Regulation Of Chemotaxis Research Articles

Screening and identification of susceptibility genes for osteosarcoma based on bioinformatics analysis.

Gene play an important role in malignant tumors. However, there is still insufficient research on genetic variations in osteosarcoma (OS) patients. Therefore, we aimed to analyze the gene expression profile of OS using bioinformatics and to explore the pathogenesis of OS at the molecular level. The gene chip dataset of OS samples was downloaded from the Gene Expression Omnibus (GEO) database for screening differentially expressed genes (DEGs). The R language clusterProfiler software package was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs. The central node proteins of the protein interaction network were analyzed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database, Cytoscape, and its plug-ins cytoHubba and NetworkAnalyzer to find the key genes. A total of 631 DEGs were obtained, including 362 upregulated genes and 269 downregulated genes. DEGs were mainly involved in the regulation of leukocyte chemotaxis and migration, vascular development, and other biological processes (BPs); mediation of receptor ligand activity, growth factor binding, growth factor activity, integrin binding, and other molecular functions (MFs); and were enriched in the extracellular matrix (ECM). DEGs in the ECM and growth factors play a key role in the development of OS. The leukocyte transendothelial migration pathway and the PI3K-AKT pathway are closely related to OS, and the related molecular mechanism is worthy of further study.

FlgC gene is involved in the virulence regulation of Pseudomonas plecoglossicida and affects the immune response of Epinephelus coioides

As a pathogen of cultured teleosts, Pseudomonas plecoglossicida has caused significant economic losses. flgC plays an important role in encoding flagellar basal-body rod proteins. Our previous studies revealed the high expression of P. plecoglossicida flgC in infected Epinephelus coioides. To explore the role of flgC in the virulence of P. plecoglossicida and the immune response of E. coioides to the infection of P. plecoglossicida, flgC gene of P. plecoglossicida was knocked down by RNA interference (RNAi). The results showed that the flgC gene in all four mutants of P. plecoglossicida was significantly knocked down, and the mutant with the best knockdown efficiency of 94.3% was selected for subsequent studies. Compared with the NZBD9 strain of P. plecoglossicida, the flgC-RNAi strain showed a significantly decrease in chemotaxis, motility, adhesion, and biofilm formation. Furthermore, compared with the E. coioides infected with the NZBD9 strain, the infection of flgC-RNAi strain resulted in the infected E. coioides a 1.5-day delay in the time of first death and an 80% increase in survival rate, far fewer white nodules upon the spleen surfaces, and lower pathogen load in the spleens. RNAi of flgC significantly influenced the metabolome and transcriptome of the spleen in infected E. coioides. KEGG enrichment analysis exhibited that the Toll-like receptor signaling pathway was the most enriched immune pathway; the most significantly enriched metabolic pathways were associated with Linoleic acid metabolism, Choline metabolism in cancer, and Glycerophospholipid metabolism. Further combined analysis of transcriptome and metabolome indicated significant correlations among pantothenate and CoA biosynthesis, beta-alanine metabolism, lysosome metabolites, and related genes. These results suggested that flgC was a pathogenic gene of P. plecoglossicida; flgC was associated with the regulation of chemotaxis, motility, biofilm formation, and adhesion; flgC influenced the immune response of E. coioides to the infection of P. plecoglossicida.

Immunogenetic basis of chicken’s heterophil to lymphocyte ratio revealed by genome-wide indel variants analysis

Enhancing host immunity is an effective way to reduce morbidity in chickens. Heterophil to lymphocyte ratio (H/L) is associated with host disease resistance in birds. Chickens with different H/L levels show different disease resistances. However, the utility of the H/L as an indicator of immune function needs to be further analyzed. In this study, a H/L directional breeding chicken line (Jingxing yellow chicken) was constructed, which has been bred for 12 generations. We compared the function of heterophils, and combined statistical analysis to explore the candidate genes and pathways related to H/L. The oxidative burst function of the heterophils isolated from the H/L selection line (G12) was increased (P=0.044) compared to the non-selection line (NS). The 22.44 Mb genomic regions which annotated 300 protein-coding genes were selected in the genome of G9 (n=92) compared to NS (n=92) based on a genome-wide selective sweep. Several selective regions were identified containing genes like interferon induced with helicase C domain 1 (IFIH1) and moesin (MSN) associated with the intracellular receptor signaling pathway, C–C motif chemokine receptor 6 (CCR6), dipeptidyl peptidase 4 (DPP4) and hemolytic complement (HC) associated with the negative regulation of leukocyte chemotaxis and tight junction protein 1 (TJP1) associated with actin cytoskeleton organization. In addition, 45 genome-wide significant indels containing 29 protein-coding genes were also identified as associated with the H/L based on genome-wide association study (GWAS). The expression of protein tyrosine phosphatase non-receptor type 5 (PTPN5) (r=0.75, P=0.033) and oxysterol binding protein like 5 (OSBPL5) (r=0.89, P=0.0027) were positively correlated with H/L. Compared to the high H/L group, the expressions of PTPN5 and OSBPL5 were decreased (P<0.05) in the low H/L group of Beijing you chicken. The A/A allelic frequency of indel 5_13108985 (P=3.85E–06) within OSBPL5 gradually increased from the NS to G5 and G9, and the individuals with A/A exhibited lower H/L than individuals with heterozygote A/ATCT (P=4.28E–04) and homozygous ATCT/ATCT (P=3.40E–05). Above results indicated oxidative burst function of heterophils were enhanced, and 22.44 Mb genomic regions were selected with the directional selection of H/L. In addition, PTPN5 and OSBPL5 genes were identified as H/L-related candidate genes. These findings revealed the complex genetic mechanism of H/L related to immunity and will allow selection for improving chicken immunity based on the H/L.

Identification of lncRNA-miRNA-mRNA networks in late-onset pre-eclampsia

Objective: Long non-coding RNAs (lncRNAs) are implicated in multiple pathophysiological processes in placenta-related disorders; however, their expression and function in late-onset pre-eclampsia (LOPE) remain unclear. This study aimed to investigate the expression of lncRNAs in LOPE, construct a competing endogenous RNA (ceRNA) network, and identify the pathways associated with LOPE pathogenesis. Methods: We performed lncRNA and mRNAs microarray profiling to identify the differential expression profiles of lncRNAs and mRNAs in LOPE compared to those in normal pregnancy. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to validate differentially expressed genes. Subsequently, we generated an interaction network between lncRNAs, (micro-RNAs) miRNAs, and mRNAs based on the Pearson’s correlation coefficient between lncRNAs and mRNAs. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to understand the functional significance of differentially expressed lncRNAs (DElncRNAs) in LOPE. Results: We identified 29 DElncRNAs (25 upregulated and four downregulated) and 212 differentially expressed mRNAs (DEmRNAs; 203 upregulated and nine downregulated) in LOPE placentas. Within them, six lncRNAs and four mRNAs were verified by qRT-PCR. GO and KEGG analyses revealed the potential pathways affected by these mRNAs, such as positive regulation of leukocyte chemotaxis, chemokine signaling pathway, and response to hypoxia. Finally, we constructed a ceRNA network including three DElncRNAs and 124 DEmRNAs, whose competing interactions may be mediated by 17 miRNAs. Two DElncRNAs, ENST00000515376 and ENST00000520544, were found to be hub genes, as they interacted with most miRNAs and mRNAs. ENST00000515376 is most likely related to the metabolic process of arachidonic acid, whereas ENST00000520544 is more likely related to the coagulation system, such as the regulation of blood coagulation and platelet degranulation. Conclusion: Differential expression profile of lncRNAs and the lncRNA-miRNA-mRNA network in LOPE provide potential therapeutic targets for this disease.

MiR-148a-3p attenuates apoptosis and inflammation by targeting CNTN4 in atherosclerosis

Atherosclerosis (AS) seriously affects human health. The role of microRNAs (miRNAs) in the pathogenesis and progression of AS has become a focus of research. Our goal was to identify the biological effect of differentially expressed miRNAs (DE-miRNAs) in AS. To analyze differentially expressed genes (DEGs), including differentially expressed mRNAs (DE-mRNAs) and DE-miRNAs, in AS by using the Gene Expression Omnibus (GEO) database and limma package. DEGs protein-protein interaction (PPI) network and functional enrichment analysis were constructed by using the search tool for the retrieval of interacting genes/proteins (STRING) database, Cytoscape software and Cytoscape plugin "ClueGO2.5.6". We established a coexpression network of dysregulated miRNAs and mRNAs to predict the function of miRNAs by using miRWalk database and Pearson correlation coefficient (PCC) analysis. Cellular experiments were used to validate the results of bioinformatics. First, 69 common DEGs were obtained from datasets GSE43292 and GSE97210 using the limma package in R. Next, a DEG PPI network was constructed. Functional enrichment analysis of DEGs showed that 11 functional pathways were significantly enriched, such as positive regulation of monocyte chemotaxis. Seven common DE-miRNAs were obtained from the GSE99685 dataset and DE-mRNAs predicted miRNAs through the miRWalk database. The miRNA-mRNA network constructed using Cytoscape software suggested that miR-148a-3p targeted contactin 4 (CNTN4). Quantitative real-time polymerase chain reaction (qRT-PCR) assay results indicated that miR-148a-3p was downregulated and CNTN4 was upregulated in the THP-1 + phorbol 12-myristate 13-acetate (PMA) + oxidized low-density lipoprotein (oxLDL) group compared with the THP-1 + PMA group. qRT-PCR, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) found that upregulated miR-148a-3p significantly inhibited the expression of CNTN4, cell apoptosis, and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) concentrations in oxLDL-induced THP-1 macrophages. In addition, a dual-luciferase reporter assay demonstrated that CNTN4 was a target gene of miR-148a-3p. Overall, these findings suggested that miR-148a-3p inhibited oxLDL-induced cell apoptosis and inflammation via targeting CNTN4 in THP-1 macrophages.

STAG2 expression is associated with adverse survival outcomes and regulates cell phenotype in muscle-invasive bladder cancer.

STAG2 (Stromal Antigen 2), in healthy somatic cells, functions in sister chromatid cohesion, DNA damage repair, and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. Here, using whole-exome and targeted sequencing (n=119 bladder cancer clinical samples), we found several STAG2 mutations in MIBC that correlate with loss of protein expression. The analysis of a bladder cancer tissue microarray (n=346) revealed that decreased STAG2 protein expression is associated with improved overall and progression-free survival for MIBC patients. In mouse xenograft studies, STAG2 knockdown (KD) decelerated MIBC tumor growth, whereas STAG2 overexpression accelerated tumor growth. In cell line studies, STAG2 loss augmented treatment with cisplatin, a first-line therapy for MIBC. STAG2 KD or overexpression did not alter degree of aneuploidy, copy number variations, or cell cycle distribution. However, unbiased RNA sequencing analysis revealed that STAG2 KD altered gene expression. STAG2 KD led to significant downregulation of several gene sets, such as collagen containing extracellular matrix, external encapsulating structure organization, and regulation of chemotaxis. Therefore, we investigated the effect of STAG2 KD on cell migration and invasion in vitro. We found that STAG2 KD minimized cell speed, displacement, and invasion. Altogether, our results present a non-canonical function of STAG2 in promoting cell motility and invasion of MIBC cells. This work forms the basis for additional investigation into the role of STAG2 in transcriptional regulation and how it becomes dysregulated in STAG2-mutant MIBC.

Left ventricular assist device bioinformatics identify possible hubgenes and regulatory networks involved in the myocardium of patients with left ventricular assist device.

ObjectiveThe aim of this study was to clarify the changes of myocardial gene expression profile after left ventricular assist device (LVAD) implantation and the related molecular biological significance.MethodsA thorough bioinformatic analysis to evaluate the changes in gene expression profile in patients pre-LVAD and post-LVAD was conducted. Four relevant gene expression datasets—GSE430, GSE974, GSE21610, and GSE52601 from Gene Expression Omnibus (GEO) database were downloaded. Analysis of GEO2R, Gene Ontology (GO), protein-protein interaction (PPI) were used to determine differentially expressed genes (DEGs) and their function, respectively.ResultsA total of 37 DEGs were identified, including 26 down-regulated and 11 up-regulated genes. The molecular function of DEGs were enriched in “cytokine activity,” “neurotransmitter binding,” “receptor ligand activity.” The gene set enrichment analysis (GSEA) revealed an overall marked increase of neutrophil degranulation signaling, closely correlated with the G protein coupled receptor (GPCR)—ligand binding process after LVAD assistance. 16 hubgenes in these DEGs were further selected and the biological process involved is mainly related to positive regulation of leukocyte chemotaxis mediated by chemokines.ConclusionInflammatory signaling pathway is crucial for the pathophysiology after LVAD implantation. Chemokines mediate cardiac inflammatory response and tissue remodeling after LVAD implantation through GPCR—ligand binding.

Transcriptomic and metabolomic insights into the role of fliS in the pathogenicity of Pseudomonas plecoglossicida against Epinephelus coioides

Pseudomonas plecoglossicida is responsible for visceral white spot disease in economically valuable marine fish such as Larimichthys crocea and Epinephelus coioides. Based on RNA sequencing, we previously showed that P. plecoglossicida fliS gene expression is significantly up-regulated in E. coioides spleens during infection. Here, to explore the role of this gene in pathogenicity, RNA interference (RNAi) was performed to silence fliS in P. plecoglossicida, and the mutant with the best silencing efficiency (89%) was chosen for further studies. Results showed that fliS silencing significantly attenuated motility, chemotaxis, adhesion, and biofilm formation of P. plecoglossicida. Furthermore, E. coioides infected with the fliS-RNAi strain recorded no deaths and showed fewer pathogens in the spleen and fewer white spots on the spleen surface compared to those fish infected with the wild type P. plecoglossicida strain. RNAi of fliS significantly affected the spleen transcriptome and metabolome in infected E. coioides. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the cytokine-cytokine receptor interaction pathway was the most enriched immune-related pathway, and the arginine biosynthesis pathway was the most enriched metabolism-related pathway. These findings suggest that fliS is a virulence gene of P. plecoglossicida and is involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation, as well as the inflammatory and immune responses of E. coioides to P. plecoglossicida infection.

Expression of chemotaxis in regulators of tissues of exudative lesions of Reinke’s space

Exudative tumor-like neoplasms of the Reinke space in vocal folds are widespread in the population, more often among representatives of vocal professions, e.g., actors, singers, teachers, lecturers and represent a serious medical and social problem. In pathogenesis of such neoplasms, a key role is given to chronic phonotrauma, intoxication during smoking against the background of the nearly complete absence of lymphatic drainage in the Reinke space. Cellular factors of tissue and immune homeostasis are of great importance in morphogenesis of this disorder. Switching of immune responses aimed at maintaining tissue homeostasis is accompanied by increased production of pro-inflammatory cytokines, as well as chemotaxis regulators and growth factors. The aim of our work was to study the levels of chemokines (MIP-1, MIP-1, MIP-3, fractalkine, IL-8, I-TAC) in the tissues of exudative lesions from the Reinkes space (EPPR).&#x0D; Forty tissue samples of exudative lesions from Reinkes space, in particular, vocal fold polyps, vocal nodules and neoplasms presenting as Reinkes edema were taken as biological material for the study. The samples were taken intraoperatively when the neoplasm was removed, using an Olympus TYPE 150 fiber bronchoscope (Germany) using an integrated Lumenis Acu Pulse CO2 laser (Israel). The content of chemokines was determined in the supernatants of the tissues homogenates, using multiplex analysis with MAGPIX-100 immunoanalyzer (Bio-Rad, USA). Statistical processing was carried out using Statistica 10.0 for Windows software package. The results are presented as medians (Q0.25-Q0.75).&#x0D; Chronic phonotrauma and/or exposure to toxic factors leads to increased permeability of the vascular endothelium, tissue edema and activation of cells involved in tissue and immune homeostasis, e.g., fibroblasts, monocytes, endotheliocytes. The study of the chemokine expression in the tissues of various exudative lesions of Reinkes space enabled us to reveal the following features: (1) predominance of the CXC-chemokine content produced mainly by fibroblasts in dense neoplastic tissues, thus reflecting participation of the latter in the genesis of this pathology. (2) In the tissues of soft neoplasms with large proportion of liquid component, we have revealed increased concentrations of SS-chemokines and fractalkine. The latters are produced mainly by macrophages and endotheliocytes, thus, probably, reflecting a predominant role of these cells in development of myxoid polyps and Reinkes edema.

Candidate genes for tick resistance in cattle: a systematic review combining post-GWAS analyses with sequencing data

ABSTRACT Rhipicephalus microplus causes huge losses in cattle. Host genetic background greatly affects the immune efficiency in resistance or susceptibility to tick infestation, which is one of the many factors that play a role on that trait. We performed a systematic review of genome-wide association studies (GWAS) for tick resistance in cattle resulting in 1353 candidate genes for post-GWAS analyses. From those, genes showing possible structural variants from the bovine genome were classified by the Variant Effect Predictor from Ensembl. Ninety-two candidate genes showed potential structural variants in 5′ UTR and coding region and were used for functional annotation. Enriched biological processes (e.g. regulation of eosinophil chemotaxis, RIG-I signalling pathway and monocyte differentiation) and candidate genes (e.g. DAPK2, PUM1, ACIN1, INPP5D) linked with immune system function were identified and thus associated with tick resistance. Besides, gene-transcription factors (TFs) networks were obtained from TFs associated with immune system (FOXO3, PPARG, STAT3, NFKB1, GATA3 and ARNT) and the candidate genes associated with tick resistance in cattle highlighted (e.g. OR4L1, PNP, LRRIQ1, GIMAP8, MYO6, MEP1A and LRFN2). Thus, promising candidate genes with a possible functional role for tick resistance in cattle are presented for further in vitro and/or in vivo analyses.

Abstract 2023: Spatial transcriptomic evidence for an inflammatory, pro tumorigenic microenvironment in normal human dense breast tissue

Abstract Breast cancer affects over 2,000,000 women worldwide, with more than 680,000 deaths per year. Increased breast density is one of the risk factors for breast cancer, albeit less than the risk associated with increased age and genetic mutation. It has been suggested that high breast density accounts for 15% of the breast cancers diagnosed. On radiographic examination, breast density appears as opaque regions associated with increased cellularity and matrix, which in some circumstances contribute to difficulty observing small tumors. Dense regions in the breast have been associated with both morphological changes as well as molecular changes in the tissue. How these changes may play a role in increased risk for tumorigenesis are not well described. In this study, we examine the partial spatial transcriptomes of specific regions in normal breast tissue containing dense regions, including regions of normal density, the interface of dense and non-dense regions and the dense regions themselves using the GeoMx Digital Spatial Profiling system (DSP). Multi-label immunofluorescence was used to distinguish tissue morphology in FFPE samples from two health patients. We selected 24 regions of interest (ROIs) from each tissue section included dense, interface and non-dense breast. Using barcoded DNA oligos attached in situ hybridization probes (for RNA) via ultraviolet (UV)- photocleavable linkers, we screened over 1800 genes from Cancer Transcriptome Atlas panel. Our results indicated an elevated expression of CD68 and CD33, markers for macrophages and myeloid-derived suppressor cells (MDSCs) in dense breast regions as compared to the non-dense areas. Identifying markers for myeloid lineage cells was followed by an increased expression of the genes EOMES, TIGIT and RORA, which are engaged in the immunosuppressive phenotype. This could be hypothesized to support a pro-tumorigenesis microenvironment preventing T cells from recognizing newly arisen tumor cells. In addition, we also observed a significant expression of cytokines (IL12A and IL26) and chemokines (CCL19 and CXCL10) in the dense breast, which are involved in the regulation of inflammatory response and chemotaxis of monocytes and T-lymphocytes cells. Both CCL19 and IL26 have been demonstrated to play a role in cell proliferation. Thus, these initial studies suggest that the dense breast microenvironment is potentially a pro-tumorigenic environment that, with sufficient factors, gives rise to tumorigenesis. These data also suggest potential nodes of therapeutic intervention that may lower such risk. Citation Format: Ana Karina de Oliveira, Patcharin Pramoonjago, Christopher A. Moskaluk, Jay W. Fox. Spatial transcriptomic evidence for an inflammatory, pro tumorigenic microenvironment in normal human dense breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2023.

Abstract 1245: Comprehensive analysis of non-small cell lung cancer identifies molecular genotype-immunophenotype associations and candidate biomarkers predictive of response to immunotherapy

Abstract Lung cancer has the second highest incidence and leads cancer mortality worldwide, being Non-Small Cell Lung Cancer (NSCLC) the most prevalent subtype. Immunotherapy with checkpoint blockers has shown outstanding benefits in a subset of NSCLC patients, which are not accurately identified due to the lack of robust biomarkers of response. We hypothesize that specific molecular alterations of NSCLC tumor cells impact the immune microenvironment. Defining this association could improve the prediction of response to immunotherapy. This work aims to identify a multiparametric biomarker signature. We studied 178 tumor samples from a cohort of early stage NSCLC with complete clinical annotation. We analyzed their molecular aberrations, calculated their Tumor Mutational Burden and described their immune landscape by immunohistochemistry and a dedicated RNAseq panel. We defined novel subgroups of NSCLC tumors with specific immune, molecular and clinical features, showing an association between their molecular genotype and immune phenotype. Based on these data, subgroups were proposed as pro-immunogenic, pro-tumorigenic or mixed. Adenocarcinomas clustered in 4 groups. The pro-tumorigenic group had a significant higher proportion of alterations in ARID1A, FGF10, ROS1, TP53. One pro-immunogenic group had a significant higher proportion of MET alterations and lower proportion of EGFR alterations. An immune mixed group significantly accrued never smokers and had no alterations in FGF10. Squamous cell carcinomas clustered in 4 groups. The pro-tumorigenic group had a significant higher proportion of alterations in CCND1, FGF3, FGF10, FGF19, NOTCH1, PIK3CA, PIK3CB, TFRC. A pro-immunogenic group had a significant higher infiltration of T CD4+, T CD8+ and B cells and a higher overall survival. Another pro-immunogenic group had a significant absence of MYCN and NF1 alterations. Taken together we identified a set of candidate predictive biomarkers of response to immunotherapy in NSCLC. Genes overexpressed in pro-immunogenic tumors are related to adaptive immune response stimulation (CD28, FCRLA, JCHAIN, LY9, MS4A1, SLAMF7, TNFRSF9, TNFRSF17), antigen presentation (CD1C, CD1D, HLA-A), apoptosis (FAS), immune chemotaxis (CCL20, CCR4, CCR6), and immune regulation (CD53, PDCD1, SH2D1A, ZAP70). On the other hand, genes upregulated in pro-tumorigenic tumors are involved in angiogenesis (VEGFA), cell cycle regulation (BUB1, CCNB2, FOXM1, MAD2L1, TOP2A), cell growth/survival (IGF1R), DNA replication/repair (KIAA0101) and iron metabolism (HMBS, TFRC). These findings are being validated by Digital Spatial Profiling in a cohort of patients with advanced stage NSCLC that were treated with checkpoint blockers. If confirmed, these results could remarkably improve patient selection and the benefit upon immunotherapy. Citation Format: Javier Ramos-Paradas, David Gomez-Sanchez, Aranzazu Rosado, Alvaro Conrado Ucero, Nuria Carrizo, Ana Belen Enguita, Maria Teresa Muñoz, Esther Conde, Luis Paz-Ares, Eva Maria Garrido-Martin. Comprehensive analysis of non-small cell lung cancer identifies molecular genotype-immunophenotype associations and candidate biomarkers predictive of response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1245.

Cryo‐EM structures of pentameric autoinducer‐2 exporter from Escherichia coli reveal its transport mechanism

Bacteria utilize small extracellular molecules to communicate in order to collectively coordinate their behaviors in response to the population density. Autoinducer‐2 (AI‐2), a universal molecule for both intra‐ and inter‐species communication, is involved in the regulation of biofilm formation, virulence, motility, chemotaxis, and antibiotic resistance. While many studies have been devoted to understanding the biosynthesis and sensing of AI‐2, very little information is available on its export. The protein TqsA from Escherichia coli, which belongs to the AI‐2 exporter superfamily, has been shown to export AI‐2. Here, we report the cryogenic electron microscopic structures of two AI‐2 exporters (TqsA and YdiK) from E. coli at 3.35 Å and 2.80 Å resolutions, respectively. Our structures suggest that the AI‐2 exporter exists as a homo‐pentameric complex. In silico molecular docking and native mass spectrometry experiments were employed to demonstrate the interaction between AI‐2 and TqsA, and the results highlight the functional importance of two helical hairpins in substrate binding. We propose that each monomer works as an independent functional unit utilizing an elevator‐type transport mechanism.

Transcriptome sequencing analysis revealed the molecular mechanism of podoplanin neutralization inhibiting ischemia/reperfusion-induced microglial activation.

BackgroundAnti-podoplanin antibody (α-PDPN, clone 8.1.1) reduces microglia-mediated inflammation and decreases cerebral infarct volume in mice with stroke. However, the molecular mechanism by which this occurs is unknown. This study sought to systematically analyze the molecular mechanism of α-PDPN treatment on ischemia/reperfusion (I/R)-injured microglia.MethodsMicroglia BV2 cells were pre-cultured with α-PDPN and then exposed to oxygen-glucose deprivation and reoxygenation (OGD-R) insult. The differentially expressed genes (DEGs) underwent a transcriptome sequencing technology analysis, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Quantitative real-time polymerase chain reaction (PCR) was performed to confirm the transcriptional expression of some DEGs.ResultsThe results showed that α-PDPN downregulated 338 genes and upregulated 340 genes in the BV2 cells. The GO items of the downregulated DEGs mainly involved biological processes, such as the response to the interferon (IFN), lipopolysaccharide-mediated signaling pathway, and the regulation of cell chemotaxis and migration. The upregulated molecular function mainly involved glucocorticoid-receptor binding. Further, the KEGG pathway analysis indicated that the enriched categories for the upregulated DEGs mainly involved the adenosine triphosphate (ATP) binding cassette transporters. However, the interleukin-17 signaling pathway, IFN signaling pathway, tumor necrosis factor signaling pathway, transforming growth factor beta (TGF-ꞵ) signaling pathway, nucleotide-binding and oligomerization domain (NOD)-like receptor signaling pathway, cytokine-cytokine receptor interaction, and chemokine signaling pathway were downregulated by the α-PDPN treatment.ConclusionsNumerous inflammation-related signaling pathways were regulated by the α-PDPN treatment in the OGD-R injured BV2 cells. This study provided further insights into the protective mechanism of α-PDPN treatment in ischemic stroke.

Exosomes as Key Regulators of Neutrophil Chemotaxis

Neutrophils, the most abundant leukocytes in human blood, are the first line of defense at sites of injury or infection. Upon activation by primary chemoattractants such as the formyl peptide N‐Formyl‐methionine‐leucyl‐phenylalanine (fMLF), neutrophils secrete the secondary chemoattractant leukotriene B4 (LTB4) which serves to relay primary chemotactic signals and increase the range and persistence of migration. LTB4 is synthesized from arachidonic acid (AA) through the sequential action of the 5‐lipoxygenase (5‐LO), its associated activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H). We previously demonstrated that LTB4 and its synthesizing enzymes are packaged and released in extracellular vesicles called exosomes. Conventional exosome biogenesis originates from the plasma membrane. However, the LTB4 synthesizing machinery resides on the nuclear envelope (NE). We found that the localized activation of membrane curvature inducing proteins facilitates the selective sorting of the LTB4‐synthesis machinery on the NE and its packaging within NE‐derived exosomes. We show that chemoattractant stimulation leads to the generation of 5‐LO‐ and FLAP‐positive NE buds and cytosolic vesicles, which are also positive for the inner nuclear membrane protein lamin B receptor (LBR). Furthermore, we found that the generation of ceramide from sphingomyelin through the action of the neutral sphingomyelinase 1 (nSMase1) is essential for the generation of these NE buds and cytosolic vesicles by generating ordered lipid microdomains on the NE. Together, our findings show that the spatiotemporal orchestration of chemoattractant‐initiated events leads to the recruitment of the LTB4 biogenesis machinery on the NE followed by NE‐budding and subsequent release of cytosolic vesicles containing exosomes. We envision that this novel mechanism will represent an important means of exosome biogenesis for the release of NE‐associated components in other cells, including immune cells and invasive cancer cells.

Construction and Analysis of lncRNA-Associated ceRNA Network in Atherosclerotic Plaque Formation.

Atherosclerosis (AS) is a vascular disease with plaque formation. Unstable plaques can be expected to result in cardiovascular disease, such as myocardial infarction and stroke. Studies have verified that long noncoding RNAs (lncRNAs) play a critical role in atherosclerotic plaque formation (APF), including MALAT1, GAS5, and H19. A ceRNA network is a combination of these two interacting processes, which regulate the occurrence and progression of many diseases. However, lncRNA-associated ceRNA network in terms of APF is limited. This study sought to discover novel potential biomarkers and ceRNA network for APF. We designed a triple network based on the lncRNA-miRNA and mRNA-miRNA pairs obtained from lncRNASNP and starBase. Differentially expressed genes (DEGs) and lncRNAs in human vascular tissues derived from the Gene Expression Omnibus database (GSE43292, GSE97210) were systematically selected and analyzed. A ceRNA network was constructed by hypergeometric test, including 8 lncRNAs, 243 miRNAs, and 8 mRNAs. APF-related ceRNA structure was discovered for the first time by combining network analysis and statistical validation. Topological analysis determined the key lncRNAs with the highest centroid. GO and KEGG enrichment analysis indicated that the ceRNA network was primarily enriched in “regulation of platelet-derived growth factor receptor signaling pathway,” “negative regulation of leukocyte chemotaxis,” and “axonal fasciculation.” A functional lncRNA, HAND2-AS1, was identified in the ceRNA network, and the main miRNA (miRNA-570-3p) regulated by HAND2-AS1 was further screened. This present study elucidated the important function of lncRNA in the origination and progression of APF and indicated the potential use of these hub nodes as diagnostic biomarkers and therapeutic targets.

Oct4 dependent chromatin activation is required for chicken primordial germ cell migration.

Primordial germ cells (PGCs) are the undifferentiated progenitors of the gametes. Unlike the poor maintenance of cultured mammalian PGCs, the avian PGCs can be expanded in vitro indefinitely while preserving pluripotency and germline competence. In mammals, the Oct4 is the master transcription factor that ensures the stemness of pluripotent cells such as PGCs, but the specific function of Oct4 in chicken PGCs remains unclear. As expected, the loss of Oct4 in chicken PGCs reduced the expression of key pluripotency factors and promoted the genes involved in endoderm and ectoderm differentiation. Furthermore, the global active chromatin was reduced as shown by the depletion of the H3K27ac upon Oct4 suppression. Interestingly, the de-activated chromatin caused the down-regulation of adjacent genes which are mostly known regulators of cell junction, chemotaxis and cell migration. Consequently, the Oct4-deficient PGCs show impaired cell migration and could not colonize the gonads when re-introduced into the bloodstream of the embryo. We propose that, in addition to maintaining pluripotency, the Oct4 mediated chromatin activation is dictating chicken PGC migration.

GINS4 might be a novel prognostic immune-related biomarker of not only esophageal squamous cell carcinoma and other cancers

BackgroundImmunotherapy using immune checkpoint inhibitors (ICIs), such as antibody of programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) has showed as a promising treatment for esophageal squamous cell carcinoma (ESCC), but resistance is unavoidable. This study aimed to find more immune-related genes to promote the efficiency of immunotherapy.Materials and methodsThree datasets were downloaded from Gene Expression Omnibus (GEO) database. Gene differential analysis was performed to identify differentially expressed genes (DEGs), then ceRNA network was constructed based on differentially expressed lncRNAs and mRNAs. Next, Functional enrichment analysis and protein–protein interaction (PPI) network were built to reveal the potential function of mRNAs in ceRNA network. Survival analysis and immune cell infiltration level analysis were utilized to identify prognostic immune-related genes. Finally, pan-cancer analysis was performed to show the role of immune-related genes in other cancers.ResultsThe data of 215 samples in total were obtained from GEO database (98 normal tissues and 117 tumor tissues), and 1685 differentially expressed mRNAs (176 downregulated and 1509 upregulated) and 3 upregulated lncRNAs (MCM3AP-AS1, HCP5 and GUSBP11, all upregulated) were found. ceRNA network was constructed to reveal some special correlation. Function enrichment showed some potential functions of mRNAs in ceRNA network such as mitotic cell cycle process, negative regulation of DNA-binding transcription factor activity, ossification, VEGFA-VEGFR2 signaling pathway, epithelial to mesenchymal transition, embryonic morphogenesis and so on. PPI network showed the physical interactions between each mRNA in ceRNA network. Through survival analysis and immune cell infiltration level analysis, GINS4 was confirmed as an immune-related prognostic gene in ESCC. GSEA showed some potential functions such as negative regulation of monocyte chemotaxis, antigen processing and presentation of endogenous peptide antigen via MHC class I via ER pathway, positive regulation of antigen processing and presentation, dendritic cell antigen processing and presentation and so on. Finally, pan-cancer analysis revealed that GINS4 might be a novel immune-related prognostic gene in ESCC and other cancers.ConclusionOur study suggested that GINS4 was correlated with prognosis and immune cell infiltration level of ESCC and other cancers. It may deserve further investigation as a potential immune-related prognostic biomarker of ESCC and other cancers.

Chemokine CCL9 Is Upregulated Early in Chronic Kidney Disease and Counteracts Kidney Inflammation and Fibrosis.

Inflammation and fibrosis play an important pathophysiological role in chronic kidney disease (CKD), with pro-inflammatory mediators and leukocytes promoting organ damage with subsequent fibrosis. Since chemokines are the main regulators of leukocyte chemotaxis and tissue inflammation, we performed systemic chemokine profiling in early CKD in mice. This revealed (C-C motif) ligands 6 and 9 (CCL6 and CCL9) as the most upregulated chemokines, with significantly higher levels of both chemokines in blood (CCL6: 3–4 fold; CCL9: 3–5 fold) as well as kidney as confirmed by Enzyme-linked Immunosorbent Assay (ELISA) in two additional CKD models. Chemokine treatment in a mouse model of early adenine-induced CKD almost completely abolished the CKD-induced infiltration of macrophages and myeloid cells in the kidney without impact on circulating leukocyte numbers. The other way around, especially CCL9-blockade aggravated monocyte and macrophage accumulation in kidney during CKD development, without impact on the ratio of M1-to-M2 macrophages. In parallel, CCL9-blockade raised serum creatinine and urea levels as readouts of kidney dysfunction. It also exacerbated CKD-induced expression of collagen (3.2-fold) and the pro-inflammatory chemokines CCL2 (1.8-fold) and CCL3 (2.1-fold) in kidney. Altogether, this study reveals for the first time that chemokines CCL6 and CCL9 are upregulated early in experimental CKD, with CCL9-blockade during CKD initiation enhancing kidney inflammation and fibrosis.

PiR-121380 Is Involved in Cryo-Capacitation and Regulates Post-Thawed Boar Sperm Quality Through Phosphorylation of ERK2 via Targeting PTPN7.

Cryopreservation induces capacitation-like (cryo-capacitation) changes, similar to natural capacitation, and affects the fertility potential of post-thawed sperm. The molecular mechanism of sperm cryo-capacitation during cryopreservation remains unknown. PIWI-interacting RNAs (piRNAs) have been reported to be involved in cryo-capacitation of post-thawed sperm and regulation of sperm motility, capacitation, and chemotaxis. In this study, protein tyrosine phosphatase nonreceptor type 7 (PTPN7) was positively targeted by piR-121380 after a dual luciferase assay. The mRNA expression of PTPN7 and piR-121380 was significantly decreased (p < 0.01); however, PTPN7 protein was significantly increased (p < 0.01) in post-thawed boar sperm. Furthermore, E1RK1/2 phosphorylation was reduced during cryopreservation. Six hours after transfection with piR-121380 mimic and inhibitor, the phosphorylation of ERK2 was significantly increased and decreased (p < 0.01), respectively. Furthermore, the highest and lowest total sperm motility, forward motility, and capacitation rate were observed after piR-121380 mimic and inhibitor treatments, respectively. The concentration of intracellular calcium ([Ca2+]i) showed no significant difference after transfection with either piR-121380 mimic or inhibitor at 1, 3, and 6 h. In conclusion, we demonstrated that piR-121380 modulates ERK2 phosphorylation by targeting PTPN7, which induces sperm cryo-capacitation, and eventually affects the motility and fertility potential of post-thawed sperm.