Cell Transcription Factors Research Articles

Transcriptomic and functional analyses reveal the molecular mechanisms underlying Fe-mediated tobacco resistance to potato virus Y infection.

Potato virus Y (PVY) mainly infects Solanaceous crops, resulting in considerable losses in the yield and quality. Iron (Fe) is involved in various biological processes in plants, but its roles in resistance to PVY infection has not been reported. In this study, foliar application of Fe could effectively inhibit early infection of PVY, and a full-length transcriptome and Illumina RNA sequencing was performed to investigate its modes of action in PVY-infected Nicotiana tabacum. The results showed that 18,074 alternative splicing variants, 3,654 fusion transcripts, 3,086 long non-coding RNAs and 14,403 differentially expressed genes (DEGs) were identified. Specifically, Fe application down-regulated the expression levels of the DEGs related to phospholipid hydrolysis, phospholipid signal, cell wall biosynthesis, transcription factors (TFs) and photosystem I composition, while those involved with photosynthetic electron transport chain (PETC) were up-regulated at 1 day post inoculation (dpi). At 3 dpi, these DEGs related to photosystem II composition, PETC, molecular chaperones, protein degradation and some TFs were up-regulated, while those associated with light-harvesting, phospholipid hydrolysis, cell wall biosynthesis were down-regulated. At 9 dpi, Fe application had little effects on resistance to PVY infection and transcript profiles. Functional analysis of these potentially critical DEGs was thereafter performed using virus-induced gene silencing approaches and the results showed that NbCat-6A positively regulates PVY infection, while the reduced expressions of NbWRKY26, NbnsLTP, NbFAD3 and NbHSP90 significantly promote PVY infection in N. benthamiana. Our results elucidated the regulatory network of Fe-mediated resistance to PVY infection in plants, and the functional candidate genes also provide important theoretical bases to further improve host resistance against PVY infection.

Super-enhancer-associated transcription factors collaboratively regulate trophoblast-active gene expression programs in human trophoblast stem cells.

The placenta is an essential organ that supports the growth and development of the fetus during pregnancy. However, cell type-specific enhancers and transcription factors (TFs), and the mechanisms underlying the maintenance and differentiation of trophoblast stem cell (TSC) populations in the human placenta remain elusive. Here, using human TSCs as a model system, we identify 31,362 enhancers that are enriched with the motifs of previously reported TSC-pivotal TFs, including TEAD4, GATA2/3 and TFAP2C. Subsequently, we identify 580 super-enhancers (SEs) and 549 SE-associated genes. These genes are robustly expressed in the human placenta and include numerous TFs, implying that SE-associated TFs (SE-TFs) may play crucial roles in placental development. Additionally, we identify the global binding sites of five TSC-pivotal SE-TFs (FOS, GATA2, MAFK, TEAD4 and TFAP2C), revealing that they preferentially co-occupy enhancers, regulate each otherand form a trophoblast-active gene regulatory network. Loss-of-function studies unveil that the five TFs promote self-renewal of TSCs by activating proliferation-associated genes while repressing developmental genes. We further reveal that the five TFs exert conserved and unique functions on placental development between humans and mice. Our study provides important insights into the roles of human TSC-pivotal TFs in regulating placenta-specific gene expression programs.

Chromatin confinement enhances target search of pioneer transcription factors in live cells

Chromatin confinement enhances target search of pioneer transcription factors in live cells

Co-regulation of Sox9 and TGFβ1 transcription factors in mesenchymal stem cells regenerated the intervertebral disc degeneration.

Intervertebral disc (IVD) shows aging and degenerative changes earlier than any other body connective tissue. Its repair and regeneration provide a considerable challenge in regenerative medicine due to its high degree of infrastructure and mechanical complexity. Mesenchymal stem cells, due to their tissue resurfacing potential, represent many explanatory pathways to regenerate a tissue breakdown. This study was undertaken to evaluate the co-regulation of Sox9 and TGFβ1 in differentiating human umbilical cord mesenchymal stem cells (hUC-MSC) into chondrocytes. The combinatorial impact of Sox9 and TGFβ1 on hUC-MSCs was examined in vitro by gene expression and immunocytochemical staining. In in vivo, an animal model of IVD degeneration was established under a fluoroscopic guided system through needle puncture of the caudal disc. Normal and transfected MSCs were transplanted. Oxidative stress, pain, and inflammatory markers were evaluated by qPCR. Disc height index (DHI), water content, and gag content were analyzed. Histological examinations were performed to evaluate the degree of regeneration. hUC-MSC transfected with Sox9+TGFβ1 showed a noticeable morphological appearance of a chondrocyte, and highly expressed chondrogenic markers (aggrecan, Sox9, TGFβ1, TGFβ2, and type II collagens) after transfection. Histological observation demonstrated that cartilage regeneration, extracellular matrix synthesis, and collagen remodeling were significant upon staining with H&E, Alcian blue, and Masson's trichrome stain on day 14. Additionally, oxidative stress, pain, and inflammatory markers were positively downregulated in the animals transplanted with Sox9 and TGFβ1 transfected MSCs. These findings indicate that the combinatorial effect of Sox9 and TGFβ1 substantially accelerates the chondrogenesis in hUC-MSCs. Cartilage regeneration and matrix synthesis were significantly enhanced. Therefore, a synergistic effect of Sox9 and TGFβ1 could be an immense therapeutic combination in the tissue engineering of cartilaginous joint bio-prostheses and a novel candidate for cartilage stabilization.

A cybergenetic framework for engineering intein-mediated integral feedback control systems

The ability of biological systems to tightly regulate targeted variables, despite external and internal disturbances, is known as Robust Perfect Adaptation (RPA). Achieved frequently through biomolecular integral feedback controllers at the cellular level, RPA has important implications for biotechnology and its various applications. In this study, we identify inteins as a versatile class of genetic components suitable for implementing these controllers and present a systematic approach for their design. We develop a theoretical foundation for screening intein-based RPA-achieving controllers and a simplified approach for modeling them. We then genetically engineer and test intein-based controllers using commonly used transcription factors in mammalian cells and demonstrate their exceptional adaptation properties over a wide dynamic range. The small size, flexibility, and applicability of inteins across life forms allow us to create a diversity of genetic RPA-achieving integral feedback control systems that can be used in various applications, including metabolic engineering and cell-based therapy.

Detection of Novel Pathogenic Variants in Two Families with Recurrent Fetal Congenital Heart Defects.

Congenital heart disease (CHD) is the most common birth defect with strong genetic heterogeneity. To date, about 400 genes have been linked to CHD, including cell signaling molecules, transcription factors, and structural proteins that are important for heart development. Genetic analysis of CHD cases is crucial for clinical management and etiological analysis. Whole-exome sequencing (WES) was performed to identify the genetic variants in two independent CHD cases with DNA samples from fetuses and their parents, followed by the exclusion of aneuploidy and large copy number variations (CNVs). The WES results were verified by Sanger sequencing. In family A, a compound heterozygous variation in PLD1 gene consisting of c.1132dupA (p.I378fs) and c.1171C>T (p.R391C) was identified in the fetus. The two variants were inherited from the father (c.1132dupA) and the mother (c.1171C>T), respectively. In family B, a hemizygous variant ZIC3: c.861delG (p.G289Afs*119) was identified in the fetus, which was inherited from the heterozygous mother. We further confirmed that these variants PLD1: c.1132dupA and ZIC3: c.861delG were novel. The findings in our study identified novel variants to the mutation spectrum of CHD and provided reliable evidence for the recurrent risk and reproductive care options to the affected families. Our study also demonstrates that WES has considerable prospects of clinical application in prenatal diagnosis.

Restricting genomic actions of innate immune mediators on fetal hematopoietic progenitor cells

Innate immune signaling protects against pathogens, controls hematopoietic development, and functions in oncogenesis, yet the relationship between these mechanisms is undefined. Downregulating the GATA2 transcription factor in fetal hematopoietic progenitor cells upregulates genes encoding innate immune regulators, increases Interferon-γ (IFNγ) signaling, and disrupts differentiation. We demonstrate that deletion of an enhancer that confers GATA2 expression in fetal progenitors elevated Toll-like receptor (TLR) TLR1/2 and TLR2/6 expression and signaling. Rescue by expressing GATA2 downregulated elevated TLR signaling. IFNγ amplified TLR1/2 and TLR2/6 signaling in GATA2-deficient progenitors, synergistically activating cytokine/chemokine genes and elevating cytokine/chemokine production in myeloid cell progeny. Genomic analysis of how innate immune signaling remodels the GATA2-deficient progenitor transcriptome revealed hypersensitive responses at innate immune genes harboring motifs for signal-dependent transcription factors and factors not linked to these mechanisms. As GATA2 establishes a transcriptome that constrains innate immune signaling, insufficient GATA2 renders fetal progenitor cells hypersensitive to innate immune signaling.

MYC promotes fibroblast osteogenesis by regulating ALP and BMP2 to participate in ectopic ossification of ankylosing spondylitis

BackgroundEctopic ossification is an important cause of disability in patients with ankylosing spondylitis (AS). Whether fibroblasts can transdifferentiate into osteoblasts and contribute to ossification remains unknown. This study aims to investigate the role of stem cell transcription factors (POU5F1, SOX2, KLF4, MYC, etc.) of fibroblasts in ectopic ossification in patients with AS.MethodsPrimary fibroblasts were isolated from the ligaments of patients with AS or osteoarthritis (OA). In an in vitro study, primary fibroblasts were cultured in osteogenic differentiation medium (ODM) to induce ossification. The level of mineralization was assessed by mineralization assay. The mRNA and protein levels of stem cell transcription factors were measured by real-time quantitative PCR (q-PCR) and western blotting. MYC was knocked down by infecting primary fibroblasts with lentivirus. The interactions between stem cell transcription factors and osteogenic genes were analysed by chromatin immunoprecipitation (ChIP). Recombinant human cytokines were added to the osteogenic model in vitro to evaluate their role in ossification.ResultsWe found that MYC was elevated significantly in the process of inducing primary fibroblasts to differentiate into osteoblasts. In addition, the level of MYC was remarkably higher in AS ligaments than in OA ligaments. When MYC was knocked down, the expression of the osteogenic genes alkaline phosphatase (ALP) and bone morphogenic protein 2 (BMP2) was decreased, and the level of mineralization was reduced significantly. In addition, the ALP and BMP2 were confirmed to be the direct target genes of MYC. Furthermore, interferon-γ (IFN-γ), which showed high expression in AS ligaments, was found to promote the expression of MYC in fibroblasts in the process of ossification in vitro.ConclusionsThis study demonstrates the role of MYC in ectopic ossification. MYC may act as the critical bridge that links inflammation with ossification in AS, thus providing new insights into the molecular mechanisms of ectopic ossification in AS.

IL-9-Producing Th9 Cells Participate in the Occurrence and Development of Iodine-Induced Autoimmune Thyroiditis.

Iodine excess may cause and aggravate autoimmune thyroiditis (AIT), which is regarded as a typical kind of autoimmune disease mainly mediated by CD4+ T cells. Thus far, it is unclear whether T helper (Th) 9 cells, a novel subpopulation of CD4+ T cells, play a potential role in AIT. Therefore, in the present study, changes in Th9 cells were detected in murine models of AIT induced by excess iodine intake to explore the possible immune mechanism. Female C57BL/6 mice were divided into 7 groups (n = 8) and were supplied with water containing 0.005% sodium iodide for 0, 2, 4, 6, 8, 10, and 12weeks. With the extension of the high-iodine intake duration, the incidence of thyroiditis and the spleen index were significantly increased, and serum thyroglobulin antibody (TgAb) titers and interleukin 9 (IL-9, major cytokine from Th9 cells) concentrations were also increased. Additionally, it was revealed that the percentages of Th9 cells in spleen mononuclear cells (SMCs) and thyroid tissues were both markedly elevated and accompanied by increased mRNA and protein expression of IL-9 and key transcription factors of Th9 cells (PU.1 and IRF-4). Significantly, dynamic changes in Th9 cells were found, with a peak at 8weeks after high iodine intake, the time point when thyroiditis was the most serious. Importantly, Th9 cells were detected in the areas of infiltrating lymphocytes in thyroid sections. In conclusion, the continuously increasing proportions of Th9 cells may play an important role in the occurrence and development of AIT induced by high iodine intake.

Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation.

Quercetin 3-O-galactoside (Q3G) is a common dietary flavanol that has been shown to possess several bioactivities, including anti-melanogenesis. However, how Q3G exerts its anti-melanogenic effect has not been studied. The current study, therefore aimed to investigate the anti-melanogenesis potential of Q3G and elucidate the underlying action mechanism in α-melanocyte-stimulating hormone (α-MSH)-induced hyperpigmentation model of B16F10 murine melanoma cells. Results showed that α-MSH stimulation significantly increased tyrosinase (TYR) and melanin production, which were significantly downregulated by Q3G treatment. The treatment with Q3G suppressed the transcriptional and protein expressions of melanogenesis-related enzymes TYR, tyrosinase related protein-1 (TRP-1), and TRP-2, along with the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) in B16F10 cells. It was shown that Q3G downregulated MITF expression and suppressed its transcriptional activity by inhibiting the cAMP-dependent protein kinase A (PKA)-mediated activation of CREB and GSK3β. In addition, MAPK-regulated MITF activation signaling was also involved in the inhibition of melanin production by Q3G. The results suggest that the anti-melanogenic properties of Q3G rationalize further studies in vivo to confirm its action mechanism and consequent utilization as a cosmetic ingredient against hyperpigmentation.

JAK2 inhibitor ameliorates the progression of experimental autoimmune myasthenia gravis and balances Th17/Treg cells via regulating the JAK2/STAT3-AKT/mTOR signaling pathway.

An imbalance in Th17/regulatory T (Treg) cells is the major pathogenic mechanism underlying myasthenia gravis (MG). JAK2 inhibitors selectively inhibit JAK2 and reduce inflammatory responses. However, there have been no studies examining the therapeutic effects of JAK2 inhibitors in the context of MG. Here, an experimental autoimmune MG (EAMG) rat model was established to explore the therapeutic effect of JAK2 inhibitors on EAMG rats immunized with the AChR α-subunit (97-116 peptide). A JAK2 inhibitor was administered to EAMG rats both in vivo and in vitro. The following experimental methods were used to evaluate the effects of JAK2 inhibitors. The behavioral scores and body weights of the rats were assessed on alternate days. Serum anti-AChR (97-116) IgG and cytokine levels were detected using ELISA. CD4+ T cell subsets and related transcription factors in mononuclear cells were detected using flow cytometry and qPCR, respectively. The expression levels of protein molecules in the signaling pathway were detected by western blotting, and the neuromuscular junctions were observed using immunofluorescence. The results revealed that JAK2 inhibitors could regulate Th17/Treg balance in vivo and in vitro. JAK2 inhibitors reduced the immune response in EAMG rats (including reducing pro-inflammatory cytokines and postsynaptic membrane complement deposition), improved clinical symptoms, and increased AChR aggregation in the postsynaptic membrane. Meanwhile, this study demonstrated that JAK2 inhibitor treatment suppressed the phosphorylation of JAK2/STAT3 and AKT/mTOR pathways and decreased the expression level of the IL-23 receptor. This study reveals that there is crosstalk between the JAK2/STAT3 and AKT/mTOR pathways in EAMG rats. JAK2 inhibitors can ameliorate EAMG by regulating Th17/Treg balance by inhibiting both signaling pathways. Our study provides new potential therapeutic targets for MG immunotherapy.

Abstract WP229: Hub Genes Drive Specific Gene Expression Changes Seen In Intracerebral Hemorrhage And Ischemic Stroke.

Gene expression changes in peripheral leukocytes display distinctive profiles after intracerebral hemorrhage (ICH) and ischemic stroke (IS), differentiating both conditions at the molecular level. The breadth of data produced by high-throughput transcriptomic analyses can identify groups of genes and main gene expression drivers that have meaningful functional associations with the disease. This can help prioritize the investigation of key genes for diagnosis and treatment. Thus, we performed whole transcriptome analyses on ICH and IS samples and constructed gene networks from a genome-wide perspective. RNA-seq was performed on peripheral blood (WB) and isolated monocytes (MON) and neutrophils (NEU) (n=6 ICH, n=33 IS and n=9 vascular risk factors control (VRFC) subjects). Gene expression results were used to construct separate co-expression networks for all datasets analyzed (ICH + VRFCs, and IS + VRFCs, for MON, NEU and WB) using Weighted Gene Co-expression Network Analysis. Modules of genes significantly associated with ICH in the ICH + VRFCs network, and with IS in the IS + VRFCs network, were identified. The most highly interconnected genes in each of these modules were identified, representing hub genes that are potential master regulators. Functional annotation of the modules and hubs were done using gene ontology. From the significantly associated modules for ICH and IS in all sample types analyzed, there was little overlap in genes between diagnoses (≤2% in MON, ≤21 % in NEU and ≤16% in WB), and no overlap of ICH or IS hub genes from MON and NEU. In WB, ≤2% of the hubs were common to ICH and IS. It is plausible that these potential master regulators drive diagnosis-specific gene expression profiles. ICH hubs were associated with RNA splicing and mRNA processing (MON), cell adhesion (NEU) and NF-κβ signaling (WB). IS hubs were associated with cell migration (MON), T cell chemotaxis (NEU) and transcription factor activity (WB). In addition, most hubs in IS MON were noncoding RNA. The gene networks and their respective hub genes provide novel cell-specific pathophysiological insights and could represent potential key pharmacological targets and biomarkers.

Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes.

Drosophila melanogaster reproductive behaviors are orchestrated by fruitless neurons. We performed single-cell RNA-sequencing on pupal neurons that produce sex-specifically spliced fru transcripts, the fru P1-expressing neurons. Uniform Manifold Approximation and Projection (UMAP) with clustering generates an atlas containing 113 clusters. While the male and female neurons overlap in UMAP space, more than half the clusters have sex differences in neuron number, and nearly all clusters display sex-differential expression. Based on an examination of enriched marker genes, we annotate clusters as circadian clock neurons, mushroom body Kenyon cell neurons, neurotransmitter- and/or neuropeptide-producing, and those that express doublesex. Marker gene analyses also show that genes that encode members of the immunoglobulin superfamily of cell adhesion molecules, transcription factors, neuropeptides, neuropeptide receptors, and Wnts have unique patterns of enriched expression across the clusters. In vivo spatial gene expression links to the clusters are examined. A functional analysis of fru P1 circadian neurons shows they have dimorphic roles in activity and period length. Given that most clusters are comprised of male and female neurons indicates that the sexes have fru P1 neurons with common gene expression programs. Sex-specific expression is overlaid on this program, to build the potential for vastly different sex-specific behaviors.

The immunomodulatory effect of intradermal allogeneic PBMC therapy in patients with recurrent spontaneous abortion

BackgroundThere has been limited study on the impact of PBMC therapy in RSA patients with immunological disorders such as Th17 and Treg cell dysregulation, as well as their associated factors. This study aimed to assess the efficacy of PBMC therapy in modulating immune cell frequency, cytokine production, transcription factors, and miRNAs implicated in the regulation of their function, as well as their potential superiority to routine treatments. MethodsFifty RSA women who had received PBMCs and 50 matched-paired control RSA women who had received the routine treatments were recruited and followed for three months. The frequencies of Th17, Treg, NK, and B cells were assessed using flow cytometry. Thereafter, the gene expression level of the transcription factors and related miRNAs of Treg cell and Th17 cell was quantified using RT-PCR. Then ELISA was employed to assess the cytokine production of Th17 and Treg cells. Finally, the live birth rate and miscarriage rate were evaluated as clinical outcomes in this study. ResultsFlow cytometry analysis revealed that PBMC therapy significantly reduces the frequencies of Th17 and NK cells while enhancing the frequency of Treg cells. RT-PCR analysis confirmed that PBMC therapy significantly downregulates RORγt and upregulates FoxP3. Likewise, RT-PCR analysis showed that PBMC therapy reduces the expression of miR-25, miR-155, and miR-326 while increasing the expression of miR-10a. ELISA results demonstrated that PBMC therapy considerably decreases the concentration of inflammatory cytokines IL-1β, IL-17, and TNF-α and enhances the concentration of anti-inflammatory cytokines IL-10 and TGF-β. Following PBMC therapy live birth rate raised while miscarriage rate reduced. ConclusionOur findings suggested that, in contrast to routine treatments, PBMC therapy can significantly modulate the maternal immune system by enhancing the Treg/Th17 paradigm and regulating the expression of Treg and Th17 cell-associated cytokines, transcription factors, and miRNAs. This treatment also can increase the live birth rate in RSA patients.

Extracellular HSP90 promotes differentiation of lens epithelial cells to fiber cells by activating LRP1-YAP-PROX1 axis.

Capsular residual lens epithelial cells (CRLEC) undergo differentiation to fiber cells for lens regeneration or tansdifferentiation to myofibroblasts leading to posterior capsular opacification (PCO) after cataract surgery. The underlying regulatory mechanism remains unclear. Using human lens epithelial cell lines and the ex vivo cultured rat lens capsular bag model, we found that the lens epithelial cells secrete HSP90α extracellularly (eHSP90) through an autophagy-associated pathway. Administration of recombinant GST-HSP90α protein or its M-domain induces the elongation of rat CRLEC cells with concomitant upregulation of the crucial fiber cell transcriptional factor PROX1and its downstream targets, β- and γ-crystallins and structure proteins. This regulation is abolished by PROX1 siRNA. GST-HSP90α upregulates PROX1 by binding to LRP1 and activating LRP1-AKT mediated YAP degradation. The upregulation of GST-HSP90α on PROX1 expression and CRLEC cell elongation is inhibited by LRP1 and AKT inhibitors, but activated by YAP-1 inhibitor (VP). These data demonstrated that the capsular residue epithelial cells upregulate and secrete eHSP90α, which in turn drive the differentiation of lens epithelial cell to fiber cells. The recombinant HSP90α protein is a potential novel differentiation regulator during lens regeneration.

Analysis of regulatory sequences in exosomal DNA of NANOGP8.

Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined.

Mitogen-Activated Protein Kinases Mediate Adventitial Fibroblast Activation and Neointima Formation via GATA4/Cyclin D1 Axis.

Activation of mitogen-activated protein kinases (MAPKs) by pathological stimuli participates in cardiovascular diseases. Dysfunction of adventitial fibroblast has emerged as a critical regulator in vascular remodeling, while the potential mechanism remains unclear. In this study, we sought to determine the effect of different activation of MAPKs in adventitial fibroblast contributing to neointima formation. Balloon injury procedure was performed in male 12-week-old Sprague-Dawley rats. After injury, MAPK inhibitors were applied to the adventitia of injured arteries to suppress MAPK activation. Adventitial fibroblasts were stimulated by platelet-derived growth factor-BB (PDGF-BB) with or without MAPK inhibitors. RNA sequencing was performed to investigate the change of pathway and cell function. Wound healing, transwell assay, and flow cytometry were used to analyze adventitial fibroblast function. Phosphorylation of p38, c-Jun N-terminal kinase (JNK), andextracellular regulated kinases 1/2 (ERK1/2) was increased in injured arteries after balloon injury. In primary culture of adventitial fibroblasts, PDGF-BB increased phosphorylation of p38, JNK, ERK1/2, andextracellular regulated kinase 5 (ERK5) in a short time, which was normalized by their inhibitors respectively. Compared with the injury group, perivascular administration of four MAPK inhibitors significantly attenuated neointima formation by quantitative analysis of neointimal area, intima to media (I/M) ratio, and lumen area. RNA sequencing of adventitial fibroblasts treated with PDGF-BB with or without four inhibitors demonstrated differentially expressed genes involved in multiple biological processes, including cell adhesion, proliferation, migration, and inflammatory response. Wound healing and transwell assays showed that four inhibitors suppressed PDGF-BB-induced adventitial fibroblast migration. Cell cycle analysis by flow cytometry demonstrated that JNK, ERK1/2, and ERK5 but not p38 inhibitor blocked PDGF-BB-induced G1 phase release associated with decrease expression of cell cycle protein Cyclin D1 and transcription factor GATA4. Moreover, four inhibitors decreased macrophage infiltration into adventitia and monocyte chemoattractant protein-1 (MCP-1) expression. These results suggest that MAPKs differentially regulate activation of adventitial fibroblast through GATA4/Cyclin D1 axis that participates in neointima formation.

Genetic Interference of FGFR3 Impedes Invasion of Upper Tract Urothelial Carcinoma Cells by Alleviating RAS/MAPK Signal Activity.

Upper tract urothelial cancer (UTUC) is a less common disease in Western countries but has a high level of prevalence in Asian populations. Compared to bladder cancer, unique etiologic and genomic factors are involved in UTUC. Fibroblast growth factor receptor 3 (FGFR3) up-regulation has been proposed as a promising target for bladder cancer therapy. In this study, we aimed to profile the expression of FGFR3 in Asian and Caucasian UTUC tissues and to evaluate the in vitro therapeutic efficacy of small interference RNA (siRNA)-mediated FGFR3 silencing in UTUC treatment. The FGFR3 expression levels in renal pelvis tissues and microarray sections from Asian and Caucasian patients with UTUC, respectively, were measured via immunohistochemistry. The BFTC-909 and UM-UC-14 UTUC cell lines were used to examine the effects of FGFR3 silencing on proliferation, migration, epithelial-mesenchymal transition (EMT) marker expression, and signaling machinery. FGFR3 expression increased as the TNM stage increased in both Asian and Caucasian UTUC tumors, and no statistical difference was identified between the two groups. In vitro studies demonstrated that FGFR3 siRNA delivery significantly inhibited proliferation and migration and suppressed the expression of EMT markers and transcription factors in UTUC cells. Mechanistically, FGFR3 silencing alleviated the constitutive expression of RAS and the phosphorylation of MAPK signaling mediators, including ERK1/2 and JNK1/2. FGFR3 silencing elicited an apoptosis-inducing effect similar to that of FGFR inhibition. Conclusion: siRNA-targeted FGFR3 expression may impede the expansion and invasion of UTUC cells by alleviating the RAS/MAPK signaling pathway. The genetic interference of FGFR3 expression via siRNA in UTUC cells may constitute a useful therapeutic strategy.

Shifting gears: Study of immune system parameters of male habitual marathon runners.

Marathon is a running event in which athletes must cover a distance of 42.195 km. In addition to participating in marathons, marathoners have incorporated extensive running into their lifestyle. In the present study, we investigated the effect of long-term strenuous exercise in the form of marathon running on the immune system. We collected peripheral blood samples from 37 male marathoners before/after a race and 37 age/sex/body mass index (BMI)-matched healthy sedentary controls. Hematological and biochemical tests revealed race-induced leukocytosis attributable to neutrophilia and significant increases in plasma lactate dehydrogenase (LDH), creatine phosphokinase (CPK), and cortisol concentrations. Phenotypic analysis of lymphocytes revealed race-induced significant decrease in the number of lymphocytes, memory helper T (Th) cells, naive, memory and activated cytotoxic T (Tc) cells, natural killer (NK), NKT, and B1 cells, and a significant increase in the number of activated Th and regulatory Th cells (Tregs). Compared with controls, marathoners maintained significantly lower levels of memory and activated Th cells and higher levels of activated Tc and B1 cells. Measurement of plasma cytokine levels revealed a pro-inflammatory cytokine polarization that increased after the race. Examination of gene expression of cytokines and Th-cell signature transcription factors in peripheral blood mononuclear cells revealed a significant decrease in tumor necrosis factor α (TNF-α) and interleukin (IL)-17, and a significant increase in IL-6, IL-10 and forkhead box P3 (FoxP3) after the race. Compared with controls, marathoners maintained significantly higher levels of TNF-α. Assessment of the suppressive capacity of Tregs in co-cultures of isolated effector Th cells and Tregs showed significantly increased suppressive capacity of marathoners' Tregs after the race. Compared with controls, marathoners live with permanent changes in certain immune parameters. Marathoners exhibit a stable pro-inflammatory cytokine polarization that increases after the race and is counterbalanced by increased numbers of Tregs overexpressing FoxP3 and having increased suppressive capacity.

PsychArray-Based Genome Wide Association Study of Suicidal Deaths in India.

Suicide is a preventable but escalating global health crisis. Genome-wide association studies (GWAS) studies to date have been limited, and some are underpowered. In this study, we aimed to perform the PsychArray-based GWAS study to identify single nucleotide variations associated with suicide in the Indian population. We recruited unrelated subjects who died by suicide as cases (N = 313) and the non-suicidal deaths as controls (N = 294). The 607 samples were genotyped, including cases and controls using the Illumina Infinium PsychArray-24 BeadChip v1.3 Results: In our study, four single nucleotide polymorphisms (SNPs) crossed the threshold of significance level <1 × 10-5. One of them is intronic at Chromosome2:rs1901851 and three are intergenic at Chromosome12:rs3847911, Chromosome8:rs2941489, Chromosome8:rs1464092. At a significance level of 5 × 10-5, we found a few more SNPs, with the majority of them being intergenic variants. The associated genes were associated with various important functions ranging from cell signaling, GTP binding, GPCR binding, and transcription factor binding. The SNPs identified in our study were not reported earlier. To our best knowledge, this study is one of the first GWAS for suicide in the Indian population. The results indicate few novel SNPs that may be associated with suicide and require further investigation. Their clinical significance is to be studied in the future.