Varying the ratio of Lys: Met through enhancing methionine supplementation improved milk secretion ability through regulating the mRNA expression in bovine mammary epithelial cells under heat stress.

Introduction: The ratio of lysine (Lys) to methionine (Met) with 3.0: 1 is confirmed as the "ideal" profile for milk protein synthesis, but whether this ratio is suitable for milk protein synthesis under HS needs to be further studied. Methods: To evaluate the molecular mechanism by which HS and Lys to Met ratios affect mammary cell functional capacity, an immortalized bovine mammary epithelial cell line (MAC-T) is incubated with 5 doses of Met while maintaining a constant concentration of Lys. The MAC-T cells was treated for 6 h as follow: Lys: Met 3.0: 1 (control 37°C and IPAA 42°C) or treatments under HS (42°C) with different ratios of Lys: Met at 2.0: 1 (LM20), 2.5: 1 (LM25), 3.5: 1 (LM35) and 4.0: 1 (LM40). RNA sequencing was used to assess transcriptome-wide alterations in mRNA abundance. Results: The significant difference between control and other groups was observed base on PCA analysis. A total of 2048 differentially expressed genes (DEGs) were identified in the IPAA group relative to the control group. Similarly, 226, 306, 148, 157 DEGs were detected in the LM20, LM25, LM35 and LM40 groups, respectively, relative to the IPAA group. The relative mRNA abundance of HSPA1A was upregulated and anti-apoptotic genes (BCL2L1 and BCL2) was down-regulated in the IPAA group, compared to the control group (p < 0.05). Compared with the IPAA group, the relative mRNA abundance of anti-apoptotic genes and casein genes (CSN1S2 and CSN2) was up-regulated in the LM25 group (p < 0.05). The DEGs between LM25 and IPAA groups were associated with the negative regulation of transcription RNA polymerase II promoter in response to stress (GO: 0051085, DEGs of BAG3, DNAJB1, HSPA1A) as well as the mTOR signaling pathway (ko04150, DEGs of ATP6V1C2, WNT11, WNT3A, and WNT9A). Several DEGs involved in amino acids metabolism (AFMID, HYKK, NOS3, RIMKLB) and glycolysis/gluconeogenesis (AFMID and MGAT5B) were up-regulated while DEGs involved in lipolysis and beta-oxidation catabolic processes (ALOX12 and ALOX12B) were down-regulated. Conclusion: These results suggested that increasing Met supply (Lys: Met at 2.5: 1) may help mammary gland cells resist HS-induced cell damage, while possibly maintaining lactation capacity through regulation of gene expression.

Identification of immune-related genes and small-molecule drugs in hypertension-induced left ventricular hypertrophy based on machine learning algorithms and molecular docking.

Background: Left ventricular hypertrophy (LVH) is a common consequence of hypertension and can lead to heart failure. The immune response plays an important role in hypertensive LVH; however, there is no comprehensive method to investigate the mechanistic relationships between immune response and hypertensive LVH or to find novel therapeutic targets. This study aimed to screen hub immune-related genes involved in hypertensive LVH as well as to explore immune target-based therapeutic drugs. Materials and methods: RNA-sequencing data from a mouse model generated by angiotensin II infusion were subjected to weighted gene co-expression network analysis (WGCNA) to identify core expression modules. Machine learning algorithms were applied to screen immune-related LVH characteristic genes. Heart structures were evaluated by echocardiography and cardiac magnetic resonance imaging (CMRI). Validation of hub genes was conducted by RT-qPCR and western blot. Using the Connectivity Map database and molecular docking, potential small-molecule drugs were explored. Results: A total of 1215 differentially expressed genes were obtained, most of which were significantly enriched in immunoregulation and collagen synthesis. WGCNA and multiple machine learning strategies uncovered six hub immune-related genes (Ankrd1, Birc5, Nuf2, C1qtnf6, Fcgr3, and Cdca3) that may accurately predict hypertensive LVH diagnosis. Immune analysis revealed that fibroblasts and macrophages were closely correlated with hypertensive LVH, and hub gene expression was significantly associated with these immune cells. A regulatory network of transcription factor-mRNA and a ceRNA network of miRNA-lncRNA was established. Notably, six hub immune-related genes were significantly increased in the hypertensive LVH model, which were positively linked to left ventricle wall thickness. Finally, 12 small-molecule compounds with the potential to reverse the high expression of hub genes were ruled out as potential therapeutic agents for hypertensive LVH. Conclusion: This study identified and validated six hub immune-related genes that may play essential roles in hypertensive LVH, providing new insights into the potential pathogenesis of cardiac remodeling and novel targets for medical interventions.

Deciphering the spatiotemporal transcriptional landscape of intestinal diseases (Review).

The intestines are the largest barrier organ in the human body. The intestinal barrier plays a crucial role in maintaining the balance of the intestinal environment and protecting the intestines from harmful bacterial invasion. Single­cell RNA sequencing technology allows the detection of the different cell types in the intestine in two dimensions and the exploration of cell types that have not been fully characterized. The intestinal mucosa is highly complex in structure, and its proper functioning is linked to multiple structures in the proximal­distal intestinal and luminal­mucosal axes. Spatial localization is at the core of the efforts to explore the interactions between the complex structures. Spatial transcriptomics (ST) is a method that allows for comprehensive tissue analysis and the acquisition of spatially separated genetic information from individual cells, while preserving their spatial location and interactions. This approach also prevents the loss of fragile cells during tissue disaggregation. The emergence of ST technology allows us to spatially dissect enzymatic processes and interactions between multiple cells, genes, proteins and signals in the intestine. This includes the exchange of oxygen and nutrients in the intestine, different gradients of microbial populations and the role of extracellular matrix proteins. This regionally precise approach to tissue studies is gaining more acceptance and is increasingly applied in the investigation of disease mechanisms related to the gastrointestinal tract. Therefore, this review summarized the application of ST in gastrointestinal diseases.

Single-cell transcriptome sequencing reveals SPP1-CD44-mediated macrophage-tumor cell interactions drive chemoresistance in TNBC.

Triple-negative breast cancer (TNBC) is often considered one of the most aggressive subtypes of breast cancer, characterized by a high recurrence rate and low overall survival (OS). It is notorious for posing challenges related to drug resistance. While there has been progress in TNBC research, the mechanisms underlying chemotherapy resistance in TNBC remain largely elusive. We collect single-cell RNA sequencing (scRNA-seq) data from five TNBC patients susceptible to chemotherapy and five resistant cases. Comprehensive analyses involving copy number variation (CNV), pseudotime trajectory, cell-cell interactions, pseudospace analysis, as well as transcription factor and functional enrichment are conducted specifically on macrophages and malignant cells. Furthermore, we performed validation experiments on clinical samples using multiplex immunofluorescence. We identified a subset of SPP1+ macrophages that secrete SPP1 signals interacting with CD44 on malignant cell surfaces, potentially activating the PDE3B pathway within malignant cells via the integrin pathway, leading to chemotherapy resistance. The abnormally enhanced SPP1 signal between macrophages and malignant cells may serve as a factor promoting chemotherapy resistance in TNBC patients. Therefore, SPP1+ macrophages could potentially serve as a therapeutic target to reduce chemotherapy resistance.

Reusable tutorials for using cloud-based computing environments for the analysis of bacterial gene expression data from bulk RNA sequencing.

This manuscript describes the development of a resource module that is part of a learning platform named "NIGMS Sandbox for Cloud-based Learning" https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module delivers learning materials on RNA sequencing (RNAseq) data analysis in an interactive format that uses appropriate cloud resources for data access and analyses. Biomedical research is increasingly data-driven, and dependent upon data management and analysis methods that facilitate rigorous, robust, and reproducible research. Cloud-based computing resources provide opportunities to broaden the application of bioinformatics and data science in research. Two obstacles for researchers, particularly those at small institutions, are: (i) access to bioinformatics analysis environments tailored to their research; and (ii) training in how to use Cloud-based computing resources. We developed five reusable tutorials for bulk RNAseq data analysis to address these obstacles. Using Jupyter notebooks run on the Google Cloud Platform, the tutorials guide the user through a workflow featuring an RNAseq dataset from a study of prophage altered drug resistance in Mycobacterium chelonae. The first tutorial uses a subset of the data so users can learn analysis steps rapidly, and the second uses the entire dataset. Next, a tutorial demonstrates how to analyze the read count data to generate lists of differentially expressed genes using R/DESeq2. Additional tutorials generate read counts using the Snakemake workflow manager and Nextflow with Google Batch. All tutorials are open-source and can be used as templates for other analysis.

Single-Cell RNA Sequencing Reveals Transcriptional Signatures and Cell-Cell Communication in Diabetic Retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of vision loss in workingage individuals worldwide. Cell-to-cell communication between retinal cells and retinal pigment epithelial cells (RPEs) in DR is still unclear, so this study aimed to generate a single-cell atlas and identify receptor‒ligand communication between retinal cells and RPEs. METHODS: A mouse single-cell RNA sequencing (scRNA-seq) dataset was retrieved from the GEO database (GSE178121) and was further analyzed with the R package Seurat. Cell cluster annotation was performed to further analyze cell‒cell communication. The differentially expressed genes (DEGs) in RPEs were explored through pathway enrichment analysis and the protein‒ protein interaction (PPI) network. Core genes in the PPI were verified by quantitative PCR in ARPE-19 cells. RESULTS: We observed an increased proportion of RPEs in STZ mice. Although some overall intercellular communication pathways did not differ significantly in the STZ and control groups, RPEs relayed significantly more signals in the STZ group. In addition, THBS1, ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 were found to be the core DEGs of the PPI network in RPEs. qPCR results showed that the expression of ITGB1, COL9A3, ITGB8, VTN, TIMP2, and FBN1 was higher and consistent with scRNA-seq results in ARPE-19 cells under hyperglycemic conditions. CONCLUSION: Our study, for the first time, investigated how signals that RPEs relay to and from other cells underly the progression of DR based on scRNA-seq. These signaling pathways and hub genes may provide new insights into DR mechanisms and therapeutic targets.

Specific microRNAs in stallion spermatozoa are potential biomarkers of high functionality.

Males of some species, from horses to humans, require medical help for subfertility problems. There is an urgent need for novel molecular assays that reflect spermatozoal function. In the last 25 years, studies examined RNAs in spermatozoa as a window into gene expression during their development and, more recently, for their functions in early embryo development. In clinics, more dense spermatozoa are isolated by density gradient centrifugation before use in artificial insemination to increase pregnancy rates. The objectives of the current study were to discover and quantify the microRNAs in stallion spermatozoa and identify those with differential expression levels in more dense versus less dense spermatozoa. First, spermatozoa from seven stallions were separated into more dense and less dense populations by density gradient centrifugation. Next, small RNAs were sequenced from each of the 14 RNA samples. We identified 287 different mature microRNAs within the 11,824,720 total mature miRNA reads from stallion spermatozoa. The most prevalent was miR-10a/b-5p. The less dense spermatozoa had fewer mature microRNAs and more microRNA precursor sequences than more dense spermatozoa, perhaps indicating that less dense spermatozoa are less mature. Two of the most prevalent microRNAs in more dense stallion spermatozoa were predicted to target mRNAs that encode proteins that accelerate mRNA decay. Nine microRNAs were more highly expressed in more dense spermatozoa. Three of those microRNAs were predicted to target mRNAs that encode proteins involved in protein decay. Both mRNA and protein decay are very active in late spermiogenesis but not in mature spermatozoa. The identified microRNAs may be part of the mechanism to shut down those processes. The microRNAs with greater expression in more dense spermatozoa may be useful biomarkers for spermatozoa with greater functional capabilities.

Atopic Dermatitis Complicated by Recurrent Eczema Herpeticum Is Characterized by Multiple, Concurrent Epidermal Inflammatory Endotypes.

A subgroup of patients with atopic dermatitis (AD) suffers from recurrent, disseminated herpes simplex virus skin infection, termed eczema herpeticum. To determine the transcriptional mechanisms of the skin and immune system pathobiology that underlie development of AD with eczema herpeticum (ADEH), we performed RNA-sequencing analysis of nonlesional skin (epidermis, dermis) from AD patients with and without a history of ADEH (ADEH+, n = 15; ADEH-, n = 13) along with healthy controls (n = 15). We also performed RNA sequencing on participants' plasmacytoid dendritic cells infected in vitro with herpes simplex virus 1. ADEH+ patients exhibited dysregulated gene expression, limited in the dermis (14 differentially expressed genes) and more widespread in the epidermis (129 differentially expressed genes). ADEH+-upregulated epidermal differentially expressed genes were enriched in type 2 cytokine (IL4R , CCL22, CRLF2, IL7R), interferon (CXCL10, ICAM1, IFI44, IRF7), and IL-36γ (IL36G) inflammatory gene pathways. All ADEH+ participants exhibited type 2 cytokine and inteferon endotypes, and 87% were IL36G-high. In contrast, these endotypes were more variably expressed among ADEH- participants. ADEH+ skin also had dysregulated epidermal differentiation complex gene expression of the late-cornified envelope, S100A, and small proline-rich gene families, which are involved in skin barrier function and antimicrobial activities. Plasmacytoid dendritic cell transcriptional responses to herpes simplex virus 1 infection were unaltered by ADEH status. The study concluded that the pathobiology underlying ADEH+ risk is associated with a unique, multifaceted epidermal inflammation that accompanies dysregulation of epidermal differentiation complex genes. These findings will help direct future studies that define how these inflammatory patterns may drive risk of eczema herpeticum in AD.

Single nucleus RNA-sequencing integrated into risk variant colocalization discovers 17 cell-type-specific abdominal obesity genes for metabolic dysfunction-associated steatotic liver disease.

BACKGROUND: Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD). METHODS: To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants. FINDINGS: We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue. INTERPRETATION: Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis. FUNDING: NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki.

RNA-sequencing reveals differential fibroblast responses to bleomycin and pneumonectomy.

Pulmonary fibrosis is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, including increased extracellular matrix synthesis, also occur following pneumonectomy (PNX), but PNX instead results in regenerative compensatory growth of the lung. As fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing fibroblast responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regenerative versus fibrotic lung responses. RNA-sequencing was performed on flow-sorted fibroblasts freshly isolated from mouse lungs 14 days after BLM, PNX, or sham controls. RNA-sequencing analysis revealed highly similar biological processes to be involved in fibroblast responses to both BLM and PNX, including TGF-ß1 and TNF-α. Interestingly, we observed smaller changes in gene expression after PNX than BLM at Day 14, suggesting that the fibroblast response to PNX may be muted by expression of transcripts that moderate pro-fibrotic pathways. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up-regulated by PNX and not BLM. ITPKC overexpression in lung fibroblasts antagonized the pro-fibrotic effect of TGF-ß1. RNA-sequencing analysis has identified considerable overlap in transcriptional changes between fibroblasts following PNX and those overexpressing ITPKC.

Human post-implantation blastocyst-like characteristics of Muse cells isolated from human umbilical cord.

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine.

Data from collection and analysis of RNA sequencing data from pearl millet.

Pearl millet (Pennisetum glaucum, also known as Cenchrus americanus) is a cereal crop that has a C4 photosynthesis system and that can grow and develop seeds even under stressed conditions including drought-stressed, high temperature-stressed and nutrient-poor conditions. In previous studies, transcriptomes of pearl millet were studied by RNA sequencing (RNA-Seq) to understand mechanisms regulating its development and tolerance to such stressed conditions. Here, RNA-Seq reads from 565 pearl millet samples from 25 projects in the NCBI (National Center for Biotechnology Information) BioProject database were collected and mapped to the pearl millet reference genome to obtain read counts and transcripts per million (TPM) for each pearl millet gene. The count and TPM data for all the 565 samples as well as the attributes of those samples and projects were deposited in the figshare repository (https://doi.org/10.6084/m9.figshare.24902100).

Exploring the Pathogenesis of Autoimmune Liver Diseases from the Heterogeneity of Target Cells.

The incidence of autoimmune liver diseases (ALDs) and research on their pathogenesis are increasing annually. However, except for autoimmune hepatitis, which responds well to immunosuppression, primary biliary cholangitis and primary sclerosing cholangitis are insensitive to immunosuppressive therapy. Besides the known effects of the environment, genetics, and immunity on ALDs, the heterogeneity of target cells provides new insights into their pathogenesis. This review started by exploring the heterogeneity in the development, structures, and functions of hepatocytes and epithelial cells of the small and large bile ducts. For example, cytokeratin (CK) 8 and CK18 are primarily expressed in hepatocytes, while CK7 and CK19 are primarily expressed in intrahepatic cholangiocytes. Additionally, emerging technologies of single-cell RNA sequencing and spatial transcriptomic are being applied to study ALDs. This review offered a new perspective on understanding the pathogenic mechanisms and potential treatment strategies for ALDs.

Direct RNA Sequencing of Foot-and-mouth Disease Virus Genome Using a Flongle on MinION.

Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).

Characterizing defective lipid metabolism in the lateral septum of mice treated with olanzapine: implications for its side effects.

Schizophrenia significantly impacts cognitive and behavioral functions and is primarily treated with second-generation antipsychotics (SGAs) such as olanzapine. Despite their efficacy, these drugs are linked to serious metabolic side effects which can diminish patient compliance, worsen psychiatric symptoms and increase cardiovascular disease risk. This study explores the hypothesis that SGAs affect the molecular determinants of synaptic plasticity and brain activity, particularly focusing on the lateral septum (LS) and its interactions within hypothalamic circuits that regulate feeding and energy expenditure. Utilizing functional ultrasound imaging, RNA sequencing, and weighted gene co-expression network analysis, we identified significant alterations in the functional connection between the hypothalamus and LS, along with changes in gene expression in the LS of mice following prolonged olanzapine exposure. Our analysis revealed a module closely linked to increases in body weight and adiposity, featuring genes primarily involved in lipid metabolism pathways, notably Apoa1, Apoc3, and Apoh. These findings suggest that olanzapine may influence body weight and adiposity through its impact on lipid metabolism-related genes in the LS. Therefore, the neural circuits connecting the LS and LH, along with the accompanying alterations in lipid metabolism, are likely crucial factors contributing to the weight gain and metabolic side effects associated with olanzapine treatment.

Single-cell analysis of Crohn's disease: Unveiling heterogeneity and evaluating ustekinumab outcomes.

BACKGROUND: The present study aimed to dissect the cellular complexity of Crohn's disease (CD) using single-cell RNA sequencing, focusing on identifying key cell populations and their transcriptional profiles in inflamed tissue. METHODS: We applied scRNA-sequencing to compare the cellular composition of CD patients with healthy controls, utilizing Seurat for clustering and annotation. Differential gene expression analysis and protein-protein interaction networks were constructed to identify crucial genes and pathways. RESULTS: Our study identified eight distinct cell types in CD, highlighting crucial fibroblast and T cell interactions. The analysis revealed key cellular communications and identified significant genes and pathways involved in the disease's pathology. The role of fibroblasts was underscored by elevated expression in diseased samples, offering insights into disease mechanisms and potential therapeutic targets, including responses to ustekinumab treatment, thus enriching our understanding of CD at a molecular level. CONCLUSIONS: Our findings highlight the complex cellular and molecular interplay in CD, suggesting new biomarkers and therapeutic targets, offering insights into disease mechanisms and treatment implications.

Bone marrow cells contribute to seven different endothelial cell populations in the heart.

Understanding the mechanisms underlying vascular regeneration in the heart is crucial for developing novel therapeutic strategies for myocardial ischemia. This study investigates the contribution of bone marrow-derived cells to endothelial cell populations in the heart, and their role in cardiac function and coronary circulation following repetitive ischemia (RI). Chimeric rats were created by transplanting BM cells from GFP female rats into irradiated male recipients. After engraftment chimeras were subjected to RI for 17 days. Vascular growth was assessed from recovery of cardiac function and increases in myocardial blood flow during LAD occlusion. After sorting GFP+ BM cells from heart and bone of Control and RI rats, single-cell RNA sequencing was implemented to determine the fate of BM cells. Our in vivo RI model demonstrated an improvement in cardiac function and myocardial blood flow after 17 days of RI with increased capillary density in the rats subjected to RI compared to Controls. Single-cell RNA sequencing of bone marrow cells isolated from rats' hearts identified distinct endothelial cell (EC) subpopulations. These ECs exhibited heterogeneous gene expression profiles and were enriched for markers of capillary, artery, lymphatic, venous, and immune ECs. Furthermore, BM-derived ECs in the RI group showed an angiogenic profile, characterized by upregulated genes associated with blood vessel development and angiogenesis. This study elucidates the heterogeneity of bone marrow-derived endothelial cells in the heart and their response to repetitive ischemia, laying the groundwork for targeting specific subpopulations for therapeutic angiogenesis in myocardial ischemia.

The Novel-B-Cell-Related Gene Signature Predicts the Prognosis and Immune Status of Patients with Esophageal Carcinoma.

BACKGROUND: The current understanding of the prognostic significance of B cells and their role in the tumor microenvironment (TME) in esophageal carcinoma (ESCA) is limited. METHODS: We conducted a screening for B-cell-related genes through the analysis of single-cell transcriptome data. Subsequently, we developed a B-cell-related gene signature (BRGrisk) using LASSO regression analysis. Patients from The Cancer Genome Atlas cohort were divided into a training cohort and a test cohort. Patients were categorized into high- and low-risk groups based on their median BRGrisk scores. The overall survival was assessed using the Kaplan-Meier method, and a nomogram based on BRGrisk was constructed. Immune infiltration profiles between the risk groups were also compared. RESULTS: The BRGrisk prognostic model indicated significantly worse outcomes for patients with high BRGrisk scores (p < 0.001). The BRGrisk-based nomogram exhibited good prognostic performance. Analysis of immune infiltration revealed that patients in the high-BRGrisk group had notably higher levels of immune cell infiltration and were more likely to be in an immunoresponsive state. Enrichment analysis showed a strong correlation between the prognostic gene signature and cancer-related pathways. IC50 results indicated that patients in the low-BRGrisk group were more responsive to common drugs compared to those in the high-BRGrisk group. CONCLUSIONS: This study presents a novel BRGrisk that can be used to stratify the prognosis of ESCA patients and may offer guidance for personalized treatment strategies aimed at improving prognosis.

RNA sequencing reveals the potential mechanism of exercise preconditioning for cerebral ischemia reperfusion injury in rats.

INTRODUCTION: Cerebral ischemia reperfusion injury (CIRI) often leads to deleterious complications after stroke patients receive reperfusion therapy. Exercise preconditioning (EP) has been reported to facilitate brain function recovery. We aim to explore the specific mechanism of EP in CIRI. METHODS: Sprague-Dawley rats were randomized into Sham, middle cerebral artery occlusion (MCAO), and EP groups (n = 11). The rats in the EP group received adaptive training for 3 days (10 m/min, 20 min/day, with a 0° incline) and formal training for 3 weeks (6 days/week, 25 m/min, 30 min/day, with a 0° incline). Then, rats underwent MCAO surgery to establish CIRI models. After 48 h, neurological deficits and cerebral infarction of the rats were measured. Neuronal death and apoptosis in the cerebral cortices were detected. Furthermore, RNA sequencing was conducted to investigate the specific mechanism of EP on CIRI, and qPCR and Western blotting were further applied to confirm RNA sequencing results. RESULTS: EP improved neurological deficit scores and reduced cerebral infarction in MCAO rats. Additionally, pre-ischemic exercise also alleviated neuronal death and apoptosis of the cerebral cortices in MCAO rats. Importantly, 17 differentially expressed genes (DEGs) were identified through RNA sequencing, and these DEGs were mainly enriched in the HIF-1 pathway, cellular senescence, proteoglycans in cancer, and so on. qPCR and Western blotting further confirmed that EP could suppress TIMP1, SOCS3, ANGPTL4, CDO1, and SERPINE1 expressions in MCAO rats. CONCLUSION: EP can improve CIRI in vivo, the mechanism may relate to TIMP1 expression and HIF-1 pathway, which provided novel targets for CIRI treatment.

Exploring the Mechanism of Shen Qi Gui Oral Liquid against Chemotherapy-Induced Myelosuppression in Cancer Patients Based on Network Pharmacology and Molecular Docking.

BACKGROUND: Shen Qi Gui oral liquid (SQG) may be beneficial for chemotherapyinduced myelosuppression (CIM). However, the underlying mechanism of CIM treated with SQG is still lacking. METHODS: A total of 27 blood samples from cancer patients were selected to perform RNA-seq to obtain the Differentially Expressed Genes (DEGs). Then, the active components and target genes of SQG were acquired. Next, the drug targets and DEGs were intersected to obtain the intersection genes, followed by functional enrichment analysis and construction of a drug-compoundgene- disease network. Subsequently, core genes were selected. Then, immune cell infiltration, molecular docking, pharmacokinetic and toxicity prediction, and RT-qPCR were performed. RESULTS: A total of 1,341 DEGs, 51 active compounds, and 264 target genes were identified. Then, 30 intersection genes were acquired. Next, a drug-compound-gene-disease network was constructed, and 7 core genes were acquired. Immune infiltration analysis exhibited that only T follicular helper cells were significantly increased in the CIM group, which was significantly negatively correlated with MAPK1, MAPK14, MCL1, PTEN, and PTGS2. The luteolin, quercetin, and beta-sitosterol showed better affinity with core genes. Luteolin and quercetin, which satisfied Lipinski's rule of five, were likely absorbed by the gastrointestinal system. Toxicity predictions showed that neither luteolin nor quercetin exhibited carcinogenicity or hepatotoxicity. CONCLUSION: PTEN, PTGS2, CCL2, FOS, MCL1, MAPK1, and MAPK14 were identified as the core genes in CIM patients, which were involved in the MAPK and PI3K-Akt signaling pathways. Luteolin and quercetin may be the promising drugs against CIM.