Regulation Of Cell Differentiation Research Articles

Molecular Docking, Molecular Dynamics Simulation, and Analysis of EGFR-derived Peptides against the EGF

Background: The epidermal growth factor receptor (EGFR) is a member of the tyrosine kinase receptor family known as ErbB. The EGFR signaling pathway is an important regulator of cell proliferation, differentiation, division, and survival, as well as cancer development in humans. Epidermal growth factor, betacellulin, amphiregulin, transforming growth factor and heparin-binding EGF-like growth factor are high-affinity ligands of EGFR. Objective: Tumor progression can be effectively prevented by inhibiting EGF/EGFR interactions. In this study, many anti-EGF peptides targeting EGFR binding regions were designed, modeled, and evaluated. After selecting the peptides with the highest binding energy to the EGF, the interactions between the candidate peptides and all of the key EGFR ligands were investigated. Methods: To identify an EGF-binding peptide capable of blocking EGFR-EGF interactions, large-scale peptide mutation screening was performed. Using the AntiCP server, several possible peptides with anticancer properties were identified. The ClusPro analysis was performed in order to analyze the interactions between EGF and all of the library peptides. A total of five peptides with favorable docking scores were identified. The stability of three peptides with the best docking scores in complex with EGF was verified, applying molecular dynamics simulation with the help of the GROMACS software package. Finally, the interaction of candidate peptides with transforming growth factor-alpha, heparin-binding EGF-like growth factor, and betacellulin was investigated using the ClusPro server. Results: After the screening of modeled peptides by the ClusPro server and GROMACS software, two anti-EGF peptides of Pep4 and Pep5 with 31 residues were developed. Then, we demonstrate that both of these peptides can bind to the other high-affinity ligands of EGFR and block TGFA/EGFR, HBEGF/EGFR, and BTC/EGFR interactions. Conclusion: The findings suggest novel insights for developing therapies based on peptides for inhibiting the EGF, TGFA, HBEGF, and BTC signaling cascade in cancer cells. Pep4 and Pep5 designed in this work, are recommended as potentially promising anticancer peptides for further experimental evaluation.

MiR-3680-3p is a novel biomarker for the diagnosis and prognosis of liver cancer and is involved in regulating the progression of liver cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that can actively participate in post-transcriptional regulation of genes. A number of studies have shown that miRNAs can serve as important regulators of cancer cell growth, differentiation, and apoptosis. They can also act as markers for the diagnosis and prognosis of certain cancers. To explore the potential prognosis-related miRNAs in liver cancer patients, to provide theoretical basis for early diagnosis and prognosis of liver cancer, as well as to provide a new direction for the targeted therapy of liver cancer. The miRNA expression profiles of liver cancer patients in the the Cancer Genome Atlas database were comprehensively analyzed and various prognostic-related miRNAs of liver cancer were screened out. The data was further subjected to survival analysis, prognostic analysis, gene ontology and kyoto encyclopedia of genes and genomes enrichment analysis, microenvironment analysis, and drug sensitivity analysis by R Language version 4.2.0. Finally, the screened miRNAs were further validated by different experiments. Thus, miNRAs involved in liver cancer diagnosis and prognosis were identified. MiRNA-3680-3p was found to be significantly different in 10 different cancers, including liver cancer, and was significantly associated with the microenvironment, survival, and prognosis of liver cancer patients. In addition, drug sensitivity analysis revealed that miRNA-3680-3p can provide a useful reference for drug selection in targeted therapy for liver cancer. MiRNA-3680-3p can serve as a biomarker for the diagnosis and prognosis of liver cancer patients and down-regulation of miRNA-3680-3p could significantly inhibit both the proliferation and migration of liver cancer cells.

Epigenetic control of SOX9 gene by the histone acetyltransferase P300 in human Sertoli cells

BackgroundThe transcription factor SOX9 is a key regulator of male sexual development and Sertoli cell differentiation. Altered SOX9 expression has been implicated in the pathogenesis of disorders of sexual development (DSD) in mammals. However, limited information exists regarding the epigenetic mechanisms governing its transcriptional control during sexual development. MethodsThis study employed real-time PCR (qPCR), immunofluorescence (IIF), and chromatin immunoprecipitation (ChIP) assays to investigate the epigenetic mechanisms associated with SOX9 gene transcriptional control in human and mouse Sertoli cell lines. To identify the specific epigenetic enzymes involved in SOX9 epigenetic control, functional assays using siRNAs for P300, GCN5, and WDR5 were performed. ResultsThe transcriptional activation of SOX9 was associated with selective deposition of active histone modifications, such as H3K4me3 and H3K27ac, at its enhancer and promoter regions. Importantly, the histone acetyltransferase P300 was found to be significantly enriched at the SOX9 enhancers, co-localizing with the H3K27ac and the SOX9 transcription factor. Silencing of P300 led to decreased SOX9 expression and reduced H3K27ac levels at the eSR-A and e-ALDI enhancers, demonstrating the crucial role of P300-mediated histone acetylation in SOX9 transcriptional activation. Interestingly, another histone lysine acetyltransferases like GNC5 and methyltransferases as the Trithorax/COMPASS-like may also have a relevant role in male sexual differentiation. ConclusionsHistone acetylation by P300 at SOX9 enhancers, is a key mechanism governing the transcriptional control of this essential regulator of male sexual development. These findings provide important insights into the epigenetic basis of sexual differentiation and the potential pathogenesis of DSDs.

A Methylation-Gated DNAzyme Circuit for Spatially Controlled Imaging of MicroRNA in Cells and Animals.

Epigenetic modification plays an indispensable role in regulating routine molecular signaling pathways, yet it is rarely used to modulate molecular self-assembly networks. Herein, we constructed a bioorthogonal demethylase-stimulated DNA circuitry (DSC) system for high-fidelity imaging of microRNA (miRNA) in live cells and mice by eliminating undesired off-site signal leakage. The simple and robust DSC system is composed of a primary cell-specific circuitry regulation (CR) module and an ultimate signal-transducing amplifier (SA) module. After the modularly designed DSC system was delivered into target live cells, the DNAzyme of the CR module was site-specifically activated by endogenous demethylase to produce fuel strands for the subsequent miRNA-targeting SA module. Through the on-site and multiply guaranteed molecular recognitions, the lucid yet efficient DSC system realized the reliably amplified in vivo miRNA sensing and enabled the in-depth exploration of the demethylase-involved signal pathway with miRNA in live cells. Our bioorthogonally on-site-activated DSC system represents a universal and versatile biomolecular sensing platform via various demethylase regulations and shows more prospects for more different personalized theragnostics.

Mechanisms and Advances of Epigenetic Regulation in Cardiovascular Disease

Epigenetics refers to heritable changes in gene expression and function that impact nuclear processes associated with chromatin, all without altering DNA sequences. These epigenetic patterns, being heritable traits, are vital biological mechanisms that intricately regulate gene expression and heredity. The application of chemical labeling and single-cell resolution mapping strategies has significantly facilitated large-scale epigenetic modifications in nucleic acids over recent years. Notably, epigenetic modifications can induce heritable phenotypic changes, regulate cell differentiation, influence cell-specific gene expression, parentally imprint genes, activate the X chromosome, and stabilize genome structure. Given their reversibility and susceptibility to environmental factors, epigenetic modifications have gained prominence in disease diagnosis, significantly impacting clinical medicine research. Recent studies have uncovered strong links between epigenetic modifications and the pathogenesis of metabolic cardiovascular diseases, including congenital heart disease, heart failure, cardiomyopathy, hypertension, and atherosclerosis. In this review, we provide an overview of the progress in epigenetic research within the context of cardiovascular diseases, encompassing their pathogenesis, prevention, diagnosis, and treatment. Furthermore, we shed light on the potential prospects of nucleic acid epigenetic modifications as a promising avenue in clinical medicine and biomedical applications.

Single-Cell Profiling Reveals the Impact of Genetic Alterations on the Differentiation of Inflammation-Induced Murine Colon Tumors.

Genetic mutations and chronic inflammation of the colon contribute to the development of colorectal cancer (CRC). Using a murine model of inflammation-induced colon tumorigenesis, we determined how genetic mutations alter colon tumor cell differentiation. Inflammation induced by enterotoxigenic Bacteroides fragilis (ETBF) colonization of multiple intestinal neoplasia (MinApcΔ716/+) mice triggers loss of heterozygosity of Apc causing colon tumor formation. Here, we report that the addition of BRAFV600E mutation (BRAFF-V600ELgr5tm1(Cre/ERT2)CleMinApcΔ716/+, BLM) or knocking out Msh2 (Msh2LoxP/LoxPVil1-creMinApcΔ716/+, MSH2KO) in the Min model altered colon tumor differentiation. Using single-cell RNA sequencing, we uncovered the differences between BLM, Min, and MSH2KO tumors at a single-cell resolution. BLM tumors showed an increase in differentiated tumor epithelial cell lineages and a reduction in the tumor stem cell population. Interestingly, the tumor stem cell population of BLM tumors had revival colon stem cell characteristics with low WNT signaling and an increase in RevCSC marker gene expression. In contrast, MSH2KO tumors were characterized by an increased tumor stem cell population that had higher WNT signaling activity compared to Min tumors. Furthermore, overall BLM tumors had higher expression of transcription factors that drive differentiation, such as Cdx2, than Min tumors. Using RNA velocity, we identified additional potential regulators of BLM tumor differentiation such as NDRG1. The role of CDX2 and NDRG1 as putative regulators for BLM tumor cell differentiation was verified using organoids derived from BLM tumors. Our results demonstrate the critical connections between genetic mutations and cell differentiation in inflammation-induced colon tumorigenesis. Understanding such roles will deepen our understanding of inflammation-associated colon cancer.

Identification of related-genes of T cells in lung tissue of chronic obstructive pulmonary disease based on bioinformatics and experimental validation

T cells are one of the main cell types shaping the immune microenvironment in chronic obstructive pulmonary disease (COPD). They persist andplay cytotoxic roles. The purpose of this study aimed to explore the potential related-genes of T cells in lung tissue of COPD. Chip data GSE38974 and single_celldata GSE196638 were downloaded from the GEO database. Difference analyses and WGCNA of GSE38974 were performed to identify DEGs and the modules most associated with the COPD phenotype. Various cell subsets were obtained by GSE196638, and DEGs of T cells were further identified. GO, GSEA and KEGG enrichment analyses were conducted to explore the biological functions and regulatory signaling pathways of the DEGs and DEGs of T cells. The intersection of the DEGs, module genes and DEGs of T cells was assessed to acquire related-genes of T cells. The mRNA and protein expression levels of related-genes ofT cells were verified in lung tissue of mouse with emphysema model. Based on GSE38974 difference analysis, 3811 DEGs were obtained. The results of WGCNA showed that the red module had the highest correlation coefficient with the COPD phenotype. GSE196638 analysis identified 124 DEGs of T cells. The GO, GSEAand KEGG enrichment analyses mainly identified genes involved in I-kappaB kinase/NF-kappaB signaling, receptor signaling pathway via STAT, regulationof CD4-positive cells, regulation of T-helper cell differentiation, chemokine signaling pathway, Toll-likereceptor signaling pathway, CD8-positive cells, alpha–beta T cell differentiation, MAPK signaling pathway and Th17 cell differentiation. The DEGs, genes of the red module and DEGs of T cells were overlapped to acquire FOXO1 and DDX17. The results of RT-qPCR and Western Blot indicate that the mRNA and protein expression levels of FOXO1 and DDX17 in lung tissue of emphysema mice were significantly higher compared with those in air-exposed mice. FOXO1 as well as DDX17 may be related-genesof T cells in lung tissue of patient with COPD, and their participation in the biological processes of different signaling pathways may inspire further COPD research.

#1479 Single nucleus RNA-sequencing indicates functional heterogeneity of renal endothelial cells in chronic kidney disease

Abstract Background and Aims The burden of cardiovascular disease (CVD) in patients with chronic kidney disease (CKD) remains very high. Endothelial cell (EC) dysfunction is an important key player in the initiation and progression of both pathologies. More importantly, endothelial dysfunction occurs early in both disease courses and is potentially reversible, making it a promising target for effective preventive strategies. Recently, accumulating single-cell RNA sequencing-based evidence has highlighted EC heterogeneity in different pathophysiological contexts. However, while biological processes such as systemic inflammation, oxidative stress fibrosis are being defined as comm EC in CKD and CVD, a comprehensive understanding of at single-cell resolution is lacking. Here, we characterized renal EC subpopulations by single-nucleus RNA sequencing (snRNA-seq) to investigate alterations in CKD patients healthy controls and identify potential drivers of EC dysfunction in CKD. Method Droplet-based snRNA-seq (10X Genomics Chromium) was performed on cryopreserved kidney biopsy cores from 6 human patients with CKD (eGFR < 45 mL/min/1.73 m2) and 3 healthy controls. CKD patients included 2 patients with primary focal segmental glomerulosclerosis (FSGS) and 4 with secondary/maladaptive FSGS (2 patients with diabetes mellitus, 2 female patients in total); all patients had a Framingham 10-year cardiovascular risk score > 20, but no established CVD. Healthy control samples were pre-perfusion biopsies from patients undergoing living kidney donation (including 2 female patients). EC subclusters were annotated based on the expression of canonical EC marker genes. Differential gene expression analysis for patient samples vs. controls included all ECs and DEGs were filtered for log2FC > 0.25 or < −0.25 and adjusted P-value of < .05. Gene set enrichment analysis (GSEA) on the identified DEGs was performed using GO terms. Results A total of 4 081 ECs were present in the dataset, consisting of 5 EC subtypes in both the patient and control cohorts (Panel A). DEGs in CKD patients vs. controls included upregulated genes related to organogenesis and cell differentiation (MEF2C, MEIS2), vascular development (FLRT2), and angiogenesis regulators (VEGFC, PRCP, HDAC9) (Panel B). Notably, VEGFC and FLRT2, are involved in directing EC motility and vascular patterning. In accordance, GSEA showed enrichment for GO biological processes related to vascular endothelial growth factor signaling pathway, endothelial cell differentiation, endothelial development, regulation of angiogenesis, and endothelial cell migration (Panel C). This suggests a potential link between these genes and aberrant angiogenesis, analogous to previous findings in diabetic nephropathy. Some but not all of these changes were also reported for renal ECs of early diabetic nephropathy patients. Of note, the majority of patients studied in our cohort were non-diabetic, warranting further investigation into renal endothelial cell transcriptional alterations that are general to renal disease or specific to particular renal conditions such as diabetic nephropathy. Differential gene expression and GSEA analysis of the EC subclusters in CKD indicated involvement of the peritubular capillary and ascending vasa recta ECs in EC migration / angiogenesis-related responses, overlapping with the overall analysis described above. Glomerular capillary ECs showed upregulation of immune-related genes and processes, reflecting the inflammatory status of the kidney disease. Conclusion snRNA-seq analysis revealed heterogeneity and subtype-specific gene expression alterations of renal ECs from CKD patients compared to healthy controls. Differential functions include enhanced angiogenic (potentially compensatory) and immune-related responses. These findings suggest that specific biological functions may direct different roles of the renal EC subtypes in CKD and will be further validated for their cluster-specificity and for their therapeutic relevance.

BACH2: The Future of Induced T-Regulatory Cell Therapies.

BACH2 (BTB Domain and CNC Homolog 2) is a transcription factor that serves as a central regulator of immune cell differentiation and function, particularly in T and B lymphocytes. A picture is emerging that BACH2 may function as a master regulator of cell fate that is exquisitely sensitive to cell activation status. In particular, BACH2 plays a key role in stabilizing the phenotype and suppressive function of transforming growth factor-beta (TGF-β)-derived human forkhead box protein P3 (FOXP3)+ inducible regulatory T cells (iTregs), a cell type that holds great clinical potential as a cell therapeutic for diverse inflammatory conditions. As such, BACH2 potentially could be targeted to overcome the instability of the iTreg phenotype and suppressive function that has hampered their clinical application. In this review, we focus on the role of BACH2 in T cell fate and iTreg function and stability. We suggest approaches to modulate BACH2 function that may lead to more stable and efficacious Treg cell therapies.

ShRNA-mediated gene silencing of HDAC11 empowers CAR-T cells against prostate cancer.

Epigenetic mechanisms are involved in several cellular functions, and their role in the immune system is of prime importance. Histone deacetylases (HDACs) are an important set of enzymes that regulate and catalyze the deacetylation process. HDACs have been proven beneficial targets for improving the efficacy of immunotherapies. HDAC11 is an enzyme involved in the negative regulation of T cell functions. Here, we investigated the potential of HDAC11 downregulation using RNA interference in CAR-T cells to improve immunotherapeutic outcomes against prostate cancer. We designed and tested four distinct short hairpin RNA (shRNA) sequences targeting HDAC11 to identify the most effective one for subsequent analyses. HDAC11-deficient CAR-T cells (shD-NKG2D-CAR-T) displayed better cytotoxicity than wild-type CAR-T cells against prostate cancer cell lines. This effect was attributed to enhanced activation, degranulation, and cytokine release ability of shD-NKG2D-CAR-T when co-cultured with prostate cancer cell lines. Our findings reveal that HDAC11 interference significantly enhances CAR-T cell proliferation, diminishes exhaustion markers PD-1 and TIM3, and promotes the formation of T central memory TCM populations. Further exploration into the underlying molecular mechanisms reveals increased expression of transcription factor Eomes, providing insight into the regulation of CAR-T cell differentiation. Finally, the shD-NKG2D-CAR-T cells provided efficient tumor control leading to improved survival of tumor-bearing mice in vivo as compared to their wild-type counterparts. The current study highlights the potential of HDAC11 downregulation in improving CAR-T cell therapy. The study will pave the way for further investigations focused on understanding and exploiting epigenetic mechanisms for immunotherapeutic outcomes.

Regulation of mesenchymal stem cell differentiation by autophagy.

Autophagy, a process that isolates intracellular components and fuses them with lysosomes for degradation, plays an important cytoprotective role by eliminating harmful intracellular substances and maintaining cellular homeostasis. Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity for self-renewal that can give rise to a subset of tissues and therefore have potential in regenerative medicine. However, a variety of variables influence the biological activity of MSCs following their proliferation and transplantation in vitro. The regulation of autophagy in MSCs represents a possible mechanism that influences MSC differentiation properties under the right microenvironment, affecting their regenerative and therapeutic potential. However, a deeper understanding of exactly how autophagy is mobilized to function as well as clarifying the mechanisms by which autophagy promotes MSCs differentiation is still needed. Here, we review the current literature on the complex link between MSCs differentiation and autophagy induced by various extracellular or intracellular stimuli and the molecular targets that influence MSCs lineage determination, which may highlight the potential regulation of autophagy on MSCs' therapeutic capacity, and provide a broader perspective on the clinical application of MSCs in the treatment of a wide range of diseases.

Downregulation of miR-568 in Atrial Fibrillation Leads to Increased Expression of NAPMT and TRMP7

Aim: Atrial fibrillation (AF) is known as the most common permanent cardiac arrhythmia worldwide with its incidence and prevalence gradually increase with age and cause significant morbidity and mortality. However, the epigenetic alterations underlying the development of this disease remains less understood. MicroRNAs (miRNAs), as one of the epigenetic regulators, are small non-coding RNAs that can target multiple genes to modulate proteins in different signaling pathways. Current studies have demonstrated that miRNAs, which are pivotal regulators of gene expression, may be involved in the pathophysiology of AF. The current study aims to clarify the miRNA regulated cellular signaling in atrial fibrillation. Material and Method: An AF model was generated by providing external electrical stimulation to the HL-1 mouse cardiomyocyte cell line for 24 hours in this study. To understand the molecular mechanisms of miRNAs underlying the AF model, miRNA microarray analysis was performed. The gene sets obtained from the microarray analysis and the bioinformatically obtained putative targets were intersected and pathway enrichment analysis was performed. qRT-PCR was performed for validation of the selected miRNAs and potential targets. Results: miRNA expression profile changes between the control group without external stimulation and the samples at the end of 3-, 6-, 12- and 24-hour stimulation were compared with microarray analysis. In particular, our transcriptomic analysis showed 5 distinctively expressed miRNAs (DEmiRNAs) whose target genes are associated with cardiovascular development within the stimulated groups in HL-1 cells. Additionally, our bioinformatics analysis revealed that targets of these miRNAs are concentrated in biological processes associated with cardiovascular development: smooth muscle cell proliferation, muscle cell proliferation, cell morphogenesis involved in differentiation and regulation of cell differentiation. Specifically, qPCR-based analyses confirmed the inverse correlation of miR-568 and potential targets of this miRNA. While miR-568 expression decreased with prolonged stimulation, expression of its potential targets, NAMPT and TRPM7, increased during prolonged stimulation. Conclusion: This study supported the potential regulative role of miRNAs and their targets in the development of AF.

The Role of the Gap Junction Protein Connexin in Adrenal Gland Tumorigenesis.

Gap junctions (GJs) are important in the regulation of cell growth, morphology, differentiation and migration. However, recently, more attention has been paid to their role in the pathogenesis of different diseases as well as tumorigenesis, invasion and metastases. The expression pattern and possible role of connexins (Cxs), as major GJ proteins, under both physiological and pathological conditions in the adrenal gland, were evaluated in this review. The databases Web of Science, PubMed and Scopus were searched. Studies were evaluated if they provided data regarding the connexin expression pattern in the adrenal gland, despite current knowledge of this topic not being widely investigated. Connexin expression in the adrenal gland differs according to different parts of the gland and depends on ACTH release. Cx43 is the most studied connexin expressed in the adrenal gland cortex. In addition, Cx26, Cx32 and Cx50 were also investigated in the human adrenal gland. Cx50 as the most widespread connexin, along with Cx26, Cx29, Cx32, Cx36 and Cx43, has been expressed in the adrenal medulla with distinct cellular distribution. Considerable effort has recently been directed toward connexins as therapeutically targeted molecules. At present, there exist several viable strategies in the development of potential connexin-based therapeutics. The differential and hormone-dependent distribution of gap junctions within adrenal glands, the relatively large gap junction within this gland and the increase in the gap junction size and number following hormonal treatment would indicate that gap junctions play a pivotal role in cell functioning in the adrenal gland.

Single-cell transcriptome profiling reveals the spatiotemporal distribution of triterpenoid saponin biosynthesis and transposable element activity in Gynostemma pentaphyllum shoot apexes and leaves.

Gynostemma pentaphyllum (Thunb.) Makino is an important producer of dammarene-type triterpenoid saponins. These saponins (gypenosides) exhibit diverse pharmacological benefits such as anticancer, antidiabetic, and immunomodulatory effects, and have major potential in the pharmaceutical and health care industries. Here, we employed single-cell RNA sequencing (scRNA-seq) to profile the transcriptomes of more than 50,000 cells derived from G. pentaphyllum shoot apexes and leaves. Following cell clustering and annotation, we identified five major cell types in shoot apexes and four in leaves. Each cell type displayed substantial transcriptomic heterogeneity both within and between tissues. Examining gene expression patterns across various cell types revealed that gypenoside biosynthesis predominantly occurred in mesophyll cells, with heightened activity observed in shoot apexes compared to leaves. Furthermore, we explored the impact of transposable elements (TEs) on G. pentaphyllum transcriptomic landscapes. Our findings the highlighted the unbalanced expression of certain TE families across different cell types in shoot apexes and leaves, marking the first investigation of TE expression at the single-cell level in plants. Additionally, we observed dynamic expression of genes involved in gypenoside biosynthesis and specific TE families during epidermal and vascular cell development. The involvement of TE expression in regulating cell differentiation and gypenoside biosynthesis warrant further exploration. Overall, this study not only provides new insights into the spatiotemporal organization of gypenoside biosynthesis and TE activity in G. pentaphyllum shoot apexes and leaves but also offers valuable cellular and genetic resources for a deeper understanding of developmental and physiological processes at single-cell resolution in this species.

Functional analysis of the mating type genes in Verticillium dahliae

BackgroundPopulations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood.ResultsIn this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence.ConclusionsThese findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Single Cell Profiling Reveals Unique Contributions of Senescent Macrophages across Musculoskeletal Tissues in Posttraumatic Osteoarthritis

Injury to the acute cruciate ligament (ACL) disrupts musculoskeletal tissue architecture and function, leading to atrophy and weakness associated with the development of posttraumatic osteoarthritis (PTOA). The objective of this study was to profile senescent cell burden across skeletal muscle and knee joint capsule compartments and to ascertain the effectiveness of senolytic treatment in mitigation of PTOA. Given the unique features of these tissues, we hypothesized that composition and physiological consequence of senescent cell burden would differ among muscle and joint capsule, and senolytic treatment would prevent cartilage degradation and improve muscle function. We employed a surgical ACL transection model (ACLT) and performed scRNAseq analysis on the quadriceps and joint capsule 14-days post-injury and discerned senescent status by SPiDER-βGal. We also examined the influence of senescent cells on neighboring populations in vitro. We investigated whether senolytic treatment (dasatinib and quercetin; D+Q) improves muscle and joint health 28-d post-injury. scRNAseq revealed that macrophages (MΦ) accounted for 83% of senescent cells in the quadriceps and 48% in the joint capsule. Senescent MΦ exhibited an alternatively activated phenotype (M2) . Gene ontology enrichment analysis in the quadriceps showed downregulation of positive regulation of muscle cell differentiation, and upregulation of secretory granule lumen; in the joint capsule, it revealed upregulation of glycosaminoglycan catabolic process and regulation of ossification. In vitro, we found that mesenchymal progenitors incubated with senescent MΦ conditioned media exhibited upregulation of senescence associated secretory phenotype (SASP) genes and extracellular matrix organization-related genes ( i.e.Col1a1 and Lox). We found upregulation of protein glycosylation and downregulation of chondroitin sulfate proteoglycan biosynthetic process pathways. PTOA mice exhibited significant reductions in quadriceps myofiber cross-sectional area, increased accumulation of M2-like Cd163+ MΦ, and increased fibrosis, which were blunted in D+Q-treated mice. D+Q-treated PTOA mice exhibited a trend of improved peak torque compared to vehicle-treated PTOA mice. Histochemical assessment of whole knee joints revealed compromised articular cartilage and moderate to severe PTOA in vehicle-treated mice, which was blunted in D+Q-treated mice. These findings reveal distinct transcriptional profiles of senescent macrophages isolated from unique musculoskeletal tissues, providing critical insight into the heterogeneity of senescent cell programming following traumatic injury. Further, we show that accumulation of senescent alternatively-activated MΦ likely contributes to musculoskeletal dysfunction and development of PTOA, and that senolytics may improve functional recovery. AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tumor Protein D52 Regulates Paligenosis in Exocrine Pancreatic Acinar Cells

Background: Paligenosis is a conserved cellular plasticity program in which fully differentiated cells reenter the cell cycle in order to regenerate lost tissue during injury. This process is utilized by cells that have a slow turnover rate and lack dedicated stem cells, as is the case with exocrine pancreatic acinar cells. Acinar cells, which synthesize and secrete digestive enzymes necessary for macronutrient digestion, undergo paligenosis in response to damage induced by the inflammatory disease pancreatitis. During paligenosis, acinar cells first downscale their secretory apparatus by upregulating autophagy, and then take on a mucinous-ductal-progenitor-like identity in a process called acinar to ductal metaplasia (ADM) prior to proliferation. This phenomenon can be recapitulated in rodent models in vivo utilizing repeated episodes of cerulein-induced pancreatitis or in 18-hour suspension culture during which primary isolated acinar cells dedifferentiate to a progenitor-like state. We have previously reported that markers of the endolysosomal secretory pathway in acinar cells are rapidly lost during models of experimental pancreatitis and suspension culture. Tumor Protein D52, which regulates endolysosomal traffcking and secretion in acinar cells, undergoes lysosomal degradation at the onset of experimental pancreatitis; furthermore, restoring D52 expression by adenovirus in isolated acinar cells maintains secretion during experimental pancreatitis as well as maintains acinar differentiation in suspension culture. Hypothesis: Degradation of D52, and loss of the endolysosomal secretory pathway, are pivotal events during the initial stages of paligenosis. Methods: To study the role of D52 and the regulation of paligenosis, we generated tamoxifen-inducible, acinar specific D52 knockout mice (D52KOac), and markers of paligenosis (ADM, proliferation) were assessed by immunoblot, qPCR, and immunostaining. Mice were given tamoxifen at 2-3 months of age to ensure normal pancreatic development, and tissues were harvested 21 days after tamoxifen. Results: Compared to controls, D52KOac exhibited 2.5-fold upregulation of the ductal markers Sox9 and CK19, 50% reduction of Ptf1α, and a 5-fold increase in β-catenin; these markers signify the presence of ADM and cells with progenitor-like characteristics. Cell cycle regulation and proliferation were also observed in D52KOac by a nearly 3-fold increase in cyclin D and increased Ki67+ nuclei. Conclusions: These data support that loss of D52 in pancreatic acinar cells induces spontaneous paligenosis in the absence of cellular injury and inflammation. These findings point to a previously unrecognized role for D52 in the regulation of acinar cell differentiation and plasticity. Given that paligenosis is a key pathway involved in the development of pancreatic neoplasms and adenocarcinoma, studying D52-regulated paligenosis will reveal additional insights into this critical protective mechanism. American Pancreatic Association Foundation Young Investigator Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

B7H3 (CD276) regulation of myeloid cell differentiation in pulmonary fibrosis

Abstract Myeloid-derived suppressor cells (MDSCs) are induced in cancer and fibrosis, while absent in healthy tissues. The expansions of MDSC are correlated with disease severity and lung function deterioration in the latter. B7H3 induced in lung fibrosis causes the recruitment of MDSCs. However, the mechanism underlying the biogenesis of MDSCs is unclear. To study if B7H3-induced expansion of MDSCs is due to inhibition of the differentiation of myeloid progenitors, the role of B7H3 in the regulation of BM-derived myeloid cells was studied. The results showed that B7H3 deficiency in BM LysM+ myeloid cells significantly increased differentiation to mature myeloid cells. In contrast, treatment with B7H3 inhibited such differentiation. In mice, LysM+ cells-specific B7H3 deficiency induced mature myeloid cell differentiation, but significantly decreased immature MDSCs, which was associated with less immunosuppressive capability. Lack of B7H3 caused a significant reduction of MDSC marker genes characteristic. Furthermore, the delayed B7H3 antibody treatment or by ablating of its secretion from collagen-expressing cells reduced BM-derived MDSCs and PD1-expressing progenitors but increased mature myeloid cell number, which was accompanied by attenuated lung fibrosis. These findings suggested that activated fibroblasts in lung fibrosis may be a key source of soluble B7H3 essential for the signal to BM skewing myeloid lineage fate and induced biogenesis of MDSC

Endothelial cell HDAC1 mediates aortic stiffness in diet-induced obese mice

Histone deacetylase 1 (HDAC1) is an important regulator of cell differentiation and is associated with progression of cardiovascular disease (CVD). Our lab previously showed that over-expression of HDAC1 in cultured endothelial cells reduces NO production. Other investigators have shown increased HDAC1 protein in models of CVD. However, there is a gap in knowledge of whether endothelial-specific HDAC1 mediates vascular dysfunction in models of CVD. Diet induced obesity (DIO) is a leading risk factor for CVD, thus our studies are focused on a chronic high fat feeding mouse model. Our studies utilize a mouse model of DIO where mice are fed a normal diet (ND, 10% fat) or high fat diet (DIO, 45% fat) beginning at 8 weeks of age for 20 weeks. Previously, we showed that this model of DIO in male mice significantly increases aortic stiffness (measured by pulse wave velocity, PWV) and increases aortic thickness compared to ND (PWV: unpaired T test, n=9-12, normal diet (ND) vs DIO, p=<0.0001; Aortic Thickness: unpaired T test, n=6, ND vs DIO, p=0.005). We hypothesized that endothelial-specific HDAC1 mediates aortic dysfunction in a mouse model of DIO. We generated inducible endothelial-Scl-Cre-ER-HDAC1 knockout (KO) mice (iEC-HDAC1KO), which specifically deletes HDAC1 in endothelial cells after tamoxifen injection, and the flox control mice (iEC-HDAC1flox), which does not delete HDAC1 after tamoxifen injection. We assessed aortic stiffness of iEC-HDAC1 KO and iEC-HDAC1flox in ND and DIO mice. We treated with tamoxifen for 5 days when ND mice were 20 weeks old and measured PWV 2 weeks later. Interestingly, iEC-HDAC1KO mice had significantly reduced PWV compared to iEC-HDAC1flox mice on ND (unpaired T test; n=4-5, p=0.009). At week 18 of the DIO protocol, mice were given daily tamoxifen injections for 5 days. After week 20 of the DIO protocol, iEC-HDAC1KO DIO mice had significantly reduced PWV compared to iEC-HDAC1flox DIO mice (unpaired T test; n=4-5; p=0.008). These data indicate that endothelial-specific HDAC1 mediates vascular stiffness and remodeling in high fat fed mice. Further investigation is needed to understand the relationship of endothelial HDAC1 and NO in the context of DIO and aortic health. Funding: R01 DK134562, F31HL167626. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-specific and cell-specific regulation of ER stress and neuroinflammation after traumatic brain injury in juvenile mice

Endoplasmic reticulum (ER) stress and neuroinflammation play an important role in secondary brain damage after traumatic brain injury (TBI). Due to the complex brain cytoarchitecture, multiple cell types are affected by TBI. However, cell type-specific and sex-specific responses to ER stress and neuroinflammation remain unclear. Here we investigated differential regulation of ER stress and neuroinflammatory pathways in neurons and microglia during the acute phase post-injury in a mouse model of impact acceleration TBI in both males and females. We found that TBI resulted in significant weight loss only in males, and sensorimotor impairment and depressive-like behaviors in both males and females at the acute phase post-injury. By concurrently isolating neurons and microglia from the same brain sample of the same animal, we were able to evaluate the simultaneous responses in neurons and microglia towards ER stress and neuroinflammation in both males and females. We discovered that the ER stress and anti-inflammatory responses were significantly stronger in microglia, especially in female microglia, compared with the male and female neurons. Whereas the degree of phosphorylated-tau (pTau) accumulation was significantly higher in neurons, compared with the microglia. In conclusion, TBI resulted in behavioral deficits and cell type-specific and sex-specific responses to ER stress and neuroinflammation, and abnormal protein accumulation at the acute phase after TBI in immature mice.