Project description:DNA-protein interactions play a pivotal role in a variety of cellular processes. Despite the considerable research dedicated to understanding these interactions, the specific role of single-stranded DNA (ssDNA) remains largely unexplored. Our study aimed to uncover the role of ssDNA in the interplay between neutrophil extracellular traps (NETs) and thrombin, a crucial enzyme involved in both blood clot formation and immune response. Using chromatin immunoprecipitation sequencing (ChIP-seq) and kethoxal-assisted ssDNA sequencing, we identified a specific short tandem repeat (STR) sequence, ss(ATTCC)n, that is highly enriched and forms an iI-motif structure crucial for its interaction with thrombin. We further observed that the binding affinity between ss(ATTCC)n and thrombin is increased under acidic conditions, a condition frequently linked to diseases such as cancer. Our findings not only shed light on the unexplored area of sequence-dependent ssDNA-protein interactions but also pave the way for targeted interventions in thrombosis and coagulation disorders.
Project description:Confluent human umbilical vein endothelial cells (HUVECs) were exposed to Thrombin (2 U/mL) for 2 hours. Ribosomal profiling via gradient centrifugation and fractionation was used to separate monosome, or under-translated, and polysome, or actively translated, mRNA species that were then used to probe cDNA arrays, a process known as Translation State Array Analysis (TSAA). Four samples were obtained from these experiments, Control Monosome, Control Polysome, Thrombin Monosome, and Thrombin Polysome. Using the normalized signal intensities from the GeneFilters, we calculated a translation index, or measure of movement of an mRNA molecule from the monosome to the polysome fraction upon stimulation. This calculation was made as follows: (thrombin polysome/thrombin monosome)/(control polysome/control monosome). Translational indices greater than 2.5 (upregulated) or lower than 0.4 (downregulated) were chosen for further study. TSAA data suggests that JunB is translationally regulated by thrombin stimulation. Immunocytochemistry, western blotting and RT-PCR were used to verify the results of TSAA. Keywords: Translation State Array Analysis
Project description:This SuperSeries is composed of the following subset Series: GSE32542: Murine serum reactivity to common autoantigens in response to immunization with neutrophil extracellular traps GSE32543: Human and murine serum reactivity to specific histone posttranslational modifications in neutrophil extracellular traps Refer to individual Series
Project description:Rat chondrocytes were divided into control group (RP), thrombin group (RPT), and thrombin plus inhibitor group (RPTi). The differential genes were identified by RNA-sequencing, and verified by western blot (WB), quantitative polymerase chain reaction (qPCR), cell immunofluorescence (IF), endoplasmic reticulum (ER) staining, functional enrichment analysis, and Gene Ontology (GO).
Project description:Cysteinyl leukotrienes (cysLT), i.e. LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway. The cysLT receptors cysLT1-R and cysLT2-R are expressed on different target cells and mediate inflammatory reactions in tissue- and LT-R-specific ways. Though endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be defined. To delineate cysLT2-R´s action, we stimulated human umbilical vein EC with 100 nM LTD4 for 60 min, determined gene signatures by microarrays, and characterized the resulting EC phenotypes. As controls, we compared LTD4-induced genes with those induced by 10 nM thrombin, a prototype vasoactive activator of EC that binds to protease-activated receptor 1 (PAR-1). Following application of stringent filters 37 LTD4-upregulated genes were identified (> 2.5fold stimulation). Surprisingly, most of the LTD4-regulated genes were also induced by thrombin and expression of cysLT2-R- and PAR-1-regulated genes strongly correlated (Pearson correlation coefficient: r = 0.90). Moreover, LTD4 + thrombin, when added together, augmented expression of LTD4- or thrombin-stimulated genes (Wilcoxon signed rank test: p < 0.01). Prominently induced genes that may play roles in vascular injury were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A; E-selectin; CXC ligand 2; interleukin 8 (IL-8); a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS-1); and tissue factor (TF). Transcripts of these genes peaked at approximately 60 min, were unaffected by the cysLT1-R antagonist montelukast, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus in LTD4-stimulated cells; LTD4 upregulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC pro-coagulant activity. These data show that cysLT2-R activation results in a pro-inflammatory EC phenotype through activation of immediate-early genes that resemble those induced by PAR-1. As LTD4 and thrombin are formed concomitantly during vascular injury and pro-thrombotic states, cysLT2-R and PAR-1 may collaborate in vivo to mediate vascular injury and repair. Keywords: Leukotriene Transcriptome, Thrombin Transcriptome, HUVEC, Immediate-Early Gene Expression, Cysteinyl Leukotriene 2 Receptor Gene Signature in HUVEC
Project description:A microarray analysis was conducted to investigate transcriptional differences between traps and vegetative hyphae of the nematophagous fungus Monacrosporium haptotylum. Our goal was to identify genes that show differential regulation in the two different cell types; 1) traps (knobs) and 2) vegetative hyphae (mycelium) grown in the same medium. The entire design involved nine slides; four of these were hybridisations of knobs versus mycelium and five were mycelium against mycelium, 10 labelled extracts, and 3 biological replicates. The microarray experiments were designed as two-sample comparisons (i.e. knobs versus mycelium or mycelium versus mycelium) using three independent biological replicates, which also included technical and dye-swapped control hybridisations.
Project description:Human aortic smooth muscles are quiesced for 24 hours followed by treatment with thrombin for 2 hours and 8 hours in presence or absence of cyclosporin A (10 micromolar) to analyze the effect of thrombin on expression pattern of various genes in presence of cyclosporin A.