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Background: The use of neoadjuvant chemoradiotherapy (neoCRT) followed by surgery has markedly enhanced the quality of survival in patients suffering from local advanced rectal cancer (LARC). Enhancing this treatment requires a deep understanding of its underlying mechanism. The heterogeneous nature of the tumor microenvironment (TME) significantly impacts therapeutic responses, presenting complex therapeutic challenges., Methods: In this comprehensive study, we explored the intricate cellular and molecular shifts within the TME of LARC after neoCRT administration. Using single-cell transcriptomic analysis, we meticulously examined 32,417 cells sourced from six samples, each representing different tumor regression grades (TRG: 0 versus 2). This detailed analysis enabled us to characterize the various cell subpopulations, encompassing epithelial cells, lymphocytes, myeloid cells, endothelial cells, and fibroblasts. Additionally, we identified their marker genes for deconvolution calculation in the READ cohort of the TCGA project. And we obtain their marker genes for deconvolution calculation in the READ cohort of the TCGA project., Results: Through cluster analysis and pathway comparisons of malignant tumor cells, we discerned that samples with poor tumor regression exhibit enhanced metabolic versatility and adaptability, enabling them to counteract the impacts of both radiotherapy and chemotherapy. Interestingly, within the TRG2 cohort, we observed a predominant immunosuppressive state in the TME, characterized by the activation of CD4 + regulatory T cells, maintained CD8 + T cell functionality, and a heightened M1 to M2 macrophage ratio. Moreover, the differing outcomes of neoCRT were reflected in the varying interaction dynamics between macrophages (M1 and M2) and CD4+/CD8 + T cells. Furthermore, our data reveal that neoCRT intricately modulates fibroblasts and endothelial cells, primarily through the extracellular matrix remodeling pathway, which orchestrates tumor angiogenesis. All changes were validated through immunofluorescence staining on intraoperative samples before and after treatment. To summarize, our investigation presents a comprehensive exploration of the cellular and molecular metamorphoses within the TME post-neoCRT., Conclusions: By unveiling the sophisticated interaction between the multifaceted cells within the TME and their respective reactions to neoCRT, we establish a robust platform for ensuing future investigations. This study paves the way for novel therapeutic strategies that leverage these insights to bolster the efficacy of neoCRT in managing LARC., Competing Interests: Declarations Ethics approval and consent to participate The study was conducted in accordance with the Declaration of Helsinki, as revised in 2013. Conflict of interest The authors declare there are no potential conflicts of interest., (© 2024. The Author(s).)

Background: Global warming-induced environmental stresses have diverse effects on gene expression and regulation in the life processes of various aquatic organisms. N6 adenylate methylation (m6A) modifications are known to influence mRNA transcription, localization, translation, stability, splicing, and nuclear export, which are pivotal in mediating stress responses. Apostichopus japonicus is a significant species in aquaculture and a representative of benthic organisms in ecosystems, thus there is a growing need for research on its heat stress mechanism., Results: In this study, m6A-modified whole transcriptome profiles of the respiratory tree tissues of A. japonicus in the control (T18) and high-temperature stress (T32) groups were obtained using MeRIP-seq technology. The results showed that 7,211 common m6A peaks, and 9,459 genes containing common m6A were identified in three replicates T18 and T32 groups. The m6A peaks were found to be highly enriched in the 3' untranslated region, and the common sequence of the m6A peak was also enriched, which was shown as RRACH (R = G or A; H = A, C, or U). A total of 1,200 peaks were identified as significantly differentially enriched in the T32 group compared with the T18 group. Among them, 245 peaks were upregulated and 955 were downregulated, which indicated that high temperature stress significantly altered the methylation pattern of m6A, and there were more demethylation sites in the T32 group. Conjoint analysis of the m6A methylation modification and the transcript expression level (the MeRIP-seq and RNA-seq data) showed co-differentiated 395 genes were identified, which were subsequently divided into four groups with a predominant pattern that more genes with decreased m6A modification and up-regulated expression, including HSP70IV, EIF2AK1, etc. GO enrichment and KEGG analyses of differential m6A peak related genes and co-differentiated genes showed the genes were significantly associated with transcription process and pathways such as protein processing in the endoplasmic reticulum, Wnt signaling pathway, and mTOR signaling pathway, etc. CONCLUSION: The comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification was involved in the regulation of heat stress-responsive genes and important functional pathways in A. japonicus in response to high-temperature stress. The study will contribute to elucidate the regulatory mechanism of m6A modification in the response of A. japonicus to environmental stress, as well as the conservation and utilization of sea cucumber resources in the context of environmental changes., Competing Interests: Declarations Ethics approval and consent to participate The animal study was approved by the Institutional Animal Care and Use Committee of the Ludong University (protocol number LDU-IRB20210308NXY). The study was conducted in accordance with the local legislation and institutional requirements. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The quality of Recombination signal sequences (RSSs), location, and genetics of mammalian V, D, and J genes synergistically affect the recombination frequency of genes; however, the specific regulatory mechanism and efficiency have not been elucidated. By taking advantage of single-cell RNA-sequencing (scRNA-seq) and high-throughput sequencing (HTS) to investigate V(D)J rearrangement characteristics in the CDR3 repertoire, we found that the distal and proximal V genes (or J genes) "to D" gene were involved in rearrangement significantly more frequently than the middle V genes (or J genes) in the TRB locus among various species, including Primates (human and rhesus monkey), Rodentia (BALB/c, C57BL/6, and Kunming mice), Artiodactyla (buffalo), and Chiroptera (Rhinolophus affinis). The RSS quality of the V and J genes affected their frequency in rearrangement to varying degrees, especially when the V-RSSs with recombination signal information content (RIC) score < -45 significantly reduced the recombination frequency of the V gene. The V and J genes that were "away from D" had the dual advantages of recombinant structural accessibility and relatively high-quality RSSs, which promoted their preferential utilization in rearrangement. The quality of J-RSSs formed during mammalian evolution was apparently greater than that of V-RSSs, and the D-J distance was obviously shorter than that of V-D, which may be one of the reasons for guaranteeing that the "D-to-J preceding V-to-DJ rule" occurred when rearranged. This study provides a novel perspective on the mechanism and efficiency of V-D-J rearrangement in the mammalian TRB locus, as well as the biased utilization characteristics and application of V and J genes in the initial CDR3 repertoire., (© 2024. The Author(s).)

Background: Chronic obstructive pulmonary disease (COPD) is a complex respiratory condition influenced by environmental and genetic factors. Using next-generation sequencing, we aimed to identify dysregulated genes and potential therapeutic targets for COPD., Methods: Peripheral blood leukocyte RNA profiles from COPD patients and healthy controls were analyzed using next-generation sequencing. Key genes involved in COPD pathogenesis were identified through protein-protein interaction network analysis. In vitro, bronchial epithelial cells treated with cigarette smoke extract (CSE) were used to study the effects on gene expression, cell viability, apoptosis, and ferroptosis. Additionally, Lipocalin 2 (LCN2) inhibition experiments were conducted to elucidate its role in COPD-related cellular processes., Results: Analysis of RNA profiles revealed consistent downregulation of 17 genes and upregulation of 21 genes across all COPD groups. Among these, Cathelicidin Antimicrobial Peptide(CAMP), Defensin Alpha 4(DEFA4), Neutrophil Elastase(ELANE), LCN2 and Lactotransferrin(LTF) were identified as potentially important players in COPD pathogenesis. Particularly, LCN2 exhibited a close association with COPD and was found to be involved in cellular processes. In vitro experiments demonstrated that CSE treatment significantly increased LCN2 expression in bronchial epithelial cells in a concentration-dependent manner. Moreover, CSE-induced apoptosis and ferroptosis were observed, along with alterations in cell viability, Glutathione content, Fe2 + accumulation, ROS: Reactive Oxygen Species and Malondialdehyde levels, Lactate Dehydrogenase(LDH) release and Glutathione Peroxidase 4(GPX4) expression. Inhibition of LCN2 expression partially reversed these effects, indicating the pivotal role of LCN2 in COPD-related cellular processes., Conclusion: Our study identified six candidate genes: CAMP, DEFA4, ELANE, LCN2, and LTF were upregulated, HSPA1B was downregulated. Notably, LCN2 emerges as a significant biomarker in COPD pathogenesis, exerting its effects by promoting apoptosis and ferroptosis in bronchial epithelial cells., (© 2024. The Author(s).)

Background: Chronic Kidney Disease (CKD) impacts over 10% of the global population, and recent advancements in high-throughput analytical technologies are uncovering the complex physiology underlying this condition. By integrating Genome-Wide Association Studies (GWAS), RNA sequencing (RNA-seq/RNA array), and single-cell RNA sequencing (scRNA-seq) data, our study aimed to explore the genes and cell types relevant to CKD traits., Methods: GWAS summary data for end-stage renal failure (ESRD) and decreased eGFR (CKD) with or without diabetes and (micro)proteinuria were obtained from the GWAS Catalog and the UK Biobank (UKB) database. Two gene Expression Omnibus (GEO) transcriptome datasets were used to establish glomerular and tubular gene expression differences between CKD patients and healthy individuals. Two scRNA-seq datasets were utilized to obtain the expression of key genes at the single-cell level. The expression profile, differentially expressed genes (DEGs), gene-gene interaction, and pathway enrichment were analysed for these CKD risk genes., Results: A total of 779 distinct SNPs were identified from GWAS across different CKD traits, involving 681 genes. While many of these risk genes are specific to the CKD traits of renal failure, decreased eGFR, and (micro)proteinuria, they share common pathways, including extracellular matrix (ECM). ECM modeling was enriched in upregulated glomerular and tubular DEGs from CKD kidneys compared to healthy controls, with the expression of relevant collagen genes, such as COL1A2, prevalent in fibroblasts/myofibroblasts. Additionally, immune responses, including T cell differentiation, were dysregulated in CKD kidneys. The late podocyte signature gene THSD7A was enriched in podocytes but downregulated in CKD. We also highlighted that the regulated risk genes of CKD are mainly expressed in tubular cells and immune cells in the kidney., Conclusions: Our integrated analysis highlight the genes, pathways, and relevant cell types associational with the pathogenesis of kidney traits, as a basis for further mechanistic studies to understand the pathogenesis of CKD., (© 2024. The Author(s).)

The pathogenesis of Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) remains unclear, though increasing evidence suggests inflammatory processes play key roles. In this study, single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) was used to decipher the immunometabolic profile in 4 ME/CFS patients and 4 heathy controls. We analyzed changes in the composition of major PBMC subpopulations and observed an increased frequency of total T cells and a significant reduction in NKs, monocytes, cDCs and pDCs. Further investigation revealed even more complex changes in the proportions of cell subpopulations within each subpopulation. Gene expression patterns revealed upregulated transcription factors related to immune regulation, as well as genes associated with viral infections and neurodegenerative diseases.CD4 + and CD8 + T cells in ME/CFS patients show different differentiation states and altered trajectories, indicating a possible suppression of differentiation. Memory B cells in ME/CFS patients are found early in the pseudotime, indicating a unique subtype specific to ME/CFS, with increased differentiation to plasma cells suggesting B cell overactivity. NK cells in ME/CFS patients exhibit reduced cytotoxicity and impaired responses, with reduced expression of perforin and CD107a upon stimulation. Pseudotime analysis showed abnormal development of adaptive immune cells and an enhanced cell-cell communication network converging on monocytes in particular. Our analysis also identified the estrogen-related receptor alpha (ESRRA)-APP-CD74 signaling pathway as a potential biomarker for ME/CFS in peripheral blood. In addition, data from the GSE214284 database confirmed higher ESRRA expression in the monocyte cell types of male ME/CFS patients. These results suggest a link between immune and neurological symptoms. The results support a disease model of immune dysfunction ranging from autoimmunity to immunodeficiency and point to amyloidotic neurodegenerative signaling pathways in the pathogenesis of ME/CFS. While the study provides important insights, limitations include the modest sample size and the evaluation of peripheral blood only. These findings highlight potential targets for diagnostic biomarkers and therapeutic interventions. Further research is needed to validate these biomarkers and explore their clinical applications in managing ME/CFS., (© 2024. The Author(s).)

Background: Huntington's disease (HD) is a hereditary neurological disorder caused by mutations in HTT, leading to neuronal degeneration. Traditionally, HD is associated with the misfolding and aggregation of mutant huntingtin due to an extended polyglutamine domain encoded by an expanded CAG tract. However, recent research has also highlighted the role of global transcriptional dysregulation in HD pathology. However, understanding the intricate relationship between mRNA expression and HD at the cellular level remains challenging. Our study aimed to elucidate the underlying mechanisms of HD pathology using single-cell sequencing data., Results: We used single-cell RNA sequencing analysis to determine differential gene expression patterns between healthy and HD cells. HD cells were effectively modeled using a residual neural network (ResNet), which outperformed traditional and convolutional neural networks. Despite the efficacy of our approach, the F1 score for the test set was 96.53%. Using the SHapley Additive exPlanations (SHAP) algorithm, we identified genes influencing HD prediction and revealed their roles in HD pathobiology, such as in the regulation of cellular iron metabolism and mitochondrial function. SHAP analysis also revealed low-abundance genes that were overlooked by traditional differential expression analysis, emphasizing its effectiveness in identifying biologically relevant genes for distinguishing between healthy and HD cells. Overall, the integration of single-cell RNA sequencing data and deep learning models provides valuable insights into HD pathology., Conclusion: We developed the model capable of analyzing HD at single-cell transcriptomic level., (© 2024. The Author(s).)

Background: Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the "1-kb" deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the 'major' and 'minor' transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in "healthy" samples without juvenile CLN3 disease., Methods: We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources., Results: We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated 'major' transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data., Conclusion: Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis., (© 2024. The Author(s).)

Background: Myocardial infarction (MI) induces complex transcriptional changes across diverse cardiac cell types. Single-cell RNA sequencing (scRNA-seq) provides an unparalleled ability to discern cellular diversity during infarction, yet the veracity of these discoveries necessitates confirmation. This investigation sought to elucidate MI mechanisms by integrating scRNA-seq and bulk RNA-seq data., Methods: Publicly available scRNA-seq (GSE136088) and bulk RNA-seq (GSE153485) data from mice MI models were analyzed. Cell types were annotated, and differential expression analysis conducted. Bulk RNA-seq underwent quality control, principal component analysis, and differential expression analysis., Results: In scRNA-seq data, the comparison between MI and sham groups unveiled a reduction in endothelial cell populations, but macrophages and monocytes increased. Within fibroblast subgroups, three distinct categories were discerned, with two exhibiting upregulation in MI. Notably, endothelial cells exhibited an elevated expression of genes associated with apoptosis and ferroptosis. In bulk RNA-seq analysis, distinct patterns emerged when comparing MI and sham groups. Specifically, six genes linked to endothelial ferroptosis exhibited heightened expression in MI group, thereby corroborating the scRNA-seq findings. Moreover, the examination of isolated cardiac macrophages from mice MI model revealed increased expression of Spp1, Col1a2, Col3a1, Ctsd, and Lgals3 compared to sham group, thus substantiating the dysregulation of macrophage apoptosis-related proteins following MI., Conclusion: MI altered the transcriptomic landscapes of cardiac cells with increased expression of apoptotic genes. Moreover, the upregulation of macrophage apoptosis marker was confirmed within MI models. The presence of endothelial cell depletion and ferroptosis in MI has been demonstrated., (© 2024. The Author(s).)

Single-cell technology depicts integrated tumor profiles including both tumor cells and tumor microenvironments, which theoretically enables more robust diagnosis than traditional diagnostic standards based on only pathology. However, the inherent challenges of single-cell RNA sequencing (scRNA-seq) data, such as high dimensionality, low signal-to-noise ratio (SNR), sparse and non-Euclidean nature, pose significant obstacles for traditional diagnostic approaches. The diagnostic value of single-cell technology has been largely unexplored despite the potential advantages. Here, we present a graph neural network-based framework tailored for molecular diagnosis of primary liver tumors using scRNA-seq data. Our approach capitalizes on the biological plausibility inherent in the intercellular communication networks within tumor samples. By integrating pathway activation features within cell clusters and modeling unidirectional inter-cellular communication, we achieve robust discrimination between malignant tumors (including hepatocellular carcinoma, HCC, and intrahepatic cholangiocarcinoma, iCCA) and benign tumors (focal nodular hyperplasia, FNH) by scRNA data of all tissue cells and immunocytes only. The efficacy to distinguish iCCA from HCC was further validated on public datasets. Through extending the application of high-throughput scRNA-seq data into diagnosis approaches focusing on integrated tumor microenvironment profiles rather than a few tumor markers, this framework also sheds light on minimal-invasive diagnostic methods based on migrating/circulating immunocytes., (© 2024. The Author(s).)

Age-related hearing loss (ARHL) or presbycusis is associated with irreversible progressive damage in the inner ear, where the sound is transduced into electrical signal; but the detailed mechanism remains unclear. Here, we sought to determine the potential molecular mechanism involved in the pathogeneses of ARHL with bioinformatics methods. A single-cell transcriptome sequencing study was performed on the cochlear samples from young and aged mice. Detection of identified cell type marker allowed us to screen 18 transcriptional clusters, including myeloid cells, epithelial cells, B cells, endothelial cells, fibroblasts, T cells, inner pillar cells, neurons, inner phalangeal cells, and red blood cells. Cell-cell communications were analyzed between young and aged cochlear tissue samples by using the latest integration algorithms Cellchat. A total of 56 differentially expressed genes were screened between the two groups. Functional enrichment analysis showed these genes were mainly involved in immune, oxidative stress, apoptosis, and metabolic processes. The expression levels of crucial genes in cochlear tissues were further verified by immunohistochemistry. Overall, this study provides new theoretical support for the development of clinical therapeutic drugs., (© 2024. The Author(s).)

Barkground: Circular RNAs (circRNAs) play important regulatory roles in a variety of biological processes in mammals. Multiple birth-traits in goats are affected by several factors, but the expression and function of circRNAs in follicular development of goats are not clear. In this study, we aimed to investigate the possible regulatory mechanisms of circRNA and collected five groups of large follicles (Follicle diameter > 6 mm) and small follicles (1 mm < Follicle diameter < 3 mm) from Leizhou goats in estrus for RNA sequencing., Results: RNA sequencing showed that 152 circRNAs were differentially expressed in small and large follicles. Among them, 101 circRNAs were up-regulated in large follicles and 51 circRNAs were up-regulated in small follicles. GO and KEGG enrichment analyses showed that parental genes of the differential circRNAs were significantly enriched in important pathways, such as ovarian steroidogenesis, GnRH signaling pathway, animal autophagy and oxytocin signalling pathway. BioSignal analysis revealed that 152 differentially expressed circRNAs could target 91 differential miRNAs including miR-101 family (chi-miR-101-3p, chi-miR-101-5p), miR-202 family (chi-miR-202-5p, chi-miR-202-3p),60 circRNAs with translation potential. Based on the predicted sequencing results, the ceRNA networks chicirc&#95;008762/chi-miR-338-3p/ARHGAP18 and chicirc&#95;040444/chi-miR-338-3p/STAR were constructed in this study. Importantly, the new gene circCFAP20DC was first discovered in goats. The EDU assay and flow cytometry results indicated that circCFAP20DC enhanced the proliferation of follicular granulosa cells(GCs). Real-time quantitative PCR and western blotting assays showed that circCFAP20DC activated the Retinoblastoma(RB) pathway and promoted the progression of granulosa cells from G1 to S phase., Conclusion: Differential circRNAs in goat size follicles may have important biological functions for follicular development. The novel gene circCFAP20DC activates the RB pathway, promoting the progression of GCs from G1 to S phase. This, in turn, enhances the proliferation of follicular GCs in goats., (© 2024. The Author(s).)

Background: Milk is essential for mammalian nutrition because it provides vital nutrients for growth and development. Milk composition, which is influenced by genetic and environmental factors, supports lactation, a complex process crucial for milk production and quality. Recent research has focused on noncoding RNAs, particularly microRNAs (miRNAs), which are present in body fluids and regulate gene expression post-transcriptionally. This study comprehensively characterizes miRNAs in milk of four livestock species, namely Bubalus bubalis, Capra hircus, Equus asinus, and Ovis aries and identifies potential target genes., Results: High-throughput sequencing of milk RNA resulted in distinct read counts across species: B. bubalis (8,790,441 reads), C. hircus (12,976,275 reads), E. asinus (9,385,067 reads), and O. aries (7,295,297 reads). E. asinus had the highest RNA mapping rate (94.6%) and O. aries the lowest (84.8%). A substantially greater proportion of miRNAs over other small RNAs was observed for the donkey milk sample (7.74%) compared to buffalo (0.87%), goat (1.57%), and sheep (1.12%). Shared miRNAs, which included miR-200a, miR-200b, miR-200c, and miR-23a among others, showed varying expression levels across species, confirmed by qPCR analysis. Functional annotation of predicted miRNA target genes highlighted diverse roles, with an enrichment in functions linked to metabolism and immunity. Pathway analysis identified immune response pathways as significant, with several miRNAs targeting specific genes across species, suggesting their regulatory function in milk., Conclusions: Both conserved and species-specific miRNAs were detected in milk of the investigated species. The identified target genes of these miRNAs have important roles in neonatal development, adaptation, growth, and immune response. Furthermore, they influence milk and meat production traits in livestock., (© 2024. The Author(s).)

Background: Although the patient survival rate for many malignancies has been improved with immune checkpoint inhibitors (ICIs), some patients experience various immune-related adverse events (irAEs). IrAEs impact several organ systems, including the kidney. With anti-programmed cell death protein 1 (PD-1) therapy (pembrolizumab), kidney-related adverse events occur relatively rarely compared with other irAEs. However, the occurrence of AKI usually leads to anti-PD-1 therapy interruption or discontinuation. Therefore, there is an urgent need to clarify the mechanisms of renal irAEs (R-irAEs) to facilitate early management. This study aimed to analyse the characteristics of peripheral blood mononuclear cells (PBMCs) in R-irAEs., Methods: PBMCs were collected from three patients who developed R-irAEs after anti-PD-1 therapy and three patients who did not. The PBMCs were subjected to scRNA-seq to identify cell clusters and differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) enrichment analyses were performed to investigate the most active biological processes in immune cells., Results: Fifteen cell clusters were identified across the two groups. FOS, RPS26, and JUN were the top three upregulated genes in CD4 + T cells. The DEGs in CD4 + T cells were enriched in Th17 differentiation, Th1 and Th2 cell differentiation, NF-kappa B, Nod-like receptor, TNF, IL-17, apoptosis, and NK cell-mediated cytotoxicity signaling pathways. RPS26, TRBV25-1, and JUN were the top three upregulated genes in CD8 + T cells. The DEGs in CD8 + T cells were enriched in Th17 cell differentiation, antigen processing and presentation, natural killer cell-mediated cytotoxicity, the intestinal immune network for IgA production, the T-cell receptor signalling pathway, Th1 and Th2 cell differentiation, the phagosome, and cell adhesion molecules., Conclusions: In conclusion, R-irAEs are associated with immune cell dysfunction. DEGs and their enriched pathways identified in CD4 + T cells and CD8 + T cells play important roles in the development of renal irAEs related to anti-PD-1 therapy. These findings offer fresh perspectives on the pathogenesis of renal damage caused by anti-PD-1 therapy., (© 2024. The Author(s).)

Objective and Methods: To ascertain the connection between cuproptosis-related genes (CRGs) and the prognosis of hepatocellular carcinoma (HCC) via single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) data, relevant data were downloaded from the GEO and TCGA databases. The differentially expressed CRGs (DE-CRGs) were filtered by the overlaps in differentially expressed genes (DEGs) between HCC patients and normal controls (NCs) in the scRNA-seq database, DE-CRGs between high- and low-CRG-activity cells, and DEGs between HCC patients and NCs in the TCGA database., Results: Thirty-three DE-CRGs in HCC were identified. A prognostic model (PM) was created employing six survival-related genes (SRGs) (NDRG2, CYB5A, SOX4, MYC, TM4SF1, and IFI27) via univariate Cox regression analysis and LASSO. The predictive ability of the model was validated via a nomogram and receiver operating characteristic curves. Research has employed tumor immune dysfunction and exclusion as a means to examine the influence of PM on immunological heterogeneity. Macrophage M0 levels were significantly different between the high-risk group (HRG) and the low-risk group (LRG), and a greater macrophage level was linked to a more unfavorable prognosis. The drug sensitivity data indicated a substantial difference in the half-maximal drug-suppressive concentrations of idarubicin and rapamycin between the HRG and the LRG. The model was verified by employing public datasets and our cohort at both the protein and mRNA levels., Conclusion: A PM using 6 SRGs (NDRG2, CYB5A, SOX4, MYC, TM4SF1, and IFI27) was developed via bioinformatics research. This model might provide a fresh perspective for assessing and managing HCC., (© 2024. The Author(s).)

Background: With the widespread use of antibiotics, more attention has been paid to their side effects. We paid extra attention to the impact of antibiotics on children's bodies. Therefore, we analyzed the characteristic changes in the gut microbiota of children after antibiotic treatment to explore the pathogenesis of antibiotic-associated diseases in more depth and to provide a basis for diagnosis and treatment., Methods: We recruited 28 children with bronchopneumonia in the western district of Zhuhai, China, and divided them into three treatment groups based on antibiotic type. We took stool samples from children before and 3-5 days after antibiotic treatment. 16S rRNA gene sequencing was used to analyze the effects of antibiotic therapy on the gut microbiota of children. Continuous nonparametric data are represented as median values and analyzed using the Wilcoxon rank-sum test., Results: While alpha diversity analysis found no significant changes in the mean abundance of the gut microbiota of children after a short course of antibiotic treatment, beta diversity analysis demonstrated significant changes in the composition and diversity of the gut microbiota of children even after a short course of antibiotic therapy. We also found that meloxicillin sulbactam can inhibit the growth of Proteobacteria, Bacteroidetes, and Verrucomicrobia, ceftriaxone inhibits Verrucomicrobia and Bacteroides, and azithromycin inhibits Fusobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. We further performed a comparative analysis at the genus level and found significantly different clusters in each group. Finally, we found that azithromycin had the greatest effect on the metabolic function of intestinal microbiota, followed by ceftriaxone, and no significant change in the metabolic process of intestinal microbiota after meloxicillin sulbactam treatment., Conclusions: Antibiotic treatment significantly affects the diversity of intestinal microbiota in children, even after a short course of antibiotic treatment. Different classes of antibiotics affect diverse microbiota primarily, leading to varying alterations in metabolic function. Meanwhile, we identified a series of intestinal microbiota that differed significantly after antibiotic treatment. These groups of microbiota could be used as biomarkers to provide an additional basis for diagnosing and treating antibiotic-associated diseases., (© 2024. The Author(s).)

Background: The Testis is an important reproductive organ in male mammals and the site for spermatogenesis, androgen synthesis, and secretion. Non-coding RNAs (ncRNAs) play an important regulatory role in various biological processes. However, the regulatory role of ncRNAs in the development of yak testes and spermatogenesis remains largely unclear., Result: In this study, we compared the expression profiles of circular RNAs (circRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs) in yak testicular tissue samples collected at 6 months (Y6M), 18 months (Y18M), and 4 years (Y4Y). Using RNA sequencing (RNA-Seq), we observed a significant difference in the expression patterns of ncRNAs in the samples collected at different testicular development stages. Twenty-two differentially expressed (DE) circRNAs, 69 DE miRNAs, and 64 DE mRNAs were detected in Y6M, Y18M, and Y4Y testicular samples, respectively. The results of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the source genes of DE circRNAs, predicted target genes of DE miRNAs, and DE mRNAs were specifically associated with signaling pathways and GO terms that were related to sperm synthesis, sperm vitality, and testicular development, such as cell cycle, Wnt signaling pathway, MAPK signaling pathway, GnRH signaling pathway, and spermatogenesis. The analysis of the circRNA-miRNA-mRNA network revealed that some DE ncRNAs, including miR-574, miR-449a, CDC42, and CYP11A1, among others, may be involved in testicular spermatogenesis. Concurrently, various circRNA-miRNA interaction pairs were observed., Conclusion: Our findings provide a database of circRNAs, miRNAs, and mRNAs expression profiles in testicular tissue of yaks at different developmental stages and a detailed understanding of the regulatory network of ncRNAs in yak testicular development and provide data that can help elucidate the molecular mechanisms underlying yak testicular development., (© 2024. The Author(s).)

Background: Obesity is a global health issue with detrimental effects on various human organs, including the reproductive system. Observational human data and several lines of animal experimental data suggest that maternal obesity impairs ovarian function and early embryo development, but the precise pathogenesis remains unclear., Methods: We established a high-fat diet (HFD)-induced obese female mouse model to assess systemic metabolism, ovarian morphology, and oocyte function in mice. For the first time, this study employed single-cell RNA sequencing to explore the altered transcriptomic landscape of preimplantation embryos at different stages in HFD-induced obese mice. Differential gene expression analysis, enrichment analysis and protein-protein interactions network analysis were performed., Results: HFD-induced obese female mice exhibited impaired glucolipid metabolism and insulin resistance. The ovaries of HFD mice had a reduced total follicle number, an increased proportion of atretic follicles, and irregular granulosa cell arrangement. Furthermore, the maturation rate of embryonic development by in vitro fertilization of oocytes was significantly decreased in HFD mice. Additionally, the transcriptional landscapes of preimplantation embryos at different stages in mice induced by different diets were significantly distinguished. The maternal-to-zygotic transition was also affected by the failure to remove maternal RNAs and to turn off zygotic genome expression., Conclusions: HFD-induced obesity impaired ovarian morphology and oocyte function in female mice and further led to alterations in the transcriptional landscape of preimplantation embryos at different stages of HFD mice., (© 2024. The Author(s).)

Background: Hemorrhagic enteritis, caused by Turkey Hemorrhagic Enteritis Virus (THEV), is a disease affecting turkey poults characterized by immunosuppression and bloody diarrhea. An avirulent THEV strain that retains the immunosuppressive ability is used as a live vaccine. Characterizing the splice map of THEV is an essential step that would allow studies of individual genes mediating its immunosuppressive functions. We used RNA sequencing to characterize the splice map of THEV for the first time, providing key insights into the THEV gene expression and mRNA structures., Methods: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection. Total RNA was extracted, and poly-A-tailed mRNA sequenced. Reads were mapped to the THEV genome after trimming and transcripts assembled with StringTie. We performed PCR of THEV cDNA, cloned the PCR products, and used Sanger sequencing to validate all identified splice junctions., Results: Researchers previously annotated the THEV genome as encoding 23 open reading frames (ORFs). We identified 29 spliced transcripts from our RNA sequencing data, all containing novel exons although some exons matched some previously annotated ORFs. The three annotated splice junctions were also corroborated by our data. During validation we identified five additional unique transcripts, a subset of which were further validated by 3' rapid amplification of cDNA ends (3' RACE). Thus, we report that the genome of THEV contains 34 transcripts with the coding capacity for all annotated ORFs. However, we found six of the previously annotated ORFs to be truncated ORFs on the basis of the identification of an in-frame upstream start codon or the detection of additional coding exons. We also identified three of the annotated ORFs with longer or shorter isoforms, and seven novel unannotated ORFs that could potentially be translated; although it is beyond the scope of this manuscript to investigate whether they are translated., Conclusions: Similar to human adenoviruses, all THEV transcripts are spliced and organized into five transcription units under the control of their cognate promoters. The genes are expressed under temporal regulation and THEV also produces multiple distinctly spliced transcripts that code for the same protein. Studies of the newly identified potential proteins should be urgently performed as these proteins may have roles in THEV-induced immunosuppression. Also, knowing the splicing of THEV genes should be invaluable to future research focusing on studying THEV genes, as this will allow accurate cloning of the mRNAs., (© 2024. The Author(s).)

Background: Extensive research has been conducted on embryonic developmental disorders linked to Polycystic Ovary Syndrome (PCOS), a pathological condition that affects 5-10% of women and is characterized by irregularities in the menstrual cycle and infertility. By employing RNA sequencing (RNA-seq), we performed an in-depth investigation of PCOS-related changes in gene expression patterns at the mouse blastocyst stage., Methods: The zygotes of female B6D2 mice were obtained and then differentiated into blastocysts in K + Simplex Optimised Medium (KSOM) cultures containing exo-NC (negative control for exosomes) or exo-LIPE-AS1 (a novel exosomal marker of PCOS). Subsequently, blastocysts were collected for RNA-seq. The bioinformatics was performed to analyze and compare the differences of gene expression profile between blastocysts of control and PCOS group., Results: There were 1150 differentially expressed genes (DEGs) between the two groups of mouse blastocysts; 243 genes were upregulated and 907 downregulated in the blastocysts of the exo-LIPE-AS1 group compared to those of the exo-NC group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that the genes involved in amino acid synthesis and glutathione metabolic pathways were down-regulated in exo-LIPE-AS1 group., Conclusion: This study has revealed that blastocyst developmental retardation may be associated with the downregulation of amino acid synthesis and glutathione metabolism, which may affect energy metabolism, biosynthesis, cellular osmotic pressure, antioxidant synthesis, ROS clearance or mitochondrial function, and ultimately cause blastocyst cell development abnormalities. Our research offers encouraging data on the mechanisms underlying aberrant embryonic development in patients with PCOS as well as potential treatment strategies., (© 2024. The Author(s).)

Background: Association testing between molecular phenotypes and genomic variants can help to understand how genotype affects phenotype. RNA sequencing provides access to molecular phenotypes such as gene expression and alternative splicing while DNA sequencing or microarray genotyping are the prevailing options to obtain genomic variants., Results: We genotype variants for 74 male Braunvieh cattle from both DNA (~ 13-fold coverage) and deep total RNA sequencing from testis, vas deferens, and epididymis tissue (~ 250 million reads per tissue). We show that RNA sequencing can be used to identify approximately 40% of variants (7-10 million) called from DNA sequencing, with over 80% precision. Within highly expressed coding regions, over 92% of expected variants were called with nearly 98% precision. Allele-specific expression and putative post-transcriptional modifications negatively impact variant genotyping accuracy from RNA sequencing and contribute to RNA-DNA differences. Variants called from RNA sequencing detect roughly 75% of eGenes identified using variants called from DNA sequencing, demonstrating a nearly 2-fold enrichment of eQTL variants. We observe a moderate-to-strong correlation in nominal association p-values (Spearman ρ 2  ~ 0.6), although only 9% of eGenes have the same top associated variant., Conclusions: We find hundreds of thousands of RNA-DNA differences in variants called from RNA and DNA sequencing on the same individuals. We identify several highly significant eQTL when using RNA sequencing variant genotypes which are not found with DNA sequencing variant genotypes, suggesting that using RNA sequencing variant genotypes for association testing results in an increased number of false positives. Our findings demonstrate that caution must be exercised beyond filtering for variant quality or imputation accuracy when analysing or imputing variants called from RNA sequencing., (© 2024. The Author(s).)

Background: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and second leading cause of cancer-related deaths worldwide. The heightened mortality associated with HCC is largely attributed to its propensity for metastasis, which cannot be achieved without remodeling or loss of the basement membrane (BM). Despite advancements in targeted therapies and immunotherapies, resistance and limited efficacy in late-stage HCC underscore the urgent need for better therapeutic options and early diagnostic biomarkers. Our study aimed to address these gaps by investigating and evaluating potential biomarkers to improve survival outcomes and treatment efficacy in patients with HCC., Method: In this study, we collected the transcriptome sequencing, clinical, and mutation data of 424 patients with HCC from The Cancer Genome Atlas (TCGA) and 240 from the International Cancer Genome Consortium (ICGC) databases. We then constructed and validated a prognostic model based on metastasis and basement membrane-related genes (MBRGs) using univariate and multivariate Cox regression analyses. Five immune-related algorithms (CIBERSORT, QUANTISEQ, MCP counter, ssGSEA, and TIMER) were then utilized to examine the immune landscape and activity across high- and low-risk groups. We also analyzed Tumor Mutation Burden (TMB) values, Tumor Immune Dysfunction and Exclusion (TIDE) scores, mutation frequency, and immune checkpoint gene expression to evaluate immune treatment sensitivity. We analyzed integrin subunit alpha 3 (ITGA3) expression in HCC by performing single-cell RNA sequencing (scRNA-seq) analysis using the TISCH 2.0 database. Lastly, wound healing and transwell assays were conducted to elucidate the role of ITGA3 in tumor metastasis., Results: Patients with HCC were categorized into high- and low-risk groups based on the median values, with higher risk scores indicating worse overall survival. Five immune-related algorithms revealed that the abundance of immune cells, particularly T cells, was greater in the high-risk group than in the low-risk group. The high-risk group also exhibited a higher TMB value, mutation frequency, and immune checkpoint gene expression and a lower tumor TIDE score, suggesting the potential for better immunotherapy outcomes. Additionally, scRNA-seq analysis revealed higher ITGA3 expression in tumor cells compared with normal hepatocytes. Wound healing scratch and transwell cell migration assays revealed that overexpression of the MBRG ITGA3 enhanced migration of HCC HepG2 cells., Conclusion: This study established a direct molecular correlation between metastasis and BM, encompassing clinical features, tumor microenvironment, and immune response, thereby offering valuable insights for predicting clinical outcomes and immunotherapy responses in HCC., (© 2024. The Author(s).)

Background: Sepsis is a life-threatening organ dysfunction, which seriously threatens human health. The clinical and experimental results have confirmed that Traditional Chinese medicine (TCM), such as Scutellariae Radix, has anti-inflammatory effects. This provides a new idea for the treatment of sepsis. This study systematically analyzed the mechanism of Scutellariae Radix treatment in sepsis based on network pharmacology, RNA sequencing and molecular docking., Methods: Gene expression analysis was performed using Bulk RNA sequencing on sepsis patients and healthy volunteers. After quality control of the results, the differentially expressed genes (DEGs) were analyzed. The active ingredients and targets of Scutellariae Radix were identified using The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Gene Ontology (GO) and Protein-Protein Interaction (PPI) analysis were performed for disease-drug intersection targets. With the help of GEO database, Survival analysis and Meta-analysis was performed on the cross-targets to evaluate the prognostic value and screen the core targets. Subsequently, single-cell RNA sequencing was used to determine where the core targets are located within the cell. Finally, in this study, molecular docking experiments were performed to further clarify the interrelationship between the active components of Scutellariae Radix and the corresponding targets., Results: There were 72 active ingredients of Scutellariae Radix, and 50 common targets of drug and disease. GO and PPI analysis showed that the intersection targets were mainly involved in response to chemical stress, response to oxygen levels, response to drug, regulation of immune system process. Survival analysis showed that PRKCD, EGLN1 and CFLAR were positively correlated with sepsis prognosis. Meta-analysis found that the three genes were highly expressed in sepsis survivor, while lowly in non-survivor. PRKCD was mostly found in Macrophages, while EGLN1 and CFLAR were widely expressed in immune cells. The active ingredient Apigenin regulates CFLAR expression, Baicalein regulates EGLN1 expression, and Wogonin regulates PRKCD expression. Molecular docking studies confrmed that the three active components of astragalus have good binding activities with their corresponding targets., Conclusions: Apigenin, Baicalein and Wogonin, important active components of Scutellaria Radix, produce anti-sepsis effects by regulating the expression of their targets CFLAR, EGLN1 and PRKCD., (© 2024. The Author(s).)

Background: Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP., Methods: Human metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software., Results: We identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP., Conclusions: Our findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest., (© 2024. The Author(s).)

Background: Global per capita meat consumption continues to rise, especially pork. Meat quality is influenced by the content of intramuscular fat (IMF) as a key factor. The longissimus dorsi muscle of Dahe pigs (DHM, IMF: 7.98% ± 1.96%) and Dahe black pigs (DHBM, IMF: 3.30% ± 0.64%) was studied to explore cellular heterogeneity and differentially expressed genes (DEGs) associated with IMF deposition using single-nucleus RNA sequencing (snRNA-seq). The lipid composition was then analyzed using non-targeted lipidomics., Results: A total of seven cell subpopulations were identified, including myocytes, fibroblast/fibro/adipogenic progenitors (FAPs), satellite cells, endothelial cells, macrophages, pericytes, and adipocytes. Among them, FAPs and adipocytes were more focused because they could be associated with lipid deposition. 1623 DEGs in the FAPs subpopulation of DHBM were up-regulated compared with DHM, while 1535 were down-regulated. These DEGs enriched in the glycolysis/gluconeogenesis pathway. 109 DEGs were up-regulated and 806 were down-regulated in the adipocyte subpopulation of DHBM compared with DHM, which were mainly enriched in the PPAR signaling pathway and fatty acid (FA) biosynthesis. The expression level of PPARG, ABP4, LEP, and ACSL1 genes in DHM was higher than that in DHBM. Lipidomics reveals porcine lipid composition characteristics of muscle tissue. A total of 41 lipid classes and 2699 lipid species were identified in DHM and DHBM groups. The top ten relative peak areas of lipid classes in DHM and DHBM were triglyceride (TG), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), diglyceride (DG), cardiolipin (CL), ceramides (Cer), Simple Glc series (Hex1Cer), sphingomyelin (phSM), and phosphatidylinositol (PI). The relative peak areas of 35 lipid species in DHM were lower than DHBM, and 28 lipid species that were higher. There was a significant increase in the TG fatty acyl chains C6:0, C17:0, and C11:4, and a significant decrease in C16:0, C18:1, C18:2, and C22:4 in DHBM (p < 0.05)., Conclusions: C16:0 FA may downregulate the expression level of PPARG gene, which leads to the downregulation of fat metabolism-related genes such as ACSL, PLIN2, and FABP4 in DHBM compared with DHM. This may be the reason that the lipid deposition ability of Dahe pigs is stronger than that of Dahe black pigs, which need further investigation., (© 2024. The Author(s).)

The Drosophila eye has been an important model to understand principles of differentiation, proliferation, apoptosis and tissue morphogenesis. However, a single cell RNA sequence resource that captures gene expression dynamics from the initiation of differentiation to the specification of different cell types in the larval eye disc is lacking. Here, we report transcriptomic data from 13,000 cells that cover six developmental stages of the larval eye. Our data show cell clusters that correspond to all major cell types present in the eye disc ranging from the initiation of the morphogenetic furrow to the differentiation of each photoreceptor cell type as well as early cone cells. We identify dozens of cell type-specific genes whose function in different aspects of eye development have not been reported. These single cell data will greatly aid research groups studying different aspects of early eye development and will facilitate a deeper understanding of the larval eye as a model system., (© 2024. The Author(s).)

Background: The present study aims to identify the differential miRNA expression profile in middle ear cholesteatoma and explore their potential roles in its pathogenesis., Methods: Cholesteatoma and matched normal retroauricular skin tissue samples were collected from patients diagnosed with acquired middle ear cholesteatoma. The miRNA expression profiling was performed using small RNA sequencing, which further validated by quantitative real-time PCR (qRT-PCR). Target genes of differentially expressed miRNAs in cholesteatoma were predicted. The interaction network of 5 most significantly differentially expressed miRNAs was visualized using Cytoscape. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses were processed to investigate the biological functions of miRNAs in cholesteatoma., Results: The miRNA expression profile revealed 121 significantly differentially expressed miRNAs in cholesteatoma compared to normal skin tissues, with 56 upregulated and 65 downregulated. GO and KEGG pathway enrichment analyses suggested their significant roles in the pathogenesis of cholesteatoma. The interaction network of the the 2 most upregulated (hsa-miR-21-5p and hsa-miR-142-5p) and 3 most downregulated (hsa-miR-508-3p, hsa-miR-509-3p and hsa-miR-211-5p) miRNAs identified TGFBR2, MBNL1, and NFAT5 as potential key target genes in middle ear cholesteatoma., Conclusions: This study provides a comprehensive miRNA expression profile in middle ear cholesteatoma, which may aid in identifying therapeutic targets for its management., (© 2024. The Author(s).)

Background: Plant growth and development are severely threatened by drought and salt stresses. Compared with structural genes, transcription factors (TFs) play more pivotal roles in plant growth and stress adaptation. However, the underlying mechanisms of sorghum adapting to drought and salt are insufficient, and systematic analysis of TFs in response to the above stresses is lacking., Results: In this study, TFs were identified in sorghum and model plants (Arabidopsis thaliana and rice), and gene number and conserved domain were compared between sorghum and model plants. According to syntenic analysis, the expansion of sorghum and rice TFs may be due to whole-genome duplications. Between sorghum and model plants TFs, specific conserved domains were identified and they may be related to functional diversification of TFs. Forty-five key genes in sorghum, including four TFs, were likely responsible for drought adaption based on differently expression analysis. MiR5072 and its target gene (Sobic.001G449600) may refer to the determination of sorghum drought resistance according to small RNA and degradome analysis. Six genes were associated with drought adaptation of sorghum based on weighted gene co-expression network analysis (WGCNA). Similarly, the core genes in response to salt were also characterized using the above methods. Finally, 15 candidate genes, particularly two TFs (Sobic.004G300300, HD-ZIP; Sobic.003G244100, bZIP), involved in combined drought and salt resistance of sorghum were identified., Conclusions: In summary, the findings in this study help clarify the molecular mechanisms of sorghum responding to drought and salt. We identified candidate genes and provide important genetic resource for potential development of drought-tolerant and salt-tolerant sorghum plants., (© 2024. The Author(s).)

In recent years, single-cell analyses have revealed the heterogeneity of the tumour microenvironment (TME) at the genomic, transcriptomic, and proteomic levels, further improving our understanding of the mechanisms of tumour development. Single-cell RNA sequencing (scRNA-seq) technology allow analysis of the transcriptome at the single-cell level and have unprecedented potential for exploration of the characteristics involved in tumour development and progression. These techniques allow analysis of transcript sequences at higher resolution, thereby increasing our understanding of the diversity of cells found in the tumour microenvironment and how these cells interact in complex tumour tissue. Although scRNA-seq has emerged as an important tool for studying the tumour microenvironment in recent years, it cannot be used to analyse spatial information for cells. In this regard, spatial transcriptomics (ST) approaches allow researchers to understand the functions of individual cells in complex multicellular organisms by understanding their physical location in tissue sections. In particular, in related research on tumour heterogeneity, ST is an excellent complementary approach to scRNA-seq, constituting a new method for further exploration of tumour heterogeneity, and this approach can also provide unprecedented insight into the development of treatments for pancreatic cancer (PC). In this review, based on the methods of scRNA-seq and ST analyses, research progress on the tumour microenvironment and treatment of pancreatic cancer is further explained., (© 2024. The Author(s).)

Background: The issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility., Materials and Methods: Our research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual's genotype., Results: We analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as "protein localization to endosomes" and "xenobiotic transport." Overrepresented molecular function terms in up-regulated genes included "voltage-gated calcium channel activity," "growth hormone-releasing hormone receptor activity," and "sialic acid transmembrane transporter activity." Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36., Conclusions: These genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility., (© 2024. The Author(s).)

Background: Flea bites could trigger a series of complex molecular responses in the host. However, our understanding of the responses at the molecular level is still relatively limited. This study quantifies the changes in gene expression in mice after flea bites by RNA sequencing (RNA-seq) from their spleens, revealing the potential biological effects of host response to flea bites., Methods: RNA-seq was used for transcriptome analysis to screen for differentially expressed genes (DEGs) between the control mice group and the flea bite mice group. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed on DEGs. Protein-protein interaction (PPI) network analysis on DEGs related to immune processes was performed. Finally, we randomly selected several genes from the screened DEGs to validate the results from the transcriptome data by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR)., Results: A total of 521 DEGs were identified, including 277 upregulated and 244 downregulated. There were 258 GO terms significantly enriched by upregulated DEGs and 419 GO terms significantly enriched by downregulated DEGs. Among the upregulated DEGs, 22 GO terms were associated with immune cells (e.g., B cells and T cells) and immune regulatory processes, while among the downregulated DEGs, 58 GO terms were associated with immune cells and immune regulatory processes. Through PPI analysis, we found that CD40 molecules with significantly downregulated expression levels after flea bites may play an important role in host immune regulation. Through KEGG pathway enrichment analysis, a total of 26 significantly enriched KEGG pathways were identified. The RT-qPCR analysis results indicated that the transcriptome sequencing results were reliable., Conclusions: Through in-depth analysis of transcriptome changes in mice caused by flea bites, we revealed that flea bites could stimulate a series of biological and immunological responses in mice. These findings not only provided a deeper understanding of the impact of flea bites on the host but also provided a basis for further research on the interaction between ectoparasites and the host. We believe that digging deeper into the significance of these transcriptome changes will help reveal more about the adaptive response of the host to ectoparasites., (© 2024. The Author(s).)

Background: Sperm storage capacity (SSC) determines the duration of fertility in hens and is an important reproduction trait that cannot be ignored in production. Currently, the genetic mechanism of SSC is still unclear in hens. Therefore, to explore the genetic basis of SSC, we analyzed the uterus-vagina junction (UVJ) of hens with different SSC at different times after insemination by RNA-seq and Ribo-seq., Results: Our results showed that 589, 596, and 527 differentially expressed genes (DEGs), 730, 783, and 324 differentially translated genes (DTGs), and 804, 625, and 467 differential translation efficiency genes (DTEGs) were detected on the 5th, 10th, and 15th days after insemination, respectively. In transcription levels, we found that the differences of SSC at different times after insemination were mainly reflected in the transmission of information between cells, the composition of intercellular adhesion complexes, the regulation of ion channels, the regulation of cellular physiological activities, the composition of cells, and the composition of cell membranes. In translation efficiency (TE) levels, the differences of SSC were mainly related to the physiological and metabolic activities in the cell, the composition of the organelle membrane, the physiological activities of oxidation, cell components, and cell growth processes. According to pathway analysis, SSC was related to neuroactive ligand-receptor interaction, histidine metabolism, and PPAR signaling pathway at the transcriptional level and glutathione metabolism, oxidative phosphorylation, calcium signaling pathway, cell adhesion molecules, galactose metabolism, and Wnt signaling pathway at the TE level. We screened candidate genes affecting SSC at transcriptional levels (COL4A4, MUC6, MCHR2, TACR1, AVPR1A, COL1A1, HK2, RB1, VIPR2, HMGCS2) and TE levels(COL4A4, MUC6, CYCS, NDUFA13, CYTB, RRM2, CAMK4, HRH2, LCT, GCK, GALT). Among them, COL4A4 and MUC6 were the key candidate genes differing in transcription, translation, and translation efficiency., Conclusions: Our study used the combined analysis of RNA-seq and Ribo-seq for the first time to investigate the SSC and reveal the physiological processes associated with SSC. The key candidate genes affecting SSC were screened, and the theoretical basis was provided for the analysis of the molecular regulation mechanism of SSC., (© 2024. The Author(s).)

Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is a highly heterogeneous disease. According to large-scale RNA sequencing (RNA-seq) data, B-ALL patients can be divided into more than 10 subgroups. However, many genomic defects associated with resistance mechanisms have not yet been identified. As an individual clinical tool for molecular diagnostic risk classification, RNA-seq and gene expression pattern-based therapy could be potential upcoming strategies. In this study, we retrospectively analyzed the RNA-seq gene expression profiles of 45 children whose molecular diagnostic classifications were inconsistent with the response to chemotherapy. The relationship between the transcriptome and chemotherapy response was analyzed. Fusion gene identification was conducted for the included patients who did not have known high-risk associated fusion genes or gene mutations. The most frequently detected fusion gene pair in the high-risk group was the DHRSX duplication, which is a novel finding. Fusions involving ABL1, LMNB2, NFATC1, PAX5, and TTYH3 at onset were more frequently detected in the high-risk group, while fusions involving LFNG, TTYH3, and NFATC1 were frequently detected in the relapse group. According to the pathways involved, the underlying drug resistance mechanism is related to DNA methylation, autophagy, and protein metabolism. Overall, the implementation of an RNA-seq diagnostic system will identify activated markers associated with chemotherapy response, and guide future treatment adjustments., (© 2024. The Author(s).)

Background: The hair follicle development process is regulated by sophisticated genes and signaling networks, and the hair grows from the hair follicle. The Tianzhu white yak population exhibits differences in hair length, especially on the forehead and shoulder region. However, the genetic mechanism is still unclear. Isoform sequencing (Iso-seq) technology with advantages in long reads sequencing. Hence, we combined the Iso-seq and RNA-seq methods to investigate the transcript complexity and difference between long-haired yak (LHY) and normal-haired yak (NHY)., Results: The hair length measurement result showed a significant difference between LHY and NHY on the forehead and the shoulder (P-value < 0.001). The skin samples from the forehead and the shoulder of LHY and NHY were pooled for isoform sequencing (Iso-seq). We obtained numerous long transcripts, including novel isoforms, long non-coding RNA, alternative splicing events, and alternative polyadenylation events. Combined with RNA-seq data, we performed differential isoforms (DEIs) analysis between LHY and NHY. We found that some hair follicle and skin development-related DEIs, like BMP4, KRT2, IGF2R, and COL1A2 in the forehead skin; BMP1, KRT1, FGF5, COL2A1, and IGFBP5 in the shoulder skin. Enrichment analysis revealed that DEIs in both two comparable groups significantly participated in skin and hair follicle development-related pathways, like ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. The results indicated that the hair follicle development of Tianzhu white yak may influence the hair length difference. Besides, the protein-protein interaction (PPI) network of DEIs showed COL2A1 and COL3A1 exhibited a high degree of centrality, and these two genes were suggested as potential candidates for the hair length growth of Tianzhu white yak., Conclusions: The results provided a comprehensive analysis of the transcriptome complexity and identified differential transcripts that enhance our understanding of the molecular mechanisms underlying the variation in hair length growth in Tianzhu white yak., (© 2024. The Author(s).)

Background: Immunotherapy has brought about a paradigm shift in the treatment of cancer. However, the majority of patients exhibit resistance or become refractory to immunotherapy, and the underlying mechanisms remain to be explored., Methods: Sing-cell RNA sequencing (scRNA‑seq) datasets derived from 1 pretreatment and 1 posttreatment achieving pathological complete response (pCR) patient with lung adenocarcinoma (LUAD) who received neoadjuvant immunotherapy were collected, and pySCENIC was used to find the gene regulatory network (GRN) between cell types and immune checkpoint inhibitor (ICI) response. A regulon predicting ICI response was identified and validated using large‑scale pan-cancer data, including a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, The Cancer Genome Atlas (TCGA) pan-cancer cohort, and 5 ICI transcriptomic cohorts. Symphony reference mapping was performed to construct the myeloid cell map., Results: Thirteen major cluster cell types were identified by comparing pretreatment and posttreatment patients, and the fraction of myeloid cells was higher in the posttreatment group (19.0% vs. 11.8%). A PPARG regulon (containing 23 target genes) was associated with ICI response, and its function was validated by a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, TCGA pan-cancer cohort, and 5 ICI transcriptomic cohorts. Additionally, a myeloid cell map was developed, and cluster I, II, and III myeloid cells with high expression of PPARG were identified. Moreover, we constructed a website called PPARG ( https://pparg.online/PPARG/ or http://43.134.20.130:3838/PPARG/ ), which provides a powerful discovery tool and resource value for researchers., Conclusions: The PPARG regulon is a predictor of ICI response. The myeloid cell map enables the identification of PPARG subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value., (© 2024. The Author(s).)

Background: The cellular origin of hypopharyngeal diseases is crucial for further diagnosis and treatment, and the microenvironment in tissues may also be associated with specific cell types at the same time. Normal adjacent tissues (NATs) of hypopharyngeal carcinoma differ from non-tumor-bearing tissues, and can influenced by the tumor. However, the heterogeneity in kinds of disease samples remains little known, and the transcriptomic profile about biological information associated with disease occurrence and clinical outcome contained in it has yet to be fully evaluated. For these reasons, we should quickly investigate the taxonomic and transcriptomic information of NATs in human hypopharynx., Results: Single-cell suspensions of normal adjacent tissues (NATs) of hypopharyngeal carcinoma were obtained and single-cell RNA sequencing (scRNA-seq) was performed. We present scRNA-seq data from 39,315 high-quality cells in the hypopharyngeal from five human donors, nine clusters of normal adjacent human hypopharyngeal cells were presented, including epithelial cells, endothelial cells (ECs), mononuclear phagocyte system cells (MPs), fibroblasts, T cells, plasma cells, B cells, mural cells and mast cells. Nonimmune components in the microenvironment, including epithelial cells, endothelial cells, fibroblasts and the subpopulations of them were performed., Conclusions: Our data provide a solid basis for the study of single-cell landscape in human normal adjacent hypopharyngeal tissues biology and related diseases., (© 2024. The Author(s).)

Background: Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition., Results: A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions., Conclusions: These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants., (© 2024. The Author(s).)

Background: Sepsis ranks among the most formidable clinical challenges, characterized by exorbitant treatment costs and substantial demands on healthcare resources. Mitochondrial dysfunction emerges as a pivotal risk factor in the pathogenesis of sepsis, underscoring the imperative to identify mitochondrial-related biomarkers. Such biomarkers are crucial for enhancing the accuracy of sepsis diagnostics and prognostication., Methods: In this study, adhering to the SEPSIS 3.0 criteria, we collected peripheral blood within 24 h of admission from 20 sepsis patients at the ICU of the Southwest Medical University Affiliated Hospital and 10 healthy volunteers as a control group for RNA-seq. The RNA-seq data were utilized to identify differentially expressed RNAs. Concurrently, mitochondrial-associated genes (MiAGs) were retrieved from the MitoCarta3.0 database. The differentially expressed genes were intersected with MiAGs. The intersected genes were then subjected to GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) analyses and core genes were filtered using the PPI (Protein-Protein Interaction) network. Subsequently, relevant sepsis datasets (GSE65682, GSE28750, GSE54514, GSE67652, GSE69528, GSE95233) were downloaded from the GEO (Gene Expression Omnibus) database to perform bioinformatic validation of these core genes. Survival analysis was conducted to assess the prognostic value of the core genes, while ROC (Receiver Operating Characteristic) curves determined their diagnostic value, and a meta-analysis confirmed the accuracy of the RNA-seq data. Finally, we collected 5 blood samples (2 normal controls (NC); 2 sepsis; 1 SIRS (Systemic Inflammatory Response Syndrome), and used single-cell sequencing to assess the expression levels of the core genes in the different blood cell types., Results: Integrating high-throughput sequencing with bioinformatics, this study identified two mitochondrial genes (COX7B, NDUFA4) closely linked with sepsis prognosis. Survival analysis demonstrated that patients with lower expression levels of COX7B and NDUFA4 exhibited a higher day survival rate over 28 days, inversely correlating with sepsis mortality. ROC curves highlighted the significant sensitivity and specificity of both genes, with AUC values of 0.985 for COX7B and 0.988 for NDUFA4, respectively. Meta-analysis indicated significant overexpression of COX7B and NDUFA4 in the sepsis group in contrast to the normal group (P < 0.01). Additionally, single-cell RNA sequencing revealed predominant expression of these core genes in monocytes-macrophages, T cells, and B cells., Conclusion: The mitochondrial-associated genes (MiAGs) COX7B and NDUFA4 are intimately linked with the prognosis of sepsis, offering potential guidance for research into the mechanisms underlying sepsis., (© 2024. The Author(s).)

Background: Cassava mosaic disease (CMD), caused by Sri Lankan cassava mosaic virus (SLCMV) infection, has been identified as a major pernicious disease in Manihot esculenta Crantz (cassava) plantations. It is widespread in Southeast Asia, especially in Thailand, which is one of the main cassava supplier countries. With the aim of restricting the spread of SLCMV, we explored the gene expression of a tolerant cassava cultivar vs. a susceptible cassava cultivar from the perspective of transcriptional regulation and the mechanisms underlying plant immunity and adaptation., Results: Transcriptomic analysis of SLCMV-infected tolerant (Kasetsart 50 [KU 50]) and susceptible (Rayong 11 [R 11]) cultivars at three infection stages-that is, at 21 days post-inoculation (dpi) (early/asymptomatic), 32 dpi (middle/recovery), and 67 dpi (late infection/late recovery)-identified 55,699 expressed genes. Differentially expressed genes (DEGs) between SLCMV-infected KU 50 and R 11 cultivars at (i) 21 dpi to 32 dpi (the early to middle stage), and (ii) 32 dpi to 67 dpi (the middle stage to late stage) were then identified and validated by real-time quantitative PCR (RT-qPCR). DEGs among different infection stages represent genes that respond to and regulate the viral infection during specific stages. The transcriptomic comparison between the tolerant and susceptible cultivars highlighted the role of gene expression regulation in tolerant and susceptible phenotypes., Conclusions: This study identified genes involved in epigenetic modification, transcription and transcription factor activities, plant defense and oxidative stress response, gene expression, hormone- and metabolite-related pathways, and translation and translational initiation activities, particularly in KU 50 which represented the tolerant cultivar in this study., (© 2024. The Author(s).)

Background: Hepatocellular carcinoma represents a significant global burden in terms of cancer-related mortality, posing a substantial risk to human health. Despite the availability of various treatment modalities, the overall survival rates for patients with hepatocellular carcinoma remain suboptimal. The objective of this study was to explore the potential of novel biomarkers and to establish a novel predictive signature utilizing multiple transcriptome profiles., Methods: The GSE115469 and CNP0000650 cohorts were utilized for single cell analysis and gene identification. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) datasets were utilized in the development and evaluation of a predictive signature. The expressions of hepatocyte-specific genes were further validated using the GSE135631 cohort. Furthermore, immune infiltration results, immunotherapy response prediction, somatic mutation frequency, tumor mutation burden, and anticancer drug sensitivity were analyzed based on various risk scores. Subsequently, functional enrichment analysis was performed on the differential genes identified in the risk model. Moreover, we investigated the expression of particular genes in chronic liver diseases utilizing datasets GSE135251 and GSE142530., Results: Our findings revealed hepatocyte-specific genes (ADH4, LCAT) with notable alterations during cell maturation and differentiation, leading to the development of a novel predictive signature. The analysis demonstrated the efficacy of the model in predicting outcomes, as evidenced by higher risk scores and poorer prognoses in the high-risk group. Additionally, a nomogram was devised to forecast the survival rates of patients at 1, 3, and 5 years. Our study demonstrated that the predictive model may play a role in modulating the immune microenvironment and impacting the anti-tumor immune response in hepatocellular carcinoma. The high-risk group exhibited a higher frequency of mutations and was more likely to benefit from immunotherapy as a treatment option. Additionally, we confirmed that the downregulation of hepatocyte-specific genes may indicate the progression of hepatocellular carcinoma and aid in the early diagnosis of the disease., Conclusion: Our research findings indicate that ADH4 and LCAT are genes that undergo significant changes during the differentiation of hepatocytes into cancer cells. Additionally, we have created a unique predictive signature based on genes specific to hepatocytes., (© 2024. The Author(s).)

Objective: Proliferative nodular formation represents a characteristic pathological feature of benign prostatic hyperplasia (BPH) and serves as the primary cause for prostate volume enlargement and consequent lower urinary tract symptoms (LUTS). Its specific mechanism is largely unknown, although several cellular processes have been reported to be involved in BPH initiation and development and highlighted the crucial role of epithelial cells in proliferative nodular formation. However, the technological limitations hinder the in vivo investigation of BPH patients., Methods: The robust cell type decomposition (RCTD) method was employed to integrate spatial transcriptomics and single cell RNA sequencing profiles, enabling the elucidation of epithelial cell alterations during nodular formation. Immunofluorescent and immunohistochemical staining was performed for verification., Results: The alterations of epithelial cells during the formation of nodules in BPH was observed, and a distinct subgroup of basal epithelial (BE) cells, referred to as BE5, was identified to play a crucial role in driving this progression through the hypoxia-induced epithelial-mesenchymal transition (EMT) signaling pathway. BE5 served as both the initiating cell during nodular formation and the transitional cell during the transformation from luminal epithelial (LE) to BE cells. A distinguishing characteristic of the BE5 cell subgroup in patients with BPH was its heightened hypoxia and upregulated expression of FOS. Histological verification results confirmed a significant association between c-Fos expression and key biological processes such as hypoxia and cell proliferation, as well as the close relationship between hypoxia and EMT in BPH tissues. Furthermore, a strong link between c-Fos expression and the progression of BPH was also been validated. Additionally, notable functional differences were observed in glandular and stromal nodules regarding BE5 cells, with BE5 in glandular nodules exhibiting enhanced capacities for EMT and cell proliferation characterized by club-like cell markers., Conclusions: This study elucidated the comprehensive landscape of epithelial cells during in vivo nodular formation in patients, thereby offering novel insights into the initiation and progression of BPH., (© 2024. The Author(s).)

Background: The molecular mechanism of fetal cystic hygroma (CH) is still unclear, and no study has previously reported the transcriptome changes of single cells in CH. In this study, single-cell transcriptome sequencing (scRNA-seq) was used to investigate the characteristics of cell subsets in the lesion tissues of CH patients., Methods: Lymphoid tissue collected from CH patients and control donors for scRNA-seq analysis. Differentially expressed gene enrichment in major cell subpopulations as well as cell-cell communication were analyzed. At the same time, the expression and interactions of important VEGF signaling pathway molecules were analyzed, and potential transcription factors that could bind to KDR (VEGFR2) were predicted., Results: The results of scRNA-seq showed that fibroblasts accounted for the largest proportion in the lymphatic lesions of CH patients. There was a significant increase in the proportion of lymphatic endothelial cell subsets between the cases and controls. The VEGF signaling pathway is enriched in lymphatic endothelial cells and participates in the regulation of cell-cell communication between lymphatic endothelial cells and other cells. The key regulatory gene KDR in the VEGF signaling pathway is highly expressed in CH patients and interacts with other differentially expressed EDN1, TAGLN, and CLDN5 Finally, we found that STAT1 could bind to the KDR promoter region, which may play an important role in promoting KDR up-regulation., Conclusion: Our comprehensive delineation of the cellular composition in tumor tissues of CH patients using single-cell RNA-sequencing identified the enrichment of lymphatic endothelial cells in CH and highlighted the activation of the VEGF signaling pathway in lymphoid endothelial cells as a potential modulator. The molecular and cellular pathogenesis of fetal cystic hygroma (CH) remains largely unknown. This study examined the distribution and gene expression signature of each cell subpopulation and the possible role of VEGF signaling in lymphatic endothelial cells in regulating the progression of CH by single-cell transcriptome sequencing. The enrichment of lymphatic endothelial cells in CH and the activation of the VEGF signaling pathway in lymphatic endothelial cells provide some clues to the pathogenesis of CH from the perspective of cell subpopulations., (© 2024. The Author(s).)

Background: Tumor cells exhibit a heightened susceptibility to lysosomal-dependent cell death (LCD) compared to normal cells. However, the role of LCD-related genes (LCD-RGs) in Osteosarcoma (OS) remains unelucidated. This study aimed to elucidate the role of LCD-RGs and their mechanisms in OS using several existing OS related datasets, including TCGA-OS, GSE16088, GSE14359, GSE21257 and GSE162454., Results: Analysis identified a total of 8,629 DEGs1, 2,777 DEGs2 and 21 intersection genes. Importantly, two biomarkers (ATP6V0D1 and HDAC6) linked to OS prognosis were identified to establish the prognostic model. Significant differences in risk scores for OS survival were observed between high and low-risk cohorts. Additionally, scores of dendritic cells (DC), immature DCs and γδT cells differed significantly between the two risk cohorts. Cell annotations from GSE162454 encompassed eight types (myeloid cells, osteoblastic OS cells and plasma cells). ATP6V0D1 was found to be significantly over-expressed in myeloid cells and osteoclasts, while HDAC6 was under-expressed across all cell types. Moreover, single-cell trajectory mapping revealed that myeloid cells and osteoclasts differentiated first, underscoring their pivotal role in patients with OS. Furthermore, ATP6V0D1 expression progressively decreased with time., Conclusions: A new prognostic model for OS, associated with LCD-RGs, was developed and validated, offering a fresh perspective for exploring the association between LCD and OS., (© 2024. The Author(s).)

Clear cell renal cell carcinoma (ccRCC) is the prevailing histological subtype of renal cell carcinoma and has unique metabolic reprogramming during its occurrence and development. Cell senescence is one of the newly identified tumor characteristics. However, there is a dearth of methodical and all-encompassing investigations regarding the correlation between the broad-ranging alterations in metabolic processes associated with aging and ccRCC. We utilized a range of analytical methodologies, such as protein‒protein interaction network analysis and least absolute shrinkage and selection operator (LASSO) regression analysis, to form and validate a risk score model known as the senescence-metabolism-related risk model (SeMRM). Our study demonstrated that SeMRM could more precisely predict the OS of ccRCC patients than the clinical prognostic markers in use. By utilizing two distinct datasets of ccRCC, ICGC-KIRC (the International Cancer Genome Consortium) and GSE29609, as well as a single-cell dataset (GSE156632) and real patient clinical information, and further confirmed the relationship between the senescence-metabolism-related risk score (SeMRS) and ccRCC patient progression. It is worth noting that patients who were classified into different subgroups based on the SeMRS exhibited notable variations in metabolic activity, immune microenvironment, immune cell type transformation, mutant landscape, and drug responsiveness. We also demonstrated that PTGER4, a key gene in SeMRM, regulated ccRCC cell proliferation, lipid levels and the cell cycle in vivo and in vitro. Together, the utilization of SeMRM has the potential to function as a dependable clinical characteristic to increase the accuracy of prognostic assessment for patients diagnosed with ccRCC, thereby facilitating the selection of suitable treatment strategies., (© 2024. The Author(s).)

Background: Studies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC., Methods: In this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization., Results: In the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells., Conclusions: CCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages., (© 2024. The Author(s).)

Background: Post COVID-19 condition (PCC) is a complication of SARS-COV-2 infection and can lead to long-term disability., Methods: The present study was designed to analyse the gene expression patterns of PCC through bulk RNA sequencing of whole blood and to explore the potential molecular mechanisms of PCC. Whole blood was collected from 80 participants enrolled in a prospective cohort study following SARS-CoV-2 infected and non-infected individuals for 6 months after recruitment and was used for bulk RNA sequencing. Identification of differentially expressed genes (DEG), pathway enrichment and immune cell deconvolution was performed to explore potential biological pathways involved in PCC., Results: We have found 13 differentially expressed genes associated with PCC. Enriched pathways were related to interferon-signalling and anti-viral immune processes., Conclusion: The PCC transcriptome is characterized by a modest overexpression of interferon-stimulated genes, pointing to a subtle ongoing inflammatory response., (© 2024. The Author(s).)

Japanese encephalitis virus (JEV) is a neurotropic pathogen that causes lethal encephalitis. The high susceptibility and massive proliferation of JEV in neurons lead to extensive neuronal damage and inflammation within the central nervous system. Despite extensive research on JEV pathogenesis, the effect of JEV on the cellular composition and viral tropism towards distinct neuronal subtypes in the brain is still not well comprehended. To address these issues, we performed single-cell RNA sequencing (scRNA-seq) on cells isolated from the JEV-highly infected regions of mouse brain. We obtained 88,000 single cells and identified 34 clusters representing 10 major cell types. The scRNA-seq results revealed an increasing amount of activated microglia cells and infiltrating immune cells, including monocytes & macrophages, T cells, and natural killer cells, which were associated with the severity of symptoms. Additionally, we observed enhanced communication between individual cells and significant ligand-receptor pairs related to tight junctions, chemokines and antigen-presenting molecules upon JEV infection, suggesting an upregulation of endothelial permeability, inflammation and antiviral response. Moreover, we identified that Baiap2-positive neurons were highly susceptible to JEV. Our findings provide valuable clues for understanding the mechanism of JEV induced neuro-damage and inflammation as well as developing therapies for Japanese encephalitis., (© 2024. The Author(s).)

Background: Tumor-associated macrophages (TAMs) play a pivotal role in reshaping the tumor microenvironment following radiotherapy. The mechanisms underlying this reprogramming process remain to be elucidated., Methods: Subcutaneous Lewis lung carcinoma (LLC) murine model was treated with hypofrationated radiotherapy (8 Gy × 3F). Single-cell RNA sequencing was utilized to identify subclusters and functions of TAMs. Multiplex assay and enzyme-linked immunosorbent assay (ELISA) were employed to measure serum chemokine levels. Bindarit was used to inhibit CCL8, CCL7, and CCL2. The infiltration of TAMs after combination treatment with hypofractionated radiotherapy and Bindarit was quantified with flow cytometry, while the influx of CD206 and CCL8 was assessed by immunostaining., Results: Transcriptome analysis identified a distinct subset of M2-like macrophages characterized by elevated Ccl8 expression level following hypofractionated radiotherapy in LLC-bearing mice. Remarkbly, hypofractionated radiotherapy not only promoted CCL8 high macrophages infiltration but also reprogrammed them by upregulating immunosuppressive genes, thereby fostering an immunosuppressive tumor microenvironment. Additioinally, hypofractionated radiotherapy enhanced the CCL signaling pathway, augmenting the pro-tumorigenic functions of CCL8 high macrophages and boosting TAMs recruitment. The adjunctive treatment combining hypofractionated radiotherapy with Bindarit effectively reduced M2 macrophages infiltration and prolonged the duration of local tumor control., Conclusions: Hypofractionated radiotherapy enhances the infiltration of CCL8 high macrophages and amplifies their roles in macrophage recruitment through the CCL signaling pathway, leading to an immunosuppressive tumor microenvironment. These findings highlight the potential of targeting TAMs and introduces a novel combination to improve the efficacy of hypofractionated radiotherapy., (© 2024. The Author(s).)

Background: Crohn's disease (CD) is a disease characterized by intestinal immune dysfunction, often accompanied by metabolic abnormalities. Disturbances in lactate metabolism have been found in the intestine of patients with CD, but studies on the role of lactate and related Lactylation in the pathogenesis of CD are still unknown., Methods: We identified the core genes associated with Lactylation by downloading and merging three CD-related datasets (GSE16879, GSE75214, and GSE112366) from the GEO database, and analyzed the functions associated with the hub genes and the correlation between their expression levels and immune infiltration through comprehensive analysis. We explored the Lactylation levels of different immune cells using single-cell data and further analyzed the differences in Lactylation levels between inflammatory and non-inflammatory sites., Results: We identified six Lactylation-related hub genes that are highly associated with CD. Further analysis revealed that these six hub genes were highly correlated with the level of immune cell infiltration. To further clarify the effect of Lactylation on immune cells, we analyzed single-cell sequencing data of immune cells from inflammatory and non-inflammatory sites in CD patients and found that there were significant differences in the levels of Lactylation between different types of immune cells, and that the levels of Lactylation were significantly higher in immune cells from inflammatory sites., Conclusions: These results suggest that Lactylation-related genes and their functions are closely associated with changes in inflammatory cells in CD patients., (© 2024. The Author(s).)

Background: Recently, long non-coding RNAs (lncRNAs) have been demonstrated as essential roles in tumor immune microenvironments (TIME). Nevertheless, researches on the clinical significance of TIME-related lncRNAs are limited in lung adenocarcinoma (LUAD)., Methods: Single-cell RNA sequencing and bulk RNA sequencing data are integrated to identify TIME-related lncRNAs. A total of 1368 LUAD patients are enrolled from 6 independent datasets. An integrative machine learning framework is introduced to develop a TIME-related lncRNA signature (TRLS)., Results: This study identified TIME-related lncRNAs from integrated analysis of single‑cell and bulk RNA sequencing data. According to these lncRNAs, a TIME-related lncRNA signature was developed and validated from an integrative procedure in six independent cohorts. TRLS exhibited a robust and reliable performance in predicting overall survival. Superior prediction performance barged TRLS to the forefront from comparison with general clinical features, molecular characters, and published signatures. Moreover, patients with low TRLS displayed abundant immune cell infiltration and active lipid metabolism, while patients with high TRLS harbored significant genomic alterations, high PD-L1 expression, and elevated DNA damage repair (DDR) relevance. Notably, subclass mapping analysis of nine immunotherapeutic cohorts demonstrated that patients with high TRLS were more sensitive to immunotherapy., Conclusions: This study developed a promising tool based on TIME-related lncRNAs, which might contribute to tailored treatment and prognosis management of LUAD patients., (© 2024. The Author(s).)