/+ ). In resting platelets, P-selectin abundance, αllbβ3 inte-grin activation, caspase-3 activity and phosphatidylserine exposure were negligible in both genotypes and Orai1 protein abundance, [Ca2+]i, and volume were similar in cd44-/- and cd44+/+ platelets. Platelet degranulation and αllbβ3 integrin activation were significantly increased by thrombin (0.02 U/ml), collagen related peptide (CRP, 2 µg/ml and Ca2+-store depletion with thapsigargin (1 µM), effects more pronounced in cd44-/- than in cd44+/+ platelets. Thrombin (0.02 U/ml) increased platelet [Ca2+]i, caspase-3 activity, phosphatidylserine exposure and Orai1 surface abundance, effects again significantly stronger in cd44-/- than in cd44+/+ platelets. Thrombin further decreased forward scatter in cd44-/- and cd44+/+ platelets, an effect which tended to be again more pronounced in cd44-/- than in cd44+/+ platelets. Platelet adhesion and in vitro thrombus formation under high arterial shear rates (1,700 s-1) were significantly augmented in cd44-/- mice. In conclusion, genetic deficiency of CD44 augments activation, apoptosis and prothrombotic potential of platelets.

Background Cardiovascular disease (CVD) describes the pathological conditions of the heart and blood vessels. Despite the large number of studies on CVD and its etiology, its key modulators remain largely unknown. To this end, we performed a comprehensive proteomic analysis of blood plasma, with the scope to identify disease-associated changes after placing them in the context of existing knowledge, and generate a well characterized dataset for further use in CVD multi-omics integrative analysis. Methods LC–MS/MS was employed to analyze plasma from 32 subjects (19 cases of various CVD phenotypes and 13 controls) in two steps: discovery (13 cases and 8 controls) and test (6 cases and 5 controls) set analysis. Following label-free quantification, the detected proteins were correlated to existing plasma proteomics datasets (plasma proteome database; PPD) and functionally annotated (Cytoscape, Ingenuity Pathway Analysis). Differential expression was defined based on identification confidence (≥ 2 peptides per protein), statistical significance (Mann–Whitney p value ≤ 0.05) and a minimum of twofold change. Results Peptides detected in at least 50% of samples per group were considered, resulting in a total of 3796 identified proteins (838 proteins based on ≥ 2 peptides). Pathway annotation confirmed the functional relevance of the findings (representation of complement cascade, fibrin clot formation, platelet degranulation, etc.). Correlation of the relative abundance of the proteins identified in the discovery set with their reported concentrations in the PPD was significant, confirming the validity of the quantification method. The discovery set analysis revealed 100 differentially expressed proteins between cases and controls, 39 of which were verified (≥ twofold change) in the test set. These included proteins already studied in the context of CVD (such as apolipoprotein B, alpha-2-macroglobulin), as well as novel findings (such as low density lipoprotein receptor related protein 2 [LRP2], protein SZT2) for which a mechanism of action is suggested. Conclusions This proteomic study provides a comprehensive dataset to be used for integrative and functional studies in the field. The observed protein changes reflect known CVD-related processes (e.g. lipid uptake, inflammation) but also novel hypotheses for further investigation including a potential pleiotropic role of LPR2 but also links of SZT2 to CVD. Electronic supplementary material The online version of this article (10.1186/s12967-018-1476-9) contains supplementary material, which is available to authorized users.

Background Preoperative treatment of anti-vascular endothelial growth factor (VEGF) agents is extensively used in proliferative diabetic retinopathy (PDR), but the molecular mechanism is not fully understood. The objective of this research is to observe change of protein profile induced by ranibizumab (an anti-VEGF agent) in vitreous humor from PDR patients and reveal the effects of anti-VEGF treatment on PDR. Methods A proteomic method was used to identify differentially expressed proteins in vitreous humor. Untreated PDR patients were defined as PDR group, while those who treated with intravitreal injection of ranibizumab (IVR) were defined as IVR. Gene Ontology (GO) annotation and REACTOME pathways were obtained from DAVID Bioinformatics Resources. Intravitreal level of apolipoprotein C-I (APOC1), serpin peptidase inhibitor clade A member 5 (SERPINA5), tissue inhibitor of metalloproteinases (TIMP2), and keratin 1 (KRT1) were determined by enzyme-linked immuno sorbent assay (ELISA). Results 339 differentially expressed proteins were identified in response to IVR. The most notable GO annotation describes the altered proteins was “innate immune response”. The most notable REACTOME pathway was “platelet degranulation”. ELISA result showed increased level of APOC1, SERPINA5, KRT1 and a decreased level of TIMP2 in PDR group compared with IVR. Conclusions In addition to decreasing VEGF level, ranibizumab is associated with change of human vitreous protein profile in patients with PDR, in which the differential proteins are involved in immune response, platelet degranulation, complement activation etc., suggesting that the effects of VEGF are involved in these signaling pathways. Electronic supplementary material The online version of this article (10.1186/s12014-018-9187-z) contains supplementary material, which is available to authorized users.

Purpose. To examine the difference in the vitreal protein profiles of patients with proliferative diabetic retinopathy (PDR) with and without preoperative intravitreal conbercept (IVC) treatment.Methods. Liquid chromatography-tandem mass spectrometry- (LC-MS/MS-) based proteomic methods were used to determine the protein profiles of the vitreous humor in patients with PDR treated with (IVC group;n=9) and without (PDR group;n=8) preoperative IVC. Gene ontology (GO) annotation and REACTOME pathway analysis were obtained to overview differentially expressed proteins between each group. Intravitreal levels of apolipoprotein A-II (APOA2) and ceruloplasmin (CP) were measured using enzyme-linked immunosorbent assays.Results. 307 proteins were expressed differentially between PDR and IVC groups, including 218 proteins downregulated in response to IVC. The most notable GO annotations in level 3 and REACTOME pathways describing the differentially expressed proteins were “innate immune response” and “platelet degranulation.” The intravitreal levels of APOA2 and CP were lower in the IVC group than in the PDR group (p<0.01).Conclusions. In addition to decreasing the intravitreal vascular endothelial growth factor level, IVC may alter the vitreal protein profile in patients with PDR, with the differentially regulated proteins involved in the immune response, platelet degranulation, complement activation, and inflammation.

Several circulating microRNAs (miRNAs) have been proved to serve as stable biomarkers in blood for acute ischemic stroke (AIS). However, the functions of these biomarkers remain elusive. By conducting the integration analysis of circulating miRNAs and peripheral whole-blood mRNAs using bioinformatics methods, we explored the biological role of these circulating markers in peripheral whole blood at the genome-wide level. Stroke-related circulating miRNA profile data (GSE86291) and peripheral whole-blood mRNA expression data (GSE16561) were collected from the Gene Expression Omnibus (GEO) datasets. We selected male patients to avoid any gender differences in stroke pathology. Male stroke-related miRNAs (M-miRNAs) and mRNAs (M-mRNAs) were detected using GEO2R. Nine M-miRNAs (five up- and four down-regulated) were applied to TargetScan to predict the possible target mRNAs. Next, we intersected these targets with the M-mRNAs (38 up- and three down-regulated) to obtain the male stroke-related overlapped mRNAs (Mo-mRNAs). Finally, we analyzed biological functions of Mo-mRNAs using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), and constructed networks among the Mo-mRNAs, overlapped M-miRNAs (Mo-miRNAs), and their functions. The Mo-mRNAs were enriched in functions such as platelet degranulation, immune response, and pathways associated with phagosome biology and Staphylococcus aureus infection. This study provides an integrated view of interactions among circulating miRNAs and peripheral whole-blood mRNAs involved in the pathophysiological processes of male AIS.