Single Cell Transcriptome Analysis During Development in Dictyostelium.

Dictyostelium represents a stripped-down model for understanding how cells make decisions during development. The complete life cycle takes around a day and the fully differentiated structure is composed of only two major cell types. With this apparent reduction in "complexity," single cell transcriptomics has proven to be a valuable tool in defining the features of developmental transitions and cell fate separation events, even providing causal information on how mechanisms of gene expression can feed into cell decision-making. These scientific outputs have been strongly facilitated by the ease of non-disruptive single cell isolation-allowing access to more physiological measures of transcript levels. In addition, the limited number of cell states during development allows the use of more straightforward analysis tools for handling the ensuing large datasets, which provides enhanced confidence in inferences made from the data. In this chapter, we will outline the approaches we have used for handling Dictyostelium single cell transcriptomic data, illustrating how these approaches have contributed to our understanding of cell decision-making during development.

Corrigendum: Bipartite networks represent causality better than simple networks: Evidence, algorithms, and applications.

[This corrects the article DOI: 10.3389/fgene.2024.1371607.].

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.

Temporal sex specific brain gene expression pattern during early rat embryonic development.

Background: The classical concept of brain sex differentiation suggests that steroid hormones released from the gonads program male and female brains differently. However, several studies indicate that steroid hormones are not the only determinant of brain sex differentiation and that genetic differences could also be involved. Methods: In this study, we have performed RNA sequencing of rat brains at embryonic days 12 (E12), E13, and E14. The aim was to identify differentially expressed genes between male and female rat brains during early development. Results: Analysis of genes expressed with the highest sex differences showed that Xist was highly expressed in females having XX genotype with an increasing expression over time. Analysis of genes expressed with the highest male expression identified three early genes, Sry2, Eif2s3y, and Ddx3y. Discussion: The observed sex-specific expression of genes at early development confirms that the rat brain is sexually dimorphic prior to gonadal action on the brain and identifies Sry2 and Eif2s3y as early genes contributing to male brain development.

Integrated analysis of single-cell RNA-seq, bulk RNA-seq, Mendelian randomization, and eQTL reveals T cell-related nomogram model and subtype classification in rheumatoid arthritis.

Objective: Rheumatoid arthritis (RA) is a systemic disease that attacks the joints and causes a heavy economic burden on humans worldwide. T cells regulate RA progression and are considered crucial targets for therapy. Therefore, we aimed to integrate multiple datasets to explore the mechanisms of RA. Moreover, we established a T cell-related diagnostic model to provide a new method for RA immunotherapy. Methods: scRNA-seq and bulk-seq datasets for RA were obtained from the Gene Expression Omnibus (GEO) database. Various methods were used to analyze and characterize the T cell heterogeneity of RA. Using Mendelian randomization (MR) and expression quantitative trait loci (eQTL), we screened for potential pathogenic T cell marker genes in RA. Subsequently, we selected an optimal machine learning approach by comparing the nine types of machine learning in predicting RA to identify T cell-related diagnostic features to construct a nomogram model. Patients with RA were divided into different T cell-related clusters using the consensus clustering method. Finally, we performed immune cell infiltration and clinical correlation analyses of T cell-related diagnostic features. Results: By analyzing the scRNA-seq dataset, we obtained 10,211 cells that were annotated into 7 different subtypes based on specific marker genes. By integrating the eQTL from blood and RA GWAS, combined with XGB machine learning, we identified a total of 8 T cell-related diagnostic features (MIER1, PPP1CB, ICOS, GADD45A, CD3D, SLFN5, PIP4K2A, and IL6ST). Consensus clustering analysis showed that RA could be classified into two different T-cell patterns (Cluster 1 and Cluster 2), with Cluster 2 having a higher T-cell score than Cluster 1. The two clusters involved different pathways and had different immune cell infiltration states. There was no difference in age or sex between the two different T cell patterns. In addition, ICOS and IL6ST were negatively correlated with age in RA patients. Conclusion: Our findings elucidate the heterogeneity of T cells in RA and the communication role of these cells in an RA immune microenvironment. The construction of T cell-related diagnostic models provides a resource for guiding RA immunotherapeutic strategies.

Occurrence, distribution, and genetic diversity of faba bean viruses in China.

With worldwide cultivation, the faba bean (Vicia faba L.) stands as one of the most vital cool-season legume crops, serving as a major component of food security. China leads global faba bean production in terms of both total planting area and yield, with major production hubs in Yunnan, Sichuan, Jiangsu, and Gansu provinces. The faba bean viruses have caused serious yield losses in these production areas, but previous researches have not comprehensively investigated this issue. In this study, we collected 287 faba bean samples over three consecutive years from eight provinces/municipalities of China. We employed small RNA sequencing, RT-PCR, DNA sequencing, and phylogenetic analysis to detect the presence of viruses and examine their incidence, distribution, and genetic diversity. We identified a total of nine distinct viruses: bean yellow mosaic virus (BYMV, Potyvirus), milk vetch dwarf virus (MDV, Nanovirus), vicia cryptic virus (VCV, Alphapartitivirus), bean common mosaic virus (BCMV, Potyvirus), beet western yellows virus (BWYV, Polerovirus), broad bean wilt virus (BBWV, Fabavirus), soybean mosaic virus (SMV, Potyvirus), pea seed-borne mosaic virus (PSbMV, Potyvirus), and cucumber mosaic virus (CMV, Cucumovirus). BYMV was the predominant virus found during our sampling, followed by MDV and VCV. This study marks the first reported detection of BCMV in Chinese faba bean fields. Except for several isolates from Gansu and Yunnan provinces, our sequence analysis revealed that the majority of BYMV isolates contain highly conserved nucleotide sequences of coat protein (CP). Amino acid sequence alignment indicates that there is a conserved NAG motif at the N-terminal region of BYMV CP, which is considered important for aphid transmission. Our findings not only highlight the presence and diversity of pathogenic viruses in Chinese faba bean production, but also provide target pathogens for future antiviral resource screening and a basis for antiviral breeding.

Exploring the diversity of galls on Artemisia indica induced by Rhopalomyia species through morphological and transcriptome analyses.

Plant galls generated by insects have highly organized structures, providing nutrients and shelter to the insects living within them. Most research on the physiological and molecular mechanisms of gall development has focused on single galls. To understand the diversity of gall development, we examined five galls with different morphologies generated by distinct species of Rhopalomyia (gall midge; Diptera: Cecidomyiidae) on a single host plant of Artemisia indica var. maximowiczii (Asteraceae). Vasculature developed de novo within the galls, indicating active transport of nutrients between galls and the host plant. Each gall had a different pattern of vasculature and lignification, probably due to differences in the site of gall generation and the gall midge species. Transcriptome analysis indicated that photosynthetic and cell wall-related genes were down-regulated in leaf and stem galls, respectively, compared with control leaf and stem tissues, whereas genes involved in floral organ development were up-regulated in all types of galls, indicating that transformation from source to sink organs occurs during gall development. Our results help to understand the diversity of galls on a single herbaceous host plant.

Lipid-associated macrophages reshape BAT cell identity in obesity.

Obesity and type 2 diabetes cause a loss in brown adipose tissue (BAT) activity, but the molecular mechanisms that drive BAT cell remodeling remain largely unexplored. Using a multilayered approach, we comprehensively mapped a reorganization in BAT cells. We uncovered a subset of macrophages as lipid-associated macrophages (LAMs), which were massively increased in genetic and dietary model of BAT expansion. LAMs participate in this scenario by capturing extracellular vesicles carrying damaged lipids and mitochondria released from metabolically stressed brown adipocytes. CD36 scavenger receptor drove LAM phenotype, and CD36-deficient LAMs were able to increase brown fat genes in adipocytes. LAMs released transforming growth factor ß1 (TGF-ß1), which promoted the loss of brown adipocyte identity through aldehyde dehydrogenase 1 family member A1 (Aldh1a1) induction. These findings unfold cell dynamic changes in BAT during obesity and identify LAMs as key responders to tissue metabolic stress and drivers of loss of brown adipocyte identity.

Establishment of an ovarian cancer exhausted CD8+T cells-related genes model by integrated analysis of scRNA-seq and bulk RNA-seq.

Ovarian cancer (OC) was the fifth leading cause of cancer death and the deadliest gynecological cancer in women. This was largely attributed to its late diagnosis, high therapeutic resistance, and a dearth of effective treatments. Clinical and preclinical studies have revealed that tumor-infiltrating CD8+T cells often lost their effector function, the dysfunctional state of CD8+T cells was known as exhaustion. Our objective was to identify genes associated with exhausted CD8+T cells (CD8TEXGs) and their prognostic significance in OC. We downloaded the RNA-seq and clinical data from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. CD8TEXGs were initially identified from single-cell RNA-seq (scRNA-seq) datasets, then univariate Cox regression, the least absolute shrinkage and selection operator (LASSO), and multivariate Cox regression were utilized to calculate risk score and to develop the CD8TEXGs risk signature. Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, time-dependent receiver operating characteristics (ROC), nomogram, and calibration were conducted to verify and evaluate the risk signature. Gene set enrichment analyses (GSEA) in the risk groups were used to figure out the closely correlated pathways with the risk group. The role of risk score has been further explored in the homologous recombination repair deficiency (HRD), BRAC1/2 gene mutations and tumor mutation burden (TMB). A risk signature with 4 CD8TEXGs in OC was finally built in the TCGA database and further validated in large GEO cohorts. The signature also demonstrated broad applicability across various types of cancer in the pan-cancer analysis. The high-risk score was significantly associated with a worse prognosis and the risk score was proven to be an independent prognostic biomarker. The 1-, 3-, and 5-years ROC values, nomogram, calibration, and comparison with the previously published models confirmed the excellent prediction power of this model. The low-risk group patients tended to exhibit a higher HRD score, BRCA1/2 gene mutation ratio and TMB. The low-risk group patients were more sensitive to Poly-ADP-ribose polymerase inhibitors (PARPi). Our findings of the prognostic value of CD8TEXGs in prognosis and drug response provided valuable insights into the molecular mechanisms and clinical management of OC.

Imbalance of programmed cell death patterns mediated by dendritic cell subsets in systemic lupus erythematosus and lupus nephritis. / æ ‘çªçŠ¶ç»†èƒžäºšç¾¤ä»‹å¯¼ç³»ç»Ÿæ€§çº¢æ–‘ç‹¼ç–®å’Œç‹¼ç–®è‚¾ç‚Žçš„ç¨‹åºæ€§ç»†èƒžæ­»äº¡æ¨¡å¼å¤±è¡¡ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Abnormal programmed cell death in immune cells is associated with autoimmune diseases, but the patterns of programmed cell death in systemic lupus erythematosus (SLE) and especially lupus nephritis (LN) remain unclear. This study aims to explore the association between SLE, LN, and immune cell death patterns. METHODS: Bulk RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatic analysis was conducted to explore the expression levels of genes related to 3 cell death patterns in peripheral blood mononuclear cells of SLE patients. Key cell subsets involved in the imbalance of cell death patterns were identified through scRNA-seq. Immunofluorescence was used to detect the expression levels of receptor interacting serine/threonine kinase 3 (RIPK3), mixed-lineage kinase domain-like protein (MLKL), phosphorylated MLKL (pMLKL), caspase 1 (CASP1), CD1c molecule (CD1C), C-type lectin domain containing 9A (CLEC9A), and X-C motif chemokine receptor 1 (XCR1) in dendritic cells (DC). scRNA-seq was performed on kidney tissues collected from LN patients and healthy controls (HC) at the Third Xiangya Hospital of Central South University, followed by bioinformatic analysis to identify key cell subsets involved in the imbalance of cell death patterns. Pseudotime analysis and ligand-receptor analysis were used to explore the differentiation direction and cell communication of different DC subsets. Transient transfection was used to transfect RAW264.7 cells with empty plasmid, empty plasmid+dsDNA (HSV-DNA), empty plasmid+200 µmol/L tert-butyl hydroperoxide (TBHP), stimulator of interferon genes (STING) shRNA plasmid, STING shRNA plasmid+dsDNA (HSV-DNA), and STING shRNA plasmid+200 µmol/L TBHP. Annexin V-mCherry and SYTOX Green staining were used to detect cell death in each group. Western blotting was used to detect the activation of CASP1, gasdermin D (GSDMD), RIPK3, and MLKL in each group. RESULTS: Bioinformatic analysis showed an imbalance in 3 cell death patterns in SLE and LN patients: Pro-inflammatory pyroptosis and necroptosis were activated, while anti-inflammatory apoptosis was inhibited. The key cell subsets involved were DC subsets, particularly focusing on CLEC9A+cDC1. Immunofluorescence results showed that the expression levels of RIPK3, MLKL, and CASP1 in DCs were higher in the SLE group compared to the HC group. pMLKL and CASP1 expression levels in renal cDC1 marked by CLEC9A and XCR1 were higher in the LN group than in the HC group. Pseudotime analysis and ligand-receptor analysis suggested that the CLEC9A+cDC1 subset in LN kidney tissues originated from peripheral circulation. Annexin V-mCherry and SYTOX Green staining results showed that the number of dead cells decreased in the STING shRNA transfection group compared to the empty plasmid group in RAW264.7 cells. Western blotting results showed that the activation of CASP1, GSDMD, RIPK3, and MLKL was decreased in the STING shRNA transfection group compared to the empty plasmid group. CONCLUSIONS: This study provides novel insights into the role of CLEC9A+cDC1 in the imbalance of cell death patterns in SLE and LN.

Single-cell transcriptome analysis reveals two subtypes of tumor cells of sclerosing pneumocytoma with distinct molecular features and clinical implications.

Pulmonary sclerosing pneumocytoma (PSP) is a rare, distinctive benign lung adenoma of pneumocyte origin. Despite its rarity, the tumor's unique cellular morphology has sparked ongoing debates regarding the origin of its constituent cells. This study aimed to elucidate the molecular features of PSP tumor cells and enhance our understanding of the cellular processes contributing to PSP formation and biological behavior. Tissue samples from PSP and corresponding normal lung tissues (n = 4) were collected. We employed single-cell RNA sequencing and microarray-based spatial transcriptomic analyses to identify cell types and investigate their transcriptomes, with a focus on transcription factors, enriched gene expression, and single-cell trajectory evaluations. Our analysis identified two types of tumor cells: mesenchymal epithelial dual phenotype cells (MEDPs) and a distinct subpopulation of type II alveolar epithelial cells exhibiting characteristics slightly reminiscent of type I alveolar epithelial cells (AT2Cs), corresponding to histological round stromal cells and surface cuboidal cells, respectively. MEDPs displayed weak alveolar epithelial differentiation but strong collagen production capabilities, as indicated by the expression of both TTF-1 and vimentin. These cells played a pivotal role in forming the solid and sclerotic areas of PSP. Moreover, MEDPs exhibited a pronounced propensity for epithelial-mesenchymal transition, suggesting a greater potential for metastasis compared to AT2Cs. The capillary endothelial cells of PSP displayed notable diversity. Overall, this study provides, for the first time, a comprehensive mapping of the single-cell transcriptome profile of PSP. Our findings delineate two distinct subtypes of tumor cells, MEDPs and AT2Cs, each with its own biological characteristics and spatial distribution. A deeper understanding of these cell types promises insights into the histology and biological behaviors of this rare tumor.

Identification of a subpopulation of highly adherent endothelial cells for seeding synthetic vascular grafts.

OBJECTIVE: There is an unmet clinical need for alternatives to autologous vessel grafts. Small-diameter (<6mm) synthetic vascular grafts are not suitable because of unacceptable patency rates. This mainly occurs due to the lack of an endothelial cell (EC) monolayer to prevent platelet activation, thrombosis, and intimal hyperplasia. There are no reliable methods to endothelialize small-diameter grafts, as most seeded ECs are lost due to exposure to fluid shear stress (SS) after implantation. The goal of this work is to determine if EC loss is a random process or if it is possible to predict which cells are more likely to remain adherent. METHODS: In initial studies, we sorted ECs using fluid SS and identified a subpopulation of ECs that are more likely to resist detachment. We use RNA-sequencing (RNA-seq) to examine gene expression of adherent ECs compared to the whole population. Using fluorescence activated cell sorting (FACS), we sorted ECs based on the expression level of a candidate marker and studied their retention in small-diameter vascular grafts in vitro. RESULTS: Transcriptomic analysis revealed that fibronectin leucine rich transmembrane protein 2 (FLRT2), encoding protein FLRT2, is downregulated in the ECs that are more likely to resist detachment. When seeded onto vascular grafts and exposed to SS, ECs expressing low levels of FLRT2 exhibit 59.2±7.4% retention compared to 24.5±6.1% retention for the remainder of the EC population. CONCLUSIONS: For the first time, we show EC detachment is not an entirely random process. This provides validation for the concept that we can seed small-diameter vascular grafts only with highly adherent ECs to maintain a stable endothelium and improve graft patency rates.

Deciphering brain cellular and behavioral mechanisms: Insights from single-cell and spatial RNA sequencing.

The brain is a complex computing system composed of a multitude of interacting neurons. The computational outputs of this system determine the behavior and perception of every individual. Each brain cell expresses thousands of genes that dictate the cell's function and physiological properties. Therefore, deciphering the molecular expression of each cell is of great significance for understanding its characteristics and role in brain function. Additionally, the positional information of each cell can provide crucial insights into their involvement in local brain circuits. In this review, we briefly overview the principles of single-cell RNA sequencing and spatial transcriptomics, the potential issues and challenges in their data processing, and their applications in brain research. We further outline several promising directions in neuroscience that could be integrated with single-cell RNA sequencing, including neurodevelopment, the identification of novel brain microstructures, cognition and behavior, neuronal cell positioning, molecules and cells related to advanced brain functions, sleep-wake cycles/circadian rhythms, and computational modeling of brain function. We believe that the deep integration of these directions with single-cell and spatial RNA sequencing can contribute significantly to understanding the roles of individual cells or cell types in these specific functions, thereby making important contributions to addressing critical questions in those fields. This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA in Disease and Development > RNA in Development RNA in Disease and Development > RNA in Disease.

Maize zinc uptake is influenced by arbuscular mycorrhizal symbiosis under various soil phosphorus availabilities.

The antagonistic interplay between phosphorus (P) and zinc (Zn) in plants is well established. However, the molecular mechanisms mediating those interactions as influenced by arbuscular mycorrhizal (AM) symbiosis remain unclear. We investigated Zn concentrations, root AM symbiosis, and transcriptome profiles of maize roots grown under field conditions upon different P levels. We also validated genotype-dependent P-Zn uptake in selected genotypes from a MAGIC population and conducted mycorrhizal inoculation experiments using mycorrhizal-defective mutant pht1;6 to elucidate the significance of AM symbiosis in P-Zn antagonism. Finally, we assessed how P supply affects Zn transporters and Zn uptake in extraradical hyphae within a three-compartment system. Elevated P levels led to a significant reduction in maize Zn concentration across the population, correlating with a marked decline in AM symbiosis, thus elucidating the P-Zn antagonism. We also identified ZmPht1;6 is crucial for AM symbiosis and confirmed that P-Zn antagonistic uptake is dependent on AM symbiosis. Moreover, we found that high P suppressed the expression of the fungal RiZRT1 and RiZnT1 genes, potentially impacting hyphal Zn uptake. We conclude that high P exerts systemic regulation over root and AM hyphae-mediated Zn uptake in maize. These findings hold implications for breeding Zn deficiency-tolerant maize varieties.

Prostate cancer cancer-associated fibroblasts with stable markers post-androgen deprivation therapy associated with tumor progression and castration resistant prostate cancer.

The specificity and clinical relevance of cancer-associated fibroblasts (CAFs) in prostate cancer (PCa), as well as the effect of androgen deprivation therapy (ADT) on CAFs, remain to be fully elucidated. Using cell lineage diversity and weighted gene co-expression network analysis (WGCNA), we pinpointed a unique CAF signature exclusive to PCa. The specificity of this CAF signature was validated through single-cell RNA sequencing (scRNA-seq), cell line RNA sequencing, and immunohistochemistry. This signature associates CAFs with tumor progression, elevated Gleason scores, and the emergence of castration resistant prostate cancer (CRPC). Using scRNA-seq on collected samples, we demonstrated that the CAF-specific signature is not altered by ADT, maintaining its peak signal output. Identifying a PCa-specific CAF signature and observing signaling changes in CAFs after ADT lay essential groundwork for further PCa studies.

A distinct subset of urothelial cells with enhanced EMT features promotes chemotherapy resistance and cancer recurrence by increasing COL4A1-ITGB1 mediated angiogenesis.

Drug resistance and tumor recurrence remain clinical challenges in the treatment of urothelial carcinoma (UC). However, the underlying mechanism is not fully understood. Here, we performed single-cell RNA sequencing and identified a subset of urothelial cells with epithelial-mesenchymal transition (EMT) features (EMT-UC), which is significantly correlated with chemotherapy resistance and cancer recurrence. To validate the clinical significance of EMT-UC, we constructed EMT-UC like cells by introducing overexpression of two markers, Zinc Finger E-Box Binding Homeobox 1 (ZEB1) and Desmin (DES), and examined their histological distribution characteristics and malignant phenotypes. EMT-UC like cells were mainly enriched in UC tissues from patients with adverse prognosis and exhibited significantly elevated EMT, migration and gemcitabine tolerance in vitro. However, EMT-UC was not specifically identified from tumorous tissues, certain proportion of them were also identified in adjacent normal tissues. Tumorous EMT-UC highly expressed genes involved in malignant behaviors and exhibited adverse prognosis. Additionally, tumorous EMT-UC was associated with remodeled tumor microenvironment (TME), which exhibited high angiogenic and immunosuppressive potentials compared with the normal counterparts. Furthermore, a specific interaction of COL4A1 and ITGB1 was identified to be highly enriched in tumorous EMT-UC, and in the endothelial component. Targeting the interaction of COL4A1 and ITGB1 with specific antibodies significantly suppressed tumorous angiogenesis and alleviated gemcitabine resistance of UC. Overall, our findings demonstrated that the driven force of chemotherapy resistance and recurrence of UC was EMT-UC mediated COL4A1-ITGB1 interaction, providing a potential target for future UC treatment.

RNA-binding protein AZGP1 inhibits epithelial cell proliferation by regulating the genes of alternative splicing in COPD.

BACKGROUND: Chronic Obstructive Pulmonary Disease (COPD) is characterized by high morbidity, disability, and mortality rates worldwide. RNA-binding proteins (RBPs) might regulate genes involved in oxidative stress and inflammation in COPD patients. Single-cell transcriptome sequencing (scRNA-seq) offers an accurate tool for identifying intercellular heterogeneity and the diversity of immune cells. However, the role of RBPs in the regulation of various cells, especially AT2 cells, remains elusive. MATERIALS AND METHODS: A scRNA-seq dataset (GSE173896) and a bulk RNA-seq dataset acquired from airway tissues (GSE124180) were employed for data mining. Next, RNA-seq analysis was performed in both COPD and control patients. Differentially expressed genes (DEGs) were identified using criteria of fold change (FC ≥ 1.5 or ≤ 1.5) and P value ≤ 0.05. Lastly, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and alternative splicing identification analyses were carried out. RESULTS: RBP genes exhibited specific expression patterns across different cell groups and participated in cell proliferation and mitochondrial dysfunction in AT2 cells. As an RBP, AZGP1 expression was upregulated in both the scRNA-seq and RNA-seq datasets. It might potentially be a candidate immune biomarker that regulates COPD progression by modulating AT2 cell proliferation and adhesion by regulating the expression of SAMD5, DNER, DPYSL3, GBP5, GBP3, and KCNJ2. Moreover, AZGP1 regulated alternative splicing events in COPD, particularly DDAH1 and SFRP1, holding significant implications in COPD. CONCLUSION: RBP gene AZGP1 inhibits epithelial cell proliferation by regulating genes participating in alternative splicing in COPD.

Transcriptomic analysis reveals zinc-mediated virulence and pathogenicity in multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium well known for its multidrug resistance and connection to nosocomial infections under ESKAPE pathogens. This opportunistic pathogen is ubiquitously associated with nosocomial infections, posing significant threats within healthcare environments. Its critical clinical symptoms, namely, meningitis, urinary tract infections, bloodstream infections, ventilator-associated pneumonia, and pneumonia, catalyze the imperative demand for innovative therapeutic interventions. The proposed research focuses on delineating the role of Zinc, a crucial metallo-binding protein and micronutrient integral to bacterial metabolism and virulence, to enhance understanding of the pathogenicity of A. baumannii. RNA sequencing and subsequent DESeq2 analytical methods were used to identify differential gene expressions influenced by zinc exposure. Exploiting the STRING database for functional enrichment analysis has demonstrated the complex molecular mechanisms underlying the enhancement of pathogenicity prompted by Zinc. Moreover, hub genes like gltB, ribD, AIL77834.1, sdhB, nuoI, acsA_1, acoC, accA, accD were predicted using the cytohubba tool in Cytoscape. This investigation underscores the pivotal role of Zinc in the virulence of A. baumannii elucidates the underlying molecular pathways responsible for its pathogenicity. The research further accentuates the need for innovative therapeutic strategies to combat A. baumannii infections, particularly those induced by multidrug-resistant strains.

Integrating single-nucleus RNA sequencing and spatial transcriptomics to elucidate a specialized subpopulation of astrocytes, microglia and vascular cells in brains of mouse model of lipopolysaccharide-induced sepsis-associated encephalopathy.

BACKGROUND: Understanding the mechanism behind sepsis-associated encephalopathy (SAE) remains a formidable task. This study endeavors to shed light on the complex cellular and molecular alterations that occur in the brains of a mouse model with SAE, ultimately unraveling the underlying mechanisms of this condition. METHODS: We established a murine model using intraperitoneal injection of lipopolysaccharide (LPS) in wild type and Anxa1-/- mice and collected brain tissues for analysis at 0-hour, 12-hour, 24-hour, and 72-hour post-injection. Utilizing advanced techniques such as single-nucleus RNA sequencing (snRNA-seq) and Stereo-seq, we conducted a comprehensive characterization of the cellular responses and molecular patterns within the brain. RESULTS: Our study uncovered notable temporal differences in the response to LPS challenge between Anxa1-/- (annexin A1 knockout) and wild type mice, specifically at the 12-hour and 24-hour time points following injection. We observed a significant increase in the proportion of Astro-2 and Micro-2 cells in these mice. These cells exhibited a colocalization pattern with the vascular subtype Vas-1, forming a distinct region known as V1A2M2, where Astro-2 and Micro-2 cells surrounded Vas-1. Moreover, through further analysis, we discovered significant upregulation of ligands and receptors such as Timp1-Cd63, Timp1-Itgb1, Timp1-Lrp1, as well as Ccl2-Ackr1 and Cxcl2-Ackr1 within this region. In addition, we observed a notable increase in the expression of Cd14-Itgb1, Cd14-Tlr2, and Cd14-C3ar1 in regions enriched with Micro-2 cells. Additionally, Cxcl10-Sdc4 showed broad upregulation in brain regions containing both Micro-2 and Astro-2 cells. Notably, upon LPS challenge, there was an observed increase in Anxa1 expression in the mouse brain. Furthermore, our study revealed a noteworthy increase in mortality rates following Anxa1 knockdown. However, we did not observe substantial differences in the types, numbers, or distribution of other brain cells between Anxa1-/- and wildtype mice over time. Nevertheless, when comparing the 24-hour post LPS injection time point, we observed a significant decrease in the proportion and distribution of Micro-2 and Astro-2 cells in the vicinity of blood vessels in Anxa1-/- mice. Additionally, we noted reduced expression levels of several ligand-receptor pairs including Cd14-Tlr2, Cd14-C3ar1, Cd14-Itgb1, Cxcl10-Sdc4, Ccl2-Ackr1, and Cxcl2-Ackr1. CONCLUSIONS: By combining snRNA-seq and Stereo-seq techniques, our study successfully identified a distinctive cellular colocalization, referred to as a special pathological niche, comprising Astro-2, Micro-2, and Vas-1 cells. Furthermore, we observed an upregulation of ligand-receptor pairs within this niche. These findings suggest a potential association between this cellular arrangement and the underlying mechanisms contributing to SAE or the increased mortality observed in Anxa1 knockdown mice.

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out (DKO) mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.