Gene Expression Profiles Of Cells Research Articles

PATH-22. SINGLE-CELL RNA (SCRNA) SEQUENCING OF PAIRED PRIMARY AND RECURRENT ATYPICAL TERATOID/RHABDOID TUMORS (AT/RT) REVEALED MOLECULAR DIFFERENCES AND THERAPY-RESISTANT PRIMARY TUMOR CELLS

Abstract Atypical teratoid/rhabdoid tumors (AT/RT) are the most common malignant brain tumors during infancy. They split into four molecular types. The major three (AT/RT-SHH, AT/RT-TYR, and AT/RT-MYC) all carry mutations in SMARCB1, the fourth smaller type is characterized by SMARCA4 mutations (AT/RT-SMARCA4). AT/RT are associated with a dismal outcome, and 50% of the patients suffer a progress or recurrence of the tumor. Molecular mechanisms of disease recurrence are currently unclear. Here, we identified molecular characteristics of AT/RT primaries and recurrences, which could help to improve the therapy for relapsed patients. Previously, we investigated tumor tissue from 26 patients revealing signatures of recurrences in comparison to matched primary tumor samples using histology, DNA methylation analysis, and bulk RNA sequencing. To further identify mechanisms regarding tumor progression and recurrence, we performed single-cell RNA (scRNA) sequencing for five of these primary-recurrence pairs so far using formalin-fixed paraffin-embedded (FFPE) tissue. Using Uniform Manifold Approximation and Projection (UMAP) for dimensionality reduction, we observed a distinct separation in the gene expression profiles of primary and recurrent tumor cells. In addition, we aimed to identify primary tumor cells which potentially persisted after therapy and developed to recurrences. Therefore, we identified primary tumor cells showing highest similarity to recurrent tumor cells using CIBERSORT and trajectory analysis. Differentially expressed genes of these specific primary tumor cells were investigated. They are involved in cell migration, -adhesion, -proliferation, and -stability, among others. Overall, molecular characteristics that occur in the course of the disease were identified, which may help to define new therapeutic targets for AT/RT recurrences.

TMIC-56. LONGITUDINAL SINGLE CELL RNA SEQUENCING UNVEILS PHENOTYPIC CO-OPTION AND LINEAGE TRAITS OF MESENCHYMAL GLIOBLASTOMA AND GLIOMA-ASSOCIATED FIBROBLASTS

Abstract BACKGROUND Adult gliomas exhibit a high level of heterogeneity presenting a substantial challenge hampering development of targeted therapies. Bulk RNAseq studies have identified a mesenchymal gene expression signature across the glioma continuum, based on which a mesenchymal subtype of glioblastoma (MES-GBM) has been described. However, the mesenchymal phenotype of gliomas remains inconsistently defined and the origin of the mesenchymal signature has been debated). Single cell transcriptomic profiling has allowed resolution of gene expression profiles of tumor and non-tumor cells, leading to recent identification of cancer-associated fibroblasts in gliomas and improved understanding of the mesenchymal gene expression signature. METHODS We performed longitudinal scRNAseq analysis of organotypic human glioma slices from 8 patients cultured ex vivo for up to 3 weeks. RESULTS We found 2 of the 6 IDHwt glioblastomas exhibit the mesenchymal phenotype based on Consensus Methylation Profiling (NIH) and scRNAseq analysis, annotated as Mes-1 and Mes-2. The gene expression profiles of mesenchymal glioblastomas show limited concordance with that of either the proneural or classical glioblastoma signatures or of cell types in physiological human brain, with Mes-2 deviating more extensively from non-mesenchymal gliomas. Instead, single cell transcriptomics suggests a hyper-secretory phenotype and overlaps broadly with that of cancer-associated fibroblasts (CAFs) and both express FGF2, other growth factors, and cytokines. Notably, Mes-2 cells express minimal to no levels of genes associated with brain cell lineages, such as PTPRZ1 and SCG3, which are abundantly expressed in other glioma subtypes. CONCLUSIONS Taken together, our data reveals extended depth of glioma heterogeneity that distinguishes MES-GBM from other glioma subtypes and suggests a phenotypic and lineage linkage between MES-GBM and glioma-associated fibroblasts. Thus, new diagnostic and treatment strategies leveraging auto- and paracrine mechanisms underpinning tumor and non-tumor interactions including disruption of the FGF2-FGFR1 circuitry may apply specifically to MES-GBM.

Factor 3 regulates airway engraftment by human bronchial basal cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene editing and transplantation of CFTR-gene corrected airway basal cells has the potential to cure CF lung disease. Although mouse studies established that cell transplantation was feasible, the engraftment rate was typically low and frequently less than the estimated therapeutic threshold. The purpose of this study was to identify genes and culture conditions that regulate the therapeutic potential of human bronchial basal cells. Factor 3 (F3, Tissue Factor 1) is a component of the extrinsic coagulation pathway and activates a cascade of proteases that convert fibrinogen to fibrin. Based on reports that F3 was necessary for human basal cell survival and adhesion in vitro, the present study evaluated F3 as a potential determinant of therapeutic fitness. The gene expression profile of F3 mRNA-positive human bronchial basal cells was evaluated by scRNAseq and the impact of the lung environment on F3 expression was modeled by varying in vitro culture conditions. F3 necessity for adhesion, proliferation, and differentiation was determined by CRISPR/Cas9 knockout (KO) of the F3 gene. Finally, the impact of F3 manipulation on engraftment was determined by orthotropic co-transplantation of wild-type and F3-KO cells into the airways of immunocompromised mice. In contrast with the hypothesis that F3 increases the therapeutic fitness of basal cells, F3 expression decreased engraftment. These studies guide the ongoing development of cellular therapies by showing that in vitro assessments may not predict therapeutic potential and that the lung milieu influences the functional properties of transplanted bronchial basal cells.

Gene Expression Profiling of Maslinic Acid-treated MCF-7 Breast Cancer Cells Using Nanostring nCounter Pancancer Pathway Panel

Breast cancer remains a significant global health concern, impacting millions of women every year. Maslinic acid (MA), a pentacyclic triterpene has been found to exert promising anticancer effect in various cancers, including breast cancer, yet the underlying mechanisms remain unclear. This study aims to elucidate the anticancer properties of MA via gene expression profiles in breast cancer cells. Cytotoxicity assay results revealed that MCF-7 exerts the highest sensitivity after 72 h of MA treatment followed by T-47D and MDA-MB-231. MCF-7 were then selected for in-depth analysis using the Nanostring nCounter Pancancer Pathway Panel to analyze the differential expression of genes (DEGs). Across three time points (24, 48, and 72 h), 20 significant DEGs were identified, of which 5 were upregulated and 15 were downregulated. In silico analysis indicated that these DEGs were involved in Pathway of Cancer, Focal Adhesion-PI3K-mTOR Signaling Pathway, PI3K-Akt, and Ras Signaling Pathway. The regulation of these DEGs contributes to several cellular activities such as apoptosis, inhibition of cell proliferation, cell cycle and survival, reduction of glycolysis, angiogenesis, and DNA repair. Additionally, the unfolded protein response emerged as a noteworthy biological process in this study. This study unravels the molecular mechanisms underpinning the therapeutic potential of MA against breast cancer.

Physiological and pathogenic T cell autoreactivity converge in type 1 diabetes

Autoimmune diseases result from autoantigen-mediated activation of adaptive immunity; intriguingly, autoantigen-specific T cells are also present in healthy donors. An assessment of dynamic changes of this autoreactive repertoire in both health and disease is thus warranted. Here we investigate the physiological versus pathogenic autoreactive processes in the context of Type 1 diabetes (T1D) and one of its landmark autoantigens, glutamic acid decarboxylase 65 (GAD65). Using single cell gene expression profiling and tandem T cell receptor (TCR) sequencing, we find that GAD65-specific true naïve cells are present in both health and disease, with GAD65-specific effector and memory responses showing similar ratios in healthy donors and patients. Deeper assessment of phenotype and TCR repertoire uncover differential features in GAD65-specific TCRs, including lower clonal sizes of healthy donor-derived clonotypes in patients. We thus propose a model whereby physiological autoimmunity against GAD65 is needed during early life, and that alterations of these physiological autoimmune processes in predisposed individuals trigger overt Type 1 diabetes.

VSIG4 inhibits the anti-inflammatory reaction of immune cells after cardiogenic shock

Abstract Cardiogenic shock (CS) still has a mortality rate of approximately 50%. CS leads to a significant activation of the immune system. The aim of this study was to characterize the immune cell populations and the immune cell-specific changes in CS on admission to the clinic and after revascularization. We developed a web-based platform named 'IRCaS - Immune Cell Regulations in Cardiogenic Shock' to map the temporal course of immune cell-specific processes of CS after acute myocardial infarction (AMI) from admission to 3 days after revascularization. Our tool revealed an endogenous anti-inflammatory response after CS, which is inhibited by the cell-cell receptor VSIG4. The web platform contains newly acquired single-cell sequencing data from patients with CS after AMI on admission and 24, 48, and 72 hours after revascularisation, as well as controls. In total, 171,962 immune cells derived from peripheral blood mononuclear cells (PBMCs) were individually sequenced. Analysis of the immune cell populations revealed, that 24 hours after revascularisation, monocytes increased to 62.4% (vs. 30.4% in controls), while T cells decreased from 46.3% to 25.0%. On the immune cell expression level, we observed an inflammatory response indicated by IL1B which continuously increased from 24 h (1.6+fold), over 48h (3.1+fold) to 72 h (3.5+fold) after revascularization. Interestingly, there was a significant anti-inflammatory response shortly after revascularization, as evidenced by a transient increase in the anti-inflammatory CD16+ monocyte subpopulation from 12.1% to 26.7% at 24 hours and an increase in THBS1 after revascularization, which was only short-lived. We identified a negative feedback in the increasing anti-inflammatory CD16+ cells caused by a subsequent VSIG4 (+5.2-fold) increase. Indeed, overexpression of VSIG4 in monocytes resulted in a significant decrease of anti-inflammatory CD16+ monocytes by 33.9% and a decrease in anti-inflammatory transcripts. Our data show that in addition to the strong inflammatory response in CS, there is also an opposing anti-inflammatory response, which is however only short-lived, as it is attenuated by a negative feedback via an increase in VSIG4. In addition, our webplatform offers time-dynamic gene expression profiles of immune cells, allowing users to select and visualize specific genes during the course of CS after AMI.

Selective Cytotoxicity and Gene Expression Profiles in Response to Methanolic Extract of Phyllanthus amarus Extract against MCF-7 Breast Cancer Cells

Background Breast cancer, a malignant tumor, is the most common cancer type among women worldwide. Conventional chemotherapy treatments for breast cancer often cause adverse side effects in normal tissues and can lead to treatment failure. Plant-derived natural compounds offer an effective alternative with fewer side effects for cancer patients. Phyllanthus amarus is a medicinal plant widely distributed in China, India, and Brazil and reported to be used for the treatment of various types of cancer. Purpose This study was aimed to investigate the anticancer efficacy of methanolic extract of P. amarus against MCF-7 breast cancer cells. Methods We probed the anticancer activity in MCF-7 breast cancer cells by isolating the methanolic extract of P. amarus leaf extract (MEPA). The standard chemotherapy drug docetaxel is combined with MEPA to increase the drug efficacy. Moreover, the intracellular reactive oxygen species (ROS), alterations in mitochondrial membrane potential (MMP), nuclear condensation, and apoptotic activation were investigated in MCF-7 cells. Additionally, the customized PCR array comprising of cell cycle, apoptosis, mTOR, and JAK-STAT-related genes was studied during different MEPA treatments. Results In the MCF-7 breast cancer cells, the MEPA and docetaxel combination demonstrated strong cytotoxicity, with IC50 values of 45 and 2.6 µg/mL, respectively. MEPA and docetaxel together exhibited enhanced alterations in MMP and increased generation of ROS in MCF-7 cells. Furthermore, the combination of docetaxel with MEPA induces nuclear condensation and apoptotic cell death. During MEPA treatment, the gene expression profiles in MCF-7 cells were accessed. The JAK-STAT, mTOR signaling pathway, apoptosis, and cell growth-related genes were strongly affected by the dose-dependent administration of MEPA. Conclusion This study suggests that MEPA could be a useful alternative treatment for breast cancer. When combined with traditional chemotherapy, MEPA might offer better results with fewer side effects. More in vivo studies are needed to confirm MEPA’s effectiveness in animal models.

Expression of cardiomyopathy-related genes in mononuclear blood cells predicts all-cause mortality in patients presenting with acute versus chronic coronary syndrome requiring revascularisation

Abstract Objective Despite advances in the revascularisation techniques their net clinical benefit differs substantially between patients presenting with myocardial infarction (ACS) and chronic coronary syndrome (CCS) due to obstructive coronary atherosclerosis. Considering that leukocytes represents a key pathophysiological component of myocardial ischaemia, exploration of mononuclear blood cell (PBMC) gene expression profile in CCS and ACS setting could reveal clinically relevant transcriptomic signals. The aim of the present study was to identify differences in PBMC transcriptome between ACS and CCS patients which are linked to mortality after revascularisation. Methods and Results We analysed PBMC transcriptomes from719 ACS and 486 CCS patients creating discovery cohort. The results of transcriptomic analysis were verified in replication cohort (ACS: N=682 for CCS: N=489) generated based on propensity score matching. Median time from revascularisation to blood collection (IQR) was 15.68 (-4.52, 23.23) hrs. Patients with ACS showed consistent differential expression of 8173 genes (4544 upregulated, 3629 downregulated) when compared with CCS patients. The subsequent pathway analysis also showed significant enrichment of several canonical pathways involved in major cardiovascular pathologies i.e. atherosclerosis (p = 1.22-06, enrichment = 3.39), vascular endothelial growth factor production (p = 1.16-06, enrichment = 16.73), positive regulation of vascular development (p = 2.78-06, enrichment = 5.30) and heterotopic cell adhesion including gene desmocollin 2 (DSC2, p = 3.64-04, enrichment = 6.30). In der multivariate survival analysis with differentially expressed genes, increased expression of DCS2, lamin A/C (LMNA), and UGGT2, and decreased expression of SNRNP70 predicted poor survival over the median follow-up 11.35 yrs. Consistently, controlling the false discovery rate at ≤ 5% these genes enriched pathways involved in cardiomyopathies such as arrhythmogenic right-ventricular cardiomyopathy (p = 4.19x10-4, enrichment = 58.9) and collagen disease (p = 2.29x10-3, enrichment = 23.8). Conclusion We found altered PBMC expression of multiple genes in patients requiring revascularisation for ACS versus CCS. Several differentially expressed genes included in cardiomyopathy pathways predicted long-term all-cause mortality in these patients.

Immunological characteristics of bronchoalveolar lavage fluid and blood across connective tissue disease-associated interstitial lung diseases.

Interstitial lung disease (ILD) is a serious complication of connective tissue diseases (CTDs). The heterogeneity of ILDs reflects differences in pathogenesis among diseases. This study aimed to clarify the characteristics of CTD-ILDs via a detailed analysis of the bronchoalveolar lavage fluid (BALF) and blood immune cells. BALF and blood samples were collected from 39 Japanese patients with newly diagnosed ILD: five patients with Sjögren's syndrome (SS), eight patients with dermatomyositis (DM), six patients with rheumatoid arthritis (RA), six patients with systemic sclerosis, four patients with anti-neutrophil cytoplasmic antibody-associated vasculitis, and 10 patients with idiopathic interstitial pneumonia. We performed single-cell RNA sequencing to analyze the gene expression profiles in these patients' immune cells. In patients with SS, B cells in the BALF were increased and genes associated with the innate and acquired immunity were enriched in both the BALF and blood. In contrast, patients with DM showed an upregulation of genes associated with viral infection in both the BALF and blood. In patients with RA, neutrophils in the BALF tended to increase, and their gene expression patterns changed towards inflammation. These disease-specific characteristics may help us understand the pathogenesis for each disease and discover potential biomarkers.

Endogenous Magnetic Lipid Droplet-Mediated Cascade-Targeted Sonodynamic Therapy as an Approach to Reversing Breast Cancer Multidrug Resistance.

Multidrug resistance (MDR) has emerged as a major barrier to effective breast cancer treatment, contributing to high rates of chemotherapy failure and disease recurrence. There is thus a pressing need to overcome MDR and to facilitate the efficient and precise treatment of breast cancer in a targeted manner. In this study, endogenous functional lipid droplets (IR780@LDs-Fe3O4/OA) were developed and used to effectively overcome the limited diffusion distance of reactive oxygen species owing to their amenability to cascade-targeted delivery, thereby facilitating precise and effective sonodynamic therapy (SDT) for MDR breast cancer. Initially, IR780@LDs-Fe3O4/OA was efficiently enriched within tumor sites in a static magnetic field, achieving the visualization of tumor treatment. Subsequently, the cascade-targeted SDT combined with the Fenton effect induced lysosome membrane permeabilization and relieved lysosomal sequestration, thus elevating drug concentration at the target site. This treatment approach also suppressed ATP production, thereby inhibiting P-glycoprotein-mediated chemotherapeutic drug efflux. This cascade-targeted SDT strategy significantly increased the sensitivity of MDR cells to doxorubicin, increasing the IC50 value of doxorubicin by approximately 10-fold. Moreover, the cascade-targeted SDT also altered the gene expression profiles of MDR cells and suppressed the expression of MDR-related genes. In light of these promising results, the combination of cascade-targeted SDT and conventional chemotherapy holds great clinical promise as an effective treatment modality with excellent biocompatibility that can improve MDR breast cancer patient outcomes.

ScCTS: identifying the cell type-specific marker genes from population-level single-cell RNA-seq

Single-cell RNA-sequencing (scRNA-seq) provides gene expression profiles of individual cells from complex samples, facilitating the detection of cell type-specific marker genes. In scRNA-seq experiments with multiple donors, the population level variation brings an extra layer of complexity in cell type-specific gene detection, for example, they may not appear in all donors. Motivated by this observation, we develop a statistical model named scCTS to identify cell type-specific genes from population-level scRNA-seq data. Extensive data analyses demonstrate that the proposed method identifies more biologically meaningful cell type-specific genes compared to traditional methods.

Seasonal environmental fluctuations alter the transcriptome dynamics of oocytes and granulosa cells in beef cows

BackgroundExamining the mechanistic cellular responses to heat stress could aid in addressing the increasing prevalence of decreased fertility due to elevated ambient temperatures. Here, we aimed to study the differential responses of oocytes and granulosa cells to thermal fluctuations due to seasonal differences. Dry beef cows (n = 10) were housed together, synchronized and subjected to a stimulation protocol to induce follicular growth before ovum pick-up (OPU). Two OPU’s were conducted (summer and winter) to collect cumulus-oocyte-complexes (COCs) and granulosa cells. In addition, rectal temperatures and circulating blood samples were collected during OPU. Oocytes were separated from the adherent cumulus cells, and granulosa cells were isolated from the collected OPU fluid. RNA was extracted from pools of oocytes and granulosa cells, followed by library preparation and RNA-sequencing. Blood samples were further processed for the isolation of plasma and leukocytes. The transcript abundance of HSP70 and HSP90 in leukocytes was evaluated using RT-qPCR, and plasma cortisol levels were evaluated by immunoassay. Environmental data were collected daily for three weeks before each OPU session. Data were analyzed using MIXED, Glimmix or GENMOD procedures of SAS, according to each variable distribution.ResultsAir temperatures (27.5 °C vs. 11.5 °C), average max air temperatures (33.7 °C vs. 16.9 °C), and temperature-humidity indexes, THI (79.16 vs. 53.39) were shown to contrast significantly comparing both the summer and winter seasons, respectively. Rectal temperatures (Summer: 39.2 ± 0.2 °C; Winter: 38.8 ± 0.2 °C) and leukocyte HSP70 transcript abundance (Summer: 4.18 ± 0.47 arbitrary units; Winter: 2.69 ± 0.66 arbitrary units) were shown to increase in the summer compared to the winter. No visual differences persisted in HSP90 transcript abundance in leukocytes and plasma cortisol concentrations during seasonal changes. Additionally, during the summer, 446 and 940 transcripts were up and downregulated in oocytes, while 1083 and 1126 transcripts were up and downregulated in the corresponding granulosa cells, respectively (Fold Change ≤ -2 or ≥ 2 and FDR ≤ 0.05). Downregulated transcripts in the oocytes were found to be involved in ECM-receptor interaction and focal adhesion pathways, while the upregulated transcripts were involved in protein digestion and absorption, ABC transporters, and oocyte meiosis pathways. Downregulated transcripts in the granulosa cells were shown to be involved in cell adhesion molecules, chemokine signaling, and cytokine-cytokine receptor interaction pathways, while those upregulated transcripts were involved in protein processing and metabolic pathways.ConclusionIn conclusion, seasonal changes dramatically alter the gene expression profiles of oocytes and granulosa cells in beef cows, which may in part explain the seasonal discrepancies in pregnancy success rates during diverging climatic weather conditions.

Cortical morphological changes and associated transcriptional signatures in post-traumatic stress disorder and psychological resilience.

Individuals who have experienced severe traumatic events are estimated to have a post-traumatic stress disorder (PTSD) prevalence rate ranging from 10 to 50%, while those not affected by trauma exposure are often considered to possess psychological resilience. However, the neural mechanisms underlying the development of PTSD, especially resilience after trauma, remain unclear. This study aims to investigate changes of cortical morphometric similarity network (MSN) in PTSD and trauma-exposed healthy individuals (TEHI), as well as the associated molecular alterations in gene expression, providing potential targets for the prevention and intervention of PTSD. We recruited PTSD patients and TEHI who had experienced severe earthquakes, and healthy controls who had not experienced earthquakes. We identified alterations in the whole-brain MSN changes in PTSD and TEHI, and established associations between these changes and brain-wide gene expression patterns from the Allen Human Brain Atlas microarray dataset using partial least squares regression. At the neuroimaging level, we found not only trauma-susceptible changes in TEHI same as those in PTSD, but also unique neurobiological alterations to counteract the deleterious impact of severe trauma. We identified 1444 and 2214 genes transcriptionally related to MSN changes in PTSD and TEHI, respectively. Functional enrichment analysis of weighted gene expression for PTSD and TEHI revealed distinct enrichments in Gene Ontology biological processes and Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, gene expression profiles of astrocytes, excitatory neurons, and microglial cells are highly related to MSN abnormalities in PTSD. The formation of resilience may be by an active compensatory process of the brain. The combination of macroscopic neuroimaging changes and microscopic human brain transcriptomics could offer a more direct and in-depth understanding of the pathogenesis of PTSD and psychological resilience, shedding light on new targets for the prevention and treatment of PTSD.

Reduction of myeloid-derived suppressor cells in prostate cancer murine models and patients following white button mushroom treatment.

In a previously reported Phase I trial, we observed therapy-associated declines in circulating myeloid-derived suppressor cells (MDSCs) with the administration of white button mushroom (WBM) tablets in prostate cancer (PCa) patients. These observations led us to hypothesise that WBM could mitigate PCa progression by suppressing MDSCs. We performed bidirectional translational research to examine the immunomodulatory effects of WBM consumption in both syngeneic murine PCa models and patients with PCa participating in an ongoing randomised Phase II trial (NCT04519879). In murine models, WBM treatment significantly suppressed tumour growth with a reduction in both the number and function of MDSCs, which in turn promoted antitumour immune responses mediated by T cells and natural killer (NK) cells. In patients, after consumption of WBM tablets for 3 months, we observed a decline in circulating polymorphonuclear MDSCs (PMN-MDSCs), along with an increase in cytotoxic CD8+ T and NK cells. Furthermore, single immune cell profiling of peripheral blood from WBM-treated patients showed suppressed STAT3/IRF1 and TGFβ signalling in circulating PMN-MDSCs. Subclusters of PMN-MDSCs presented transcriptional profiles associated with responsiveness to fungi, neutrophil chemotaxis, leukocyte aggregation, and regulation of inflammatory response. Finally, in mouse models of PCa, we found that WBM consumption enhanced the anticancer activity of anti-PD-1 antibodies, indicating that WBM may be used as an adjuvant therapy with immune checkpoint inhibitors. Our results from PCa murine models and patients provide mechanistic insights into the immunomodulatory effects of WBM and provide a scientific foundation for WBM as a nutraceutical intervention to delay or prevent PCa progression. White button mushroom (WBM) treatment resulted in a reduction in pro-tumoural MDSCs, notably polymorphonuclear MDSCs (PMN-MDSCs), along with activation of anti-tumoural T and NK cells. Human single immune cell gene expression profiling shed light on the molecular alterations induced by WBM, specifically on PMN-MDSCs. A proof-of-concept study combining WBM with PD-1 blockade in murine models revealed an additive effect on tumour regression and survival outcomes, highlighting the clinical relevance of WBM in cancer management.

Near-stop in spermatogonia progression and reduced sperm motility in two testis forms of captive North African catfish in Kalasin, Thailand

North African catfish (Clarias gariepinus) is integral to aquaculture, particularly in hybrid production with bighead catfish (Clarias macrocephalus). Recent declines in their production output have been linked to the quality of the parental sperm. This study aimed to evaluate the state of spermatogenesis in two morphological testis forms, normal white testes (CGA_A) and black-brown testes (CGA_B), in captive populations to understand their differences in the cytological processes of spermatogenesis and impact on sperm quality and fertility. We performed computer-assisted sperm analysis (CASA), meiotic chromosome analysis, gene expression profiling, histological examination, and identification of apoptotic cells using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Additionally, microsatellite genotyping was conducted to assess genetic diversity of the populations, showing that similar gene pool patterns were observed between the two populations with the CGA_A and the CGA_B testes. The average sperm motility was 11.15 %. Histologically, the CGA_A testes predominantly contained spermatozoa. By contrast, the CGA_B testes, characterized by black-brown color and harder texture, were composed mainly of spermatocytes and exhibited a higher incidence of apoptotic spermatogonia. Additionally, the CGA_B testes displayed reduced expression of genes essential for normal testicular development. Notably, the CGA_A testes showed significantly higher expression of GtH I, GtH II, Dazl, and Cxcr4b genes and lower levels of Caspase-3 and p53 (p < 0.05). The selective removal of deficient spermatogonia through early apoptosis in CGA_B testes suggest a mechanism contributing to male infertility due to imbalances between the number of germ cells and Sertoli cells. Despite the use of CGA_A testis for fertilization, a hatching rate in the captive populations was 36.43 % over the last three years (2021−2023), substantially lower than that previously reported, indicating the presence of genetic and environmental factors that influence reproductive success in North African catfish. Differences in spermatogenesis between testis forms highlight the need for genetic and environmental strategies to enhance reproductive outcomes in aquaculture.

PSVI-24 Gene expression profiles of granulosa and peripheral white blood cells from fertile and sub-fertile beef heifers

Abstract Beef heifer fertility issues contribute to a major economic loss in the cow-calf production industry. Therefore, identifying beef heifers with superior genetic potential for improved fertility would increase profitability. This study aimed to identify differences in the transcriptome profiles from granulosa and peripheral white blood cells (pWBCs) of beef heifers with varying reproductive potential. For this, Angus-Simmental crossbred heifers were subjected to an estrus synchronization and fixed-time artificial insemination (AI) protocol (7-D CO-Synch + CIDR) followed by exposure for 60-d to a fertile bull. Depending on the presence or absence of conceptus 120 d post-AI, heifers were classified as fertile (pregnant by AI) or sub-fertile (non-pregnant by AI or bull-breeding). Pregnancies were terminated, and all animals in both groups (fertile, n = 8; and sub-fertile, n = 5) were cycling when the blood and ovaries were collected from each heifer. Total RNA was extracted from the pWBCs and granulosa cells and subjected to library preparation and sequencing on the Nova-Seq platform. The read counts were obtained after data quality control using FastQC v0.11.9 and MultiQC v1.12 and alignment to the Ensemble’s ARS UCD1.2 Bos taurus genome reference using STAR aligner v2.7.5. The filtered data were subjected to differential expression analysis using DESeq2. We identified 1,061 and 72 significantly differentially expressed genes (DEGs) with P-values ≤ 0.05 and absolute (log2 fold change) ≥ 0.5 from pWBC and granulosa, respectively. Notably, 12 targets including 9 protein coding genes (PLCL1, DNER, GNAS, CDH3, PER1, ITGA2B, CXCL12, ENSBTAG00000048613, ENSBTAG00000051519), bta-mir-2887-1, 5-8S-rRNA and U5 were found as DEGs in both the tissues. Based on a differential co-expression analysis using PCIT, we identified GNAS and DNER as hub genes in pWBC and the granulosa cells of the sub-fertile heifer group. The 12 shared genes were over-represented for pathways such as NF kappa B and chemokine signaling, regulation of actin cytoskeleton, and platelet activation. Some of the identified genes have been previously associated with fertility, while others are novel. A detailed understanding of the underlying biological mechanisms of the top genes and a follow-up study with a larger sample size at different time points could validate the candidates identified in this study for their role as potential therapeutic targets.

Comparative analysis of selected gene expression profiles in peripheral blood mononuclear cells in patients with rosacea and healthy volunteers

Introduction. Rosacea is a chronic skin disease with about 10% prevalence in the global population. The pathogenesis of the disease is multifactorial and is not yet fully understood. The dermatosis affects the facial skin and is characterized by persistent erythema, the formation of telangiectasias, papules, pustules, and can also be complicated by ophthalmic lesions.The disease is rooted in impaired neurovascular regulation and changes in the innate immune systems. It is known that the expression of vascular endothelial growth factors (VEGF) in the skin are increased in rosacea and the level of myeloperoxidase, a lymphocyte lysosomal enzyme, is increased in the inflammatory processes occurring in the arteries.Aim. To study the level of these gene expression in blood lymphocytes in the acute stage of the disease.Materials and methods. A total of 10 patients with erythematous rosacea, 10 patients with papulopustular rosacea and 10 healthy volunteers were included in the study. The peripheral blood samples were collected from the subjects for further isolation of lymphocytes. The level of gene expression was determined by real-time polymerase chain reaction.Results. Investigators observed a 3.7-fold increase in the activity of myeloperoxidase expression in immune blood cells compared to the values of healthy volunteers. Also, the level of relative VEGFA expression increased by 6.5 times, the level of VEGFС increased by 11 times in mononuclear lymphocytes of patients compared to the values of healthy subjects. A comparative analysis in the groups of patients with erythematous and papulopustular rosacea showed a multidirectional expression pattern of vascular growth factors VEGFA and VEGFС. An increase in VEGFA expression is observed in the papulopustular rosacea, while VEGFС is higher in the erythematous form of the pathology.Conclusion. The obtained results demonstrate that a significant increase in the gene expression levels associated with vascular activity and angiogenesis in rosacea occurs not only in the skin, but also in the peripheral blood mononuclear lymphocytes of the patients. The detected differences in the expression of vascular growth factors in different forms of the disease indicate the need for differentiated treatments.

Seminal plasma inhibits Chlamydia trachomatis infection in vitro, and may have consequences on mucosal immunity

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

Cellular indexing of transcriptomes and epitopes (CITE-Seq) in hidradenitis suppurativa identifies dysregulated cell types in peripheral blood and facilitates diagnosis via machine learning.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition characterized by painful nodules, abscesses, and scarring, predominantly affecting intertriginous regions and it is often underdiagnosed. This study aimed to utilize single cell RNA and cell-surface protein sequencing (CITE-Seq) to delineate the immune composition of circulating cells in Hidradenitis suppurativa (HS) peripheral blood compared to healthy controls. CITE-Seq was used to analyze the gene and protein expression profiles of peripheral blood mononuclear cells (PBMCs) from 9 HS and 29 healthy controls. The study identified significant differences cell composition between HS patients and healthy controls, including increased proportions of CD14+ and CD16+ monocytes, cDC2, plasmablasts, and proliferating CD4+ T cells in HS patients. Differential expression analysis revealed upregulation of inflammatory markers such as TNF, IL1B, and NF-κB in monocytes, as well as chemokines and cell adhesion molecules involved in immune cell recruitment and tissue infiltration. Pathway enrichment analysis highlighted the involvement of IL-17, IL-26 and TNF signaling pathways in HS pathogenesis. Machine learning identified key markers for diagnostics and therapeutic development. The findings also support the potential for machine learning models to aid in the diagnosis of HS based on immune cell markers. These insights may inform future therapeutic strategies targeting specific immune pathways in HS.

A Boolean model explains phenotypic plasticity changes underlying hepatic cancer stem cells emergence

In several carcinomas, including hepatocellular carcinoma, it has been demonstrated that cancer stem cells (CSCs) have enhanced invasiveness and therapy resistance compared to differentiated cancer cells. Mathematical-computational tools could be valuable for integrating experimental results and understanding the phenotypic plasticity mechanisms for CSCs emergence. Based on the literature review, we constructed a Boolean model that recovers eight stable states (attractors) corresponding to the gene expression profile of hepatocytes and mesenchymal cells in senescent, quiescent, proliferative, and stem-like states. The epigenetic landscape associated with the regulatory network was analyzed. We observed that the loss of p53, p16, RB, or the constitutive activation of β-catenin and YAP1 increases the robustness of the proliferative stem-like phenotypes. Additionally, we found that p53 inactivation facilitates the transition of proliferative hepatocytes into stem-like mesenchymal phenotype. Thus, phenotypic plasticity may be altered, and stem-like phenotypes related to CSCs may be easier to attain following the mutation acquisition.