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Parkinson's Disease (PD) is a debilitating neurodegenerative disorder, characterized by motor and cognitive impairments, that affects >1% of the population over the age of 60. The pathogenesis of PD is complex and remains largely unknown. Due to the cellular heterogeneity of the human brain and changes in cell type composition with disease progression, this complexity cannot be fully captured with bulk tissue studies. To address this, we generated single-nucleus RNA sequencing and whole-genome sequencing data from 100 postmortem cases and controls, carefully selected to represent the entire spectrum of PD neuropathological severity and diverse clinical symptoms. The single nucleus data were generated from five brain regions, capturing the subcortical and cortical spread of PD pathology. Rigorous preprocessing and quality control were applied to ensure data reliability. Committed to collaborative research and open science, this dataset is available on the AMP PD Knowledge Platform, offering researchers a valuable tool to explore the molecular bases of PD and accelerate advances in understanding and treating the disease., Competing Interests: Competing interests: Panos Roussos is an Editorial Board Member for Scientific Data., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: The neutrophil-mediated generation of neutrophil extracellular traps (NETs) results in an augmented inflammatory response and cellular tissue injury during acute myocardial infarction (AMI). Through the analysis of public database information, we discovered and confirmed putative critical genes involved in NETs-mediated AMI., Methods: The AMI dataset GSE66360 and the single-cell dataset GSE163465 were downloaded from the Gene Expression Omnibus database. Key genes were screened by bioinformatics. Quantitative real-time PCR (qRT-PCR) was used to verify the key genes, and then a Mendelian randomization (MR) study was conducted on the basis of the genome-wide association study to determine the causal relationship between key genes and AMI. Dimensionality reduction clustering, pseudo-time series, and cell communication were performed on the single-cell dataset to analyze the key genes screened by bulk RNA sequencing and the dynamic evolution of NETs in the AMI process. Immunohistochemistry and Western blot were used to verify the key genes., Results: Six key genes, IL1β, S100A12, TLR2, CXCL1, CXCL2, and CCL4, were screened out through bioinformatics. qRT-PCR results showed that compared with the control group, the expression of 5 key genes was upregulated in the AMI group. In the MR study, CXCL1 and CCL4 were observed to have a causal relationship with AMI. Single-cell analysis showed that NETs-related genes CCL4, CXCL2, and IL1β were highly expressed. Combining single cells, qRT-PCR and MR, gene CCL4 was selected as the focus of the study. H9c2 cardiomyocytes simulated myocardial infarction under hypoxia, and the results showed that the expression of gene CCL4 was increased. The immunohistochemical results of gene CCL4 showed that the expression was upregulated in the AMI group., Conclusions: We found 6 key genes related to NETs-mediated cell damage during AMI. The results of MR showed that CXCL1 and CCL4 were causally related to AMI. Combining single cells, qRT-PCR and MR, gene CCL4 may play an important role in the AMI process. Our results may provide some insights into neutrophil-mediated cell damage during AMI., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Charcoal rot of soybean is caused by the hemibiotrophic fungus Macrophomina phaseolina, a global crop destroyer and an important pathogen in the midwestern USA. The quantitative nature of host resistance and the complexity of the soybean-M. phaseolina interaction at the molecular level have hampered resistance breeding. A previous study showed that L-ascorbic acid (LAA) pre-treatment before M. phaseolina inoculation reduced charcoal rot lesion length in excised soybean stems. This study aimed to elucidate the genetic underpinnings of M. phaseolina-induced senescence and the mitigating effects of ascorbic acid on this physiological process within the same pathosystem., Results: RNA was sequenced from M. phaseolina-resistant and -susceptible soybean genotypes following M. phaseolina inoculation, LAA, and hydrogen peroxide (H 2 O 2 )-an oxidative stress inducer-application followed by inoculation. More genes were down-regulated in the resistant and susceptible genotypes than up-regulated when the M. phaseolina-inoculated treatments were compared to mock-inoculated control treatments. Gene ontology (GO) term and KEGG pathways analysis detected M. phaseolina-induced up-regulation of receptor-like kinase genes. In contrast, many genes related to antioxidants, defense, and hormonal pathways were down-regulated in both genotypes. LAA pre-treatment induced genes related to photosynthesis and reactive oxygen species responses in both genotypes. H 2 O 2 pre-treatment following inoculation up-regulated many stress-response genes, while hormone signal transduction and photosynthesis-related genes were down-regulated in both genotypes., Conclusions: Results revealed transcriptional variation and genes associated with M. phaseolina-induced senescence in soybean. Ascorbic acid induced many photosynthetic genes, suggesting a complex regulation of defense and immunity in the plant against the hemibiotroph. Soybean plants also exhibited enhanced stress responsiveness when treated with H 2 O 2 followed by inoculation with M. phaseolina. This study will broaden more research avenues related to transcriptional regulation during the M. phaseolina-soybean interaction and the potential role of receptor-like kinases, oxidative stress-responsive genes, ethylene-mediated signaling and enhanced photosynthetic gene expression when mounting host resistance to this important soybean pathogen., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Candida glabrata is an important and increasingly common pathogen of humans, particularly in immunocompromised hosts. Despite this, little is known about how this fungus causes disease. Here, we applied RNA sequencing and an in vivo invasive infection model to identify the attributes that allow this organism to infect hosts. Fungal transcriptomes show a dramatic increase in the expression of Fus3 and Kss1, two mitogen-activated protein kinases (MAPKs), during invasive infection. We further demonstrate that they are both highly induced under a combination of serum and high CO 2 conditions. Deletion of both FUS3 and KSS1 , but neither gene alone, results in a reduced fungal burden in organs, as well as in the gastrointestinal tract in the DSS (Dextran Sulfate Sodium)-induced colitis model. Similarly, the defect in persistence in macrophages and attenuated adhesion to epithelial cells are observed when FUS3 and KSS1 are both disrupted. The fus3 kss1 double mutant also displays defects in the induction of virulence attributes such as genes required for iron acquisition and adhesion and in the anti-fungal drug tolerance. The putative downstream transcription factors Ste12 (1), Ste12 (2), Tec1, and Tec2 are found to be involved in the regulation of these virulence attributes. Collectively, our study indicates that an evolutionary conserved MAPK pathway, which regulates mating and filamentous growth in Saccharomyces cerevisiae , is critical for C. glabrata pathogenicity., Importance: The MAPK signaling pathway, mediated by closely related kinases Fus3 and Kss1, is crucial for controlling mating and filamentous growth in Saccharomyces cerevisiae , but this pathway does not significantly impact hyphal development and pathogenicity in Candida albicans , a commensal-pathogenic fungus of humans. Furthermore, deletion of Cpk1, the ortholog of Fus3 in pathogenic fungus Cryptococcus neoformans , has no effect on virulence. Here, we demonstrate that the MAPK pathway is crucial for the pathogenicity of Candida glabrata , a fungus that causes approximately one-third of cases of hematogenously disseminated candidiasis in the United States. This pathway regulates multiple virulence attributes including the induction of iron acquisition genes and adhesins, as well as persistence in macrophages and organs. Our work provides insights into C. glabrata pathogenesis and highlights an example in which regulatory rewiring of a conserved pathway confers a virulent phenotype in a pathogen., Competing Interests: The authors declare no conflict of interest.

The advent of droplet-based single-cell RNA-sequencing (scRNA-seq) has dramatically increased data throughput, enabling the release of a diverse array of tissue cell atlases to the public. However, we will show that prominent initiatives such as the Human Cell Atlas [1], the Tabula Sapiens [2] and the Tabula Muris [3] contain a significant amount of contamination products (frequently affecting the whole organ) in their data portals due to suboptimal quality filtering. Our work addresses a critical gap by advocating for more stringent quality filtering, highlighting the imperative for a shift from existing standards, which currently lean towards greater permissiveness. We will show the importance of incorporating cell intronic fraction in quality control -or MALAT1 expression otherwise- showcasing its informative nature and potential to elevate cell atlas data reliability. In summary, here, we unveil the hidden intronic landscape of every tissue and highlight the importance of more rigorous single-cell RNA-sequencing quality assessment in cell atlases to enhance their applicability in diverse downstream analyses., Competing Interests: Declarations. Ethics approval and consent to participate: “Not applicable”. Consent for publication: “Not applicable”. Competing interests: We declare no competing interests., (© 2024. The Author(s).)

Context: Estrogen receptor α (ERα) is a key regulator of reproductive function, particularly in ovarian development and function, yet the specifics of its role at the molecular level remain unclear., Aims: The study aims to elucidate the molecular mechanisms of ERα-regulated transcriptional dynamics in ovarian cells using ERα knockout (αERKO) mice created via CRISPR/Cas9., Methods: Single-cell RNA sequencing (scRNA-seq) was used to compare transcriptomes from individual ovarian cells in both wild type and αERKO mice. Bioinformatics analyses identified distinct cell populations and their transcriptional profiles post ERα deletion., Key Results: Distinct oocyte and granulosa cell populations were identified, with ERα deletion disrupting the regulation of genes linked to ovarian infertility, the ovulation cycle, and steroidogenesis. Greb1 expression in granulosa cells was found to be ERα-dependent., Conclusions: ERα deletion significantly alters the transcriptional landscape of ovarian cells, affecting genes and pathways central to ovarian function and the ovulation process., Implications: The findings provide an in-depth, single-cell view of ERα's role in the reproductive system, offering insights that may lead to novel treatments for ovarian disorders., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Liver fibrosis is a pathological response to liver damage induced by multiple etiologies including NASH and CCl 4 , which may further lead to cirrhosis and hepatocellular carcinoma (HCC). Despite the increasing understanding of liver fibrosis and HCC, clinical prognosis and targeted therapy remain challenging., Methods: This study integrated single-cell sequencing analysis, bulk sequencing analysis, and mouse models to identify highly expressed genes, cell subsets, and signaling pathways associated with liver fibrosis and HCC. Clinical prediction models and prognostic genes were established and verified through machine learning, survival analysis, as well as the utilization of clinical data and tissue samples from HCC patients. The expression heterogeneity of the core prognostic gene, along with its correlation with the tumor microenvironment and prognostic outcomes, was analyzed through single-cell analysis and immune infiltration analysis. In addition, the cAMP database and molecular docking techniques were employed to screen potential small molecule drugs for the treatment of liver fibrosis and HCC., Result: We identified 40 pathogenic genes, 15 critical cell subsets (especially Macrophages), and regulatory signaling pathways related to cell adhesion and the actin cytoskeleton that promote the development of liver fibrosis and HCC. In addition, 7 specific prognostic genes (CCR7, COL3A1, FMNL2, HP, PFN1, SPP1 and TENM4) were identified and evaluated, and expression heterogeneity of core gene SPP1 and its positive correlation with immune infiltration and prognostic development were interpreted. Moreover, 6 potential small molecule drugs for the treatment of liver fibrosis and HCC were provided., Conclusion: The comprehensive investigation, based on a bioinformatics and mouse model strategy, may identify pathogenic genes, cell subsets, regulatory mechanisms, prognostic genes, and potential small molecule drugs, thereby providing valuable insights into the clinical prognosis and targeted treatment of liver fibrosis and HCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Hu and Xu.)

Background: Uveal melanoma (UM), arising from melanocytes in the choroid, accounts for 3% to 5% of all melanocytic tumors and over 70% of intraocular malignancies. Despite effective local treatments, metastasis remains a significant challenge, with more than half of patients developing metastatic disease within ten years. Conventional therapies often yield poor outcomes, highlighting the urgent need for novel therapeutic strategies to enhance survival and prognosis for UM patients., Methods: We conducted a detailed analysis of the GSE139829 dataset, focusing on scRNA-seq data from eight primary UM patients and three with metastatic disease. Through clustering and marker gene expression analyses, we identified distinct subtypes of UM tumor cells and examined their transcriptional, metabolic, and intercellular communication profiles. We developed a novel prognostic model, PCOLCE TCs Risk Score (PTRS), centered on the C5 PCOLC E+ tumor cells, which was validated through in vitro functional assays. Additionally, we performed immune infiltration and metabolic pathway analyses to elucidate tumor-immune interactions and their clinical significance., Results: We identified eight distinct cell types in UM and classified tumor subpopulations into six subgroups. The C5 PCOLCE + TCs subpopulation was highlighted as crucial in UM malignancy, demonstrating high differentiation potential and a significant role in tumor progression. CellChat analysis revealed substantial communication between C5 PCOLCE + TCs and fibroblasts, suggesting their involvement in tumor growth and extracellular matrix remodeling. Metabolic pathway analysis indicated enhanced oxidative phosphorylation and glutathione metabolism in this subpopulation. Additionally, we developed a PTRS model based on C5 PCOLCE + TCs, identifying CITED1 as a high-risk gene that promotes UM cell proliferation, invasion, and migration in vitro ., Conclusion: This study provides insights into UM metastasis via single-cell analysis, identifying C5 PCOLCE + TCs as key malignancy drivers associated with oxidative phosphorylation and immune interactions. Our PTRS model highlights CITED1 as a high-risk gene that promotes UM cell proliferation, paving the way for new prognostic models and therapeutic targets to enhance patient outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Song, Shao, Xiahou, Xu and Zhang.)

Purpose: This study aimed to identify new susceptibility modules and genes by analyzing the transcriptional profiles of peri-implantitis and periodontitis within the same host environment, using weighted gene co-expression network analysis (WGCNA)., Methods: Gingival tissue samples were collected from 10 patients, each presenting with both periodontitis and peri-implantitis sites, and were used for RNA sequencing. We conducted WGCNA to identify key modules that showed distinct transcriptional expression profiles between periodontitis and peri-implantitis. Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses were carried out using R software. Genes with an adjusted P value greater than 0.05 were excluded from gene selection using the Pearson correlation method., Results: A total of 2,226 regulated genes were identified, and those with similar expression patterns were grouped into 5 color-coded functional modules using WGCNA. Among these, 3 modules showed distinct differences in expression profiles between peri-implantitis and periodontitis. The turquoise and yellow modules were associated with upregulation in peri-implantitis, while the blue module was linked to periodontitis. This finding suggests that peri-implantitis and periodontitis have significantly different transcriptional signatures. Over-representation analysis was conducted to explore the component genes of the established modules. The top-ranked genes, selected based on their network connectivity within the modules, were identified using DESeq2 and were considered hub genes., Conclusions: WGCNA revealed distinct modular gene patterns in peri-implantitis and periodontitis, highlighting transcriptional differences between the 2 conditions. Notably, we identified 10 key genes from each of the 3 modules-the blue module associated with periodontitis-dominant pathways, and the turquoise and yellow modules associated with peri-implantitis-dominant pathways. The hub genes and pathways unveiled in this research are likely key contributors to the progression of peri-implantitis and warrant further exploration as promising candidates., Competing Interests: No potential conflict of interest relevant to this article was reported., (Copyright © 2024. Korean Academy of Periodontology.)

Background: Transcriptome analysis of skin wound tissues from diabetic foot ulcer (DFU) patients to assess changes in the microenvironment during wound healing is performed by messenger RNA (mRNA) sequencing., Methods: All 5 patients with initial DFU area ≥ 3 cm 2 were selected for wound specimen collection at two time points of 0% and 50% wound healing. A total of 10 skin wound samples were obtained for mRNA sequencing. According to the sequencing results, quantitative polymerase chain reaction (qPCR) validation was performed on 12 relevant genes related to angiogenesis, fibroblast proliferation, and wound inflammation. All patients received electrospun poly (L-lactide-co-caprolactone) and formulated porcine fibrinogen (PLCL/Fg) dressing for DFU treatment., Results: The mRNA sequencing results of DFU skin specimens showed that compared to the 0% and 50% wound healing time points, there were 4347 differentially expressed genes, including 2827 upregulated genes and 1520 downregulated genes. Enrichment analysis of the differentially expressed genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the upregulated genes were mainly associated with biological processes such as cell adhesion, adhesion junctions, epidermal development, and skin barrier formation. The qPCR analysis results indicated that the increased expression of fibroblast growth factor, vascular endothelial growth factor, and CD200 gene was related to DFU healing., Conclusion: The healing process of DFU wounds involves the interaction of multiple factors, especially in inflammation control, angiogenesis, and fibroblast proliferation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Wu, Lu, Wu, Cai, Meng and Liu.)

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: The early exposure of nutrients during pubertal mammary gland development may reduce the risk of developing breast cancer later in life. Anticancer n -3 polyunsaturated fatty acids ( n -3 PUFA) are shown to modulate pubertal mammary gland development; however, the mechanisms of action remain unclear. Prior work focused on effects at the whole tissue level, and little is known at the cellular level, such as at the level of mammary epithelial cells (MECs), which are implicated in cancer development., Methods: This pilot study examined the effects of lifelong n -3 PUFA exposure on the transcriptome by RNA-Seq in the isolated MECs of pubertal (6-8-week-old) female fat-1 transgenic mice capable of de novo n -3 PUFA synthesis. edgeR and DESeq2 were used separately for the differential expression analysis of RNA sequencing data followed by the Benjamani-Hochberg procedure for multiple testing correction., Results: Nine genes were found concordant and significantly different ( p ≤ 0.05) by both the DESeq2 and edgeR methods. These genes were associated with multiple pathways, suggesting that n -3 PUFA stimulates estrogen-related signaling ( Mlltl0 , Galr3 , and Nrip1 ) and a glycolytic profile ( Soga1 , Pdpr , and Uso1 ) while offering protective effects for immune and DNA damage responses ( Glpd1 , Garre1 , and Rpa1 ) in MECs during puberty., Conclusions: This pilot study highlights the utility of RNA-Seq to better understanding the mechanistic effects of specific nutrients such as n -3 PUFA in a cell-specific manner. Thus, further studies are warranted to investigate the cell-specific mechanisms by which n -3 PUFA influences pubertal mammary gland development and breast cancer risk later in life., Competing Interests: The authors declare no conflicts of interest.

Gastroesophageal disorders and cancers impose a significant global burden. Particularly, the prevalence of esophageal adenocarcinoma (EAC) has increased dramatically in recent years. Barrett's esophagus, a precursor of EAC, features a unique tissue adaptation at the gastroesophageal squamo-columnar junction (GE-SCJ), where the esophagus meets the stomach. Investigating the evolution of GE-SCJ and understanding dysregulation in its homeostasis are crucial for elucidating cancer pathogenesis. Here, we present the technical quality of the comprehensive single-cell RNA sequencing (scRNA-seq) dataset from mice that captures the transcriptional dynamics during the development of the esophagus, stomach and the GE-SCJ at embryonic, neonatal and adult stages. Through integration with external scRNA-seq datasets and validations using organoid and animal models, we demonstrate the dataset's consistency in identified cell types and transcriptional profiles. This dataset will be a valuable resource for studying developmental patterns and associated signaling networks in the tissue microenvironment. By offering insights into cellular programs during homeostasis, it facilitates the identification of changes leading to conditions like metaplasia and cancer, crucial for developing effective intervention strategies., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Previous studies have demonstrated the clinical relevance of aberrant serum immunoglobulin G (IgG) N-glycomic profiles in liver fibrosis and the pathogenic effects of agalactosyl IgG on activating hepatic stellate cells (HSCs). However, the dynamics of gene expression changes during HSC activation by agalactosyl IgG remain poorly understood. We performed RNA sequencing to analyze the mRNAome of human LX-2 HSCs at multiple time points after treatment with agalactosyl IgG and then compared these results with those obtained after normal IgG and transforming growth factor (TGF)-β1 treatments. Gene expression changes were significantly pronounced on day 5 and subsided by day 11 after HSC activation. A high degree of similarity in gene expression patterns between HSCs treated with agalactosyl IgG and TGF-β1 was observed, of which 1796 and 1785 differentially expressed genes (DEGs) were identified, respectively. Disease ontology analyses revealed that 114 and 105 DEGs in activated HSCs following agalactosyl IgG and TGF-β1 treatments, respectively, were linked to liver cirrhosis, hepatitis, fatty liver disease, hepatitis B, and alcoholic hepatitis, with CCL5 and FAS being the most commonly affected genes. DEGs associated with liver fibrosis or aforementioned liver diseases involved in gene annotation, physiological functions, and signaling pathways regarding secretion of cytokines and chemokines, expression of fibrosis-related growth factors and their receptors, modification of extracellular matrices, and regulation of cell viability in activated HSCs. In conclusion, this study characterized the dynamics of mRNAome and gene networks and identified the liver fibrosis-related DEGs during HSC activation by agalactosyl IgG and TGF-β1., Competing Interests: Declarations Informed consent Not applicable Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: The domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing., Methods: Peripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs., Results: Unsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster., Discussion: Our study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ramarapu, Wulcan, Chang, Moore, Vernau and Keller.)

Melon thrips, Thrips palmi, represent a significant threat to plants, inducing necrosis and acting as vectors for numerous plant viruses. Entomopathogenic fungi present a promising avenue for the management of melon thrips populations resistant to conventional chemical treatments. In this work, an adult colony of melon thrips was exposed to Beauveria bassiana strain JEF-350, and the ensuing transcriptional response of the infected thrips was scrutinized to elucidate their reactions during fungal pathogenesis. Utilizing Illumina sequencing, RNA samples were extracted from untreated thrips as well as from thrips continuously infected for 2 and 4 days, each with three biological replicates. While no notable alterations in gene expression were observed between the untreated control and thrips infected for 2 days, those infected for 4 days exhibited a plethora of differentially expressed genes. Specifically, in the thrips infected for the extended period, pathways associated with lysosomal function and insect hormone biosynthesis were notably repressed, while others such as serine and glycine metabolism, Toll and Imd, and circadian rhythm pathways displayed heightened activity. Noteworthy downregulation was observed in numerous lysosomal hydrolase genes encoding glycosidases, sulfatases, and lipases, particularly glycosidases. Furthermore, certain genes related to hydrolase precursors within the Golgi apparatus exhibited heightened expression levels but failed to progress toward hydrolase biosynthesis. Upstream regulation of juvenile hormone biosynthesis was augmented, yet downstream genes were significantly downregulated, leading to a disruption in juvenile hormone production. Similarly, while cytochrome P450 genes in the downstream of ecdysone biosynthesis were upregulated, expressions of cholesterol desaturase and cytochrome P450 genes in the upstream were inhibited, consequently dampening ecdysone biosynthesis. The observed differential targeting of organs or pathways by B. bassiana JEF-350, in contrast to conventional chemicals primarily affecting neurotransmission and energy production, suggests its potential efficacy in managing resistant thrips populations. Consequently, integrating JEF-350 into the chemical spray regimen or incorporating it into tank-mix formulations with chemical insecticides emerges as a pragmatic approach within the realm of integrated pest management strategies. KEY POINTS: • Beauveria treatment inhibited lysosomal function and hormone synthesis in thrips. • Thrips serine/glycine metabolism, Toll and Imd, and circadian rhythm pathways were activated. • Upstream functions of thrips hormone biosynthesis increased, while downstream functions were suppressed. • Regarding biosynthesis of metabolites, this fungus targets other pathways with resistance management., Competing Interests: Declarations Conflict of interest The authors declare no competing interests., (© 2024. The Author(s).)

Background: Recently, serum metabolites have shown potential in predicting survival outcomes and may be related to the pathogenesis of prostate cancer. Nevertheless, the precise impact concerning the genetic effect of metabolites on prostate cancer risk remains obscure. In this context, we conducted a Mendelian randomization (MR) study aiming to explore the causality between genetically determined metabolites and the risk of prostate cancer., Methods: We conducted a two-sample MR analysis aiming to identify the underlying metabolites associated with prostate cancer. Exposure information was obtained from the largest metabolome-based genome-wide association (GWAS) data containing 7,824 Europeans. Genome-wide association analysis was utilized to detect instrumental variables (IVs) for metabolites. We applied the inverse-variance weighted (IVW) approach as the primary method, and to augment the reliability and robustness of our findings, additional analysis methods encompassing weighted median, MR-Egger, and leave-one-out analysis were utilized. MR-Egger intercept test was implemented to explore the pleiotropy. Cochran's Q test was utilized to quantify the degree of heterogeneity. Additionally, we performed metabolic pathway analysis and single-cell RNA sequencing analysis., Results: We found that three serum metabolites were causally associated with prostate cancer after utilizing rigorous screening standards. Utilizing single nucleotide polymorphisms as IVs, a 1-SD increase in fructose was associated with 77% higher risk of prostate cancer (OR:1.77, 95%CI: 1.05-2.97, P IVW =0.031), a 1-SD increase in N1-methyl-3-pyridone-4-carboxamide was associated with 29% higher risk of prostate cancer (OR:1.29, 95%CI: 1.05-1.58, P IVW =0.017), and a 1-SD increase in 12-hydroxyeicosatetraenoate (12-HETE) was associated with 18% higher risk of prostate cancer (OR:1.18, 95%CI: 1.07-1.31, P IVW =0.0008). Metabolites that were causally linked to the risk of prostate cancer were mainly enriched in the valine, leucine and isoleucine biosynthesis pathway (P=0.026) and the nicotinate and nicotinamide metabolism pathway (P=0.048)., Conclusions: Our MR analysis provided suggestive evidence supporting the causal relationships between three identified serum metabolites and prostate cancer, necessitating further investigation to elucidate the underlying mechanisms through which these blood metabolites and metabolic pathways may impact the initiation and progression of prostate cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pan, Liu, Xiao, Sun, Zhu, Wan and Xue.)

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).)

Nervous system cancers exhibit diverse transcriptional cell states influenced by normal development, injury response, and growth. However, the understanding of these states' regulation and pharmacological relevance remains limited. Here we present "single-cell regulatory-driven clustering" (scregclust), a method that reconstructs cellular regulatory programs from extensive collections of single-cell RNA sequencing (scRNA-seq) data from both tumors and developing tissues. The algorithm efficiently divides target genes into modules, predicting key transcription factors and kinases with minimal computational time. Applying this method to adult and childhood brain cancers, we identify critical regulators and suggest interventions that could improve temozolomide treatment in glioblastoma. Additionally, our integrative analysis reveals a meta-module regulated by SPI1 and IRF8 linked to an immune-mediated mesenchymal-like state. Finally, scregclust's flexibility is demonstrated across 15 tumor types, uncovering both pan-cancer and specific regulators. The algorithm is provided as an easy-to-use R package that facilitates the exploration of regulatory programs underlying cell plasticity., (© 2024. The Author(s).)

The nucleus pulposus (NP) plays a vital role in intervertebral disc degeneration (IVDD). Previous studies have revealed cellular heterogeneity in NP tissue during IVDD progression. However, the cellular and molecular alterations of diverse cell clusters during IVDD remain to be fully elucidated. NP tissues were isolated from patients with different grades of IVDD undergoing discectomy, and then subjected to single-cell RNA sequencing (scRNA-seq). Cell subsets were identified based on unbiased clustering of gene expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine the molecular features of diverse cell clusters. Monocle analysis was used to illustrate the differentiation trajectories of chondrocytes. Additionally, CellPhoneDB analysis revealed potential interactions between chondrocytes and other cells during IVDD. Based on the expression profiles of 47,610 individual cells, eight putative clusters including chondrocytes, endothelial cells, fibroblasts, macrophages, mural cells, osteoclasts, proliferating stromal cells and T cells were identified. The chondrocyte cluster was classified into three subsets, C1-C3, which were associated with stress-resistance, fibrosis and inflammatory responses, respectively. Pseudo-time trajectories suggested that chondrocytes gradually differentiated into fibroblasts during IVDD. Immune cells including cDC2s, macrophages and monocytes were identified. Further analysis showed that chondrocytes might communicate with immune cells via the MIF, TNFSF9, SPP1 and CCL4L2 signaling pathways. In addition, we found that invading endothelial cells might interact with chondrocytes through the COL4A1, CXCL12, VEGFA and SEMA3E signaling pathways. Our results reveal the cellular complexity and phenotypic characteristics of NP tissues at single-cell resolution, which will contribute to the in-depth investigation of preventative and regenerative strategies for IVDD., (© 2024. The Author(s).)

Objective: To investigate T cell subtypes and their functions in liver immune microenvironments among patients with alveolar echinococcosis (AE) using single-cell RNA sequencing (scRNA-seq)., Methods: Four AE patients that were admitted to Qinghai Provincial People's Hospital in 2023 for hepatic surgery for the first time were enrolled, and liver specimens were sampled 1 cm (peri-lesion, PL group) and > 5 cm from AE lesions (distal lesion, DL group) among each patient. Finally, a total of eight liver specimens were sampled from four AE patients for scRNA-seq analysis. Genome and transcriptome data of liver specimens were processed using the software Cell Ranger and R package. Differentially expressed genes (DEGs) and their biological functions were analyzed using gene ontology (GO) enrichment analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and the primary intercellular communication patterns and interaction mechanisms were identified among T cell subtypes in liver specimens using the CellChat package. In addition, the developmental stages of T cells were subjected to trajectory analysis with the monocle package to investigate the expression of genes associated with cell growth and tumor transformation, and to predict the developmental trajectories of T cells., Results: All four AE patients were female, with a mean age of (25.00 ± 9.06) years, and there were three cases from Jiuzhi County, Golog Tibetan Autonomous Prefecture and one case from Chengduo County, Yushu Tibetan Autonomous Prefecture, Qinghai Province. The viability of single-cell samples from eight liver specimens was 90.41% to 96.33%, and a total of 81 763 cells were analyzed, with 19 cell types annotated. Of these cell types, 13 were immune cells (87.60%), and T cells (33.13%), neutrophils (15.40%), and natural killer cells (11.92%) were the three most common cell types. Re-clustering of 27 752 T cells and proliferative T cells identified 10 distinct T cell subtypes, with CD8 + cytotoxic T cells (23.43%), CD8 + naive T cells (12.80%), and CD4 + effector memory T cells (17.73%) as dominant cell types. The proportions of T helper 2 (Th2) cells (5.19% vs. 3.63%; χ 2 = 38.35, P < 0.01) and CD4 + effector memory T cells (21.59% vs. 13.67%; χ 2 = 244.70, P < 0.01) were significantly higher in liver specimens in the PL group than in the DL group, and the proportion of CD4 + helper T cells was significantly lower in the PL group than in the DL group (7.50% vs. 14.75%; χ 2 = 330.52, P < 0.01). KEGG pathway analysis revealed that Th2 cells were significantly enriched in cell apoptosis and multiple cancer-associated pathways, and CD4 + effector memory T cells were significantly enriched in the regulation of cytokines and chronic inflammation, while CD4 + helper T cells were significantly enriched in immune responses regulation. Trajectory analysis of T cells showed that CD4 + helper T cells were at an earlier developmental stage relative to Th2 cells and CD4 + effector memory T cells, and the expression of inhibitor of DNA binding 3 ( ID3 ), thioredoxin interacting protein ( TXNIP ), Bcl2-associated athanogene 3 ( BAG3 ) and heat shock protein family B (small) member 1 ( HSPB1 ) genes appeared a tendency towards a decline over time., Conclusions: CD4 + effector memory T cells and CD8 + cytotoxic T cells are primary interacting cells in the liver specimens of AE patients. Reduced expression of Th2 cells and CD4 + helper T cells contributes to an inhibitory immune microenvironment, which promotes immune evasion by Echinococcus multilocularis , and Th2 cells are significantly enriched in multiple cancer-associated pathways, which may be linked to the invasive growth of E. multilocularis .

Key Message: The QTLs and candidate genes governing the multifoliolate phenotype were identified by combining linkage mapping with BSR-seq, revealing a possible interplay between genetics and the environment in soybean leaf development. Soybean, as a legume, is typified by trifoliolate leaves. Although multifoliolate leaves (compound leaves with more than three leaflets each) have been reported in soybean, including sporadic appearances in the first compound leaves in a recombinant inbred line (RIL) population from a cross between cultivated soybean C08 and wild soybean W05 from this study, the genetic basis of this phenomenon is still unclear. Here, we integrated quantitative trait locus (QTL) mapping with bulked segregant RNA sequencing (BSR-seq) to identify the genetic loci associated with the multifoliolate phenotype in soybean. Using linkage mapping, ten QTLs related to the multifoliolate trait were identified. Among these, a significant and major QTL, qMF-2-1 on chromosome 2 and consistently detected across biological replicates, explained more than 10% of the phenotypic variation. Together with BSR-seq analyses, which analyzed the RILs with the highest multifoliolate frequencies and those with the lowest frequencies as two distinct bulks, two candidate genes were identified: Glyma.06G204300 encoding the transcription factor TCP5, and Glyma.06G204400 encoding LONGIFOLIA 2 (LNG2). Transcriptome analyses revealed that stress-responsive genes were significantly differentially expressed between high-multifoliolate occurrence lines and low occurrence ones, indicating environmental factors probably influence the appearance of multifoliolate leaves in soybean through stress-responsive genes. Hence, this study offers new insights into the genetic mechanism behind the multifoliolate phenotype in soybean., (© 2024. The Author(s).)

Purpose: Adenoid cystic carcinoma (AdCC) of the head and neck harbors MYB/MYBL1::NFIB fusions in around 60% of cases, with unfavorable long-term survival due to frequent recurrences and metastases, currently lacking effective targeted therapy. The study aims to identify actionable alterations and to elucidate the molecular underpinnings of MYB/MYBL1::NFIB-negative AdCC using a large targeted RNA sequencing panel., Methods and Results: We retrospectively searched our MSK-Solid Fusion clinical sequencing database for head and neck AdCC sequenced between 2016 and 2023. Of a total of 55 cases, 28 showed MYB::NFIB, 7 showed MYBL1::NFIB, and one case each harbored MYB::MPDZ (case 1) and FUS::MYB (case 2). One base of tongue tumor expressed both MYB::NFIB fusion and MET exon 14 skipping transcripts due to concurrent MET splice site mutation, D1010N (case 3). One parotid tumor lacked MYB/MYBL1 rearrangement but instead showed an in-frame SEC16A::NOTCH1 fusion that preserved the secretase cleavage site (case 4). Clinical records on 4 cases with non-canonical sequencing findings were reviewed. Distant metastases were present at the initial diagnosis (case 2) or at recurrence (cases 1, 3, and 4). Disease-related mortality occurred in cases 2 and 4 despite radiotherapy and immunotherapy., Conclusions: The study improved the understanding of AdCC providing the first documentation of tumor clinical behavior associated with MYB::MPDZ and FUS::MYB fusions and reporting potentially actionable SEC16A::NOTCH1 fusion and MET exon 14 skipping mutation. Further research is needed to explore the therapeutic utility of MET inhibition and the efficacy of γ-secretase inhibitors against rare NOTCH1 fusions in AdCC., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Host iron deficiency is protective against severe malaria as the human malaria parasite Plasmodium falciparum depends on bioavailable iron from its host to proliferate. The essential pathways of iron acquisition, storage, export, and detoxification in the parasite differ from those in humans, as orthologs of the mammalian transferrin receptor, ferritin, or ferroportin, and a functional heme oxygenase are absent in P. falciparum . Thus, the proteins involved in these processes may be excellent targets for therapeutic development, yet remain largely unknown. Here, we show that parasites cultured in erythrocytes from an iron-deficient donor displayed significantly reduced growth rates compared to those grown in red blood cells from healthy controls. Sequencing of parasite RNA revealed diminished expression of genes involved in overall metabolism, hemoglobin digestion, and metabolite transport under low-iron versus control conditions. Supplementation with hepcidin, a specific ferroportin inhibitor, resulted in increased labile iron levels in erythrocytes, enhanced parasite replication, and transcriptional upregulation of genes responsible for merozoite motility and host cell invasion. Through endogenous GFP tagging of differentially expressed putative transporter genes followed by confocal live-cell imaging, proliferation assays with knockout and knockdown lines, and protein structure predictions, we identified six proteins that are likely required for ferrous iron transport in P. falciparum . Of these, we localized Pf VIT and Pf ZIPCO to cytoplasmic vesicles, Pf MRS3 to the mitochondrion, and the novel putative iron transporter Pf E140 to the plasma membrane for the first time in P. falciparum . Pf NRAMP/ Pf DMT1 and Pf CRT were previously reported to efflux Fe 2+ from the digestive vacuole. Our data support a new model for parasite iron homeostasis, in which Pf E140 is involved in iron uptake across the plasma membrane, Pf MRS3 ensures non-redundant Fe 2+ supply to the mitochondrion as the main site of iron utilization, Pf VIT transports excess iron into cytoplasmic vesicles, and Pf ZIPCO exports Fe 2+ from these organelles in case of iron scarcity. These results provide new insights into the parasite's response to differential iron availability in its environment and into the mechanisms of iron transport in P. falciparum as promising candidate targets for future antimalarial drugs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wunderlich, Kotov, Votborg-Novél, Ntalla, Geffken, Peine, Portugal and Strauss.)

Background: Cell death plays an essential role in carcinogenesis, but its function in the recurrence and postoperative prognosis of head and neck cancer (HNC), which ranks as the 7 th most common malignancy globally, remains unclear., Methods: Data from five main subtypes of HNC related single-cell RNA sequencing (scRNA-seq) were recruited to establish a single-cell atlas, and the distribution of cell death models (CDMs) across different tissues as well as cell subtypes were analyzed. Bulk RNA-seq from the Cancer Genome Atlas Program (TCGA) dataset was subjected to a machine learning-based integrative procedure for constructing a consensus cell death-related signature risk score (CDRscore) model and validated by external data. The biofunctions including different expression analysis, immune cell infiltration, genomic mutations, enrichment analysis as well as cellchat analysis were compared between the high- and low- risk score groups categorized by this CDRscore model. Finally, samples from laryngeal squamous cell cancer (LSCC) were conducted by spatial transcriptomics (ST) to further validate the results of CDRscore model., Results: T cells from HNC patients manifested the highest levels of cell death while HPV infection attenuates malignant cell death based on single-cell atlas. CDMs are positively correlated with the tumor-cell stemness, immune-related score and T cells are infiltrated. A CDRscore model was established based on the transcription of ten cell death prognostic genes ( MRPL10 , DDX19A , NDFIP1 , PCMT1 , HPRT1 , SLC2A3 , EFNB2 , HK1 , BTG3 and MAP2K7 ). It functions as an independent prognostic factor for overall survival in HNC and displays stable and powerful performance validated by GSE41613 and GSE65858 datasets. Patients in high CDRscore manifested worse overall survival, more active of epithelial mesenchymal transition, TGF-β-related pathways and hypoxia, higher transcription of T cell exhausted markers, and stronger TP53 mutation. ST from LSCC showed that spots with high-risk scores were colocalized with TGF-β and the proliferating malignant cells, additionally, the risk scores have a negative correlation with TCR signaling but positive association with LAG3 transcription., Conclusion: The CDRscore model could be utilized as a powerful prognostic indicator for HNC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Pan, Fei, Wang, Chen, Jiang, Li, Wang, Yang, Zhang and Chen.)

Macrophages induce the expression of hundreds of genes in response to immune threats. However, current technology limits our ability to capture single-cell inducible gene expression dynamics. Here, we generated high-resolution time series single-cell RNA sequencing (scRNA-seq) data from mouse macrophages responding to six stimuli, and imputed ensembles of real-time single-cell gene expression trajectories (scGETs). We found that dynamic information contained in scGETs substantially contributes to macrophage stimulus-response specificity (SRS). Dynamic information also identified correlations between immune response genes, indicating biological coordination. Furthermore, we showed that the microenvironmental context of polarizing cytokines profoundly affects scGETs, such that measuring response dynamics offered clearer discrimination of the polarization state of individual macrophage cells than single time-point measurements. Our findings highlight the important contribution of dynamic information contained in the single-cell expression responses of immune genes in characterizing the SRS and functional states of macrophages., Competing Interests: Declaration of interests K.M.S. and A.H. are authors of a patent (publication number 20230408493) that relates to the content of this manuscript., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Neuroblastoma is the most common extracranial solid tumor in children, and a leading cause of childhood cancer deaths. All neuroblastomas arise from neural crest-derived sympathetic neuronal progenitors, but numerous mutations, the most common of which is MYCN amplification, give rise to these lesions. Epigenetic aberrations also play a role in oncogenesis and tumor progression. To better understand biologic diversity of neuroblastomas, we performed joint single-nucleus ATAC sequencing and single-nucleus RNA sequencing on six neuroblastoma cell lines, three of which are MYCN amplified. After standard filtering for high-quality nuclei, we obtained chromatin accessibility and transcript abundance data from 41,733 neuroblastoma tumor cells. Preliminary analysis reveals significant diversity in chromatin landscape and gene expression across neuroblastoma cell lines. This dataset is a valuable resource for studying the transcriptional and epigenetic mechanisms of this deadly childhood disease., (© 2024. The Author(s).)

Autoimmunity underlies many painful disorders, such as enthesopathies, which localize to the enthesis. From infiltration of the synovium and axial skeleton by B cells, to disturbances in the ratio of M1/M2 enthesis macrophages, to CD8 + T cell mediated inflammation, autoimmune dysregulation is becoming increasingly well characterized in enthesopathies. Tissue resident B cells, macrophages, neutrophils, and T cells have also been localized in healthy human entheses. However, the potential developmental origins, presence, and role of immune cells (ICs) in enthesis development is not known. Here, we use single-cell RNA-sequencing analysis to describe IC subtypes present in the enthesis before, during, and after mineralization, and to infer regulatory interactions between ICs and mesenchymal cells (MCs). We report the presence of nine phenotypically distinct IC subtypes, including B cells, macrophages, neutrophils, and T cells. We find that specific IC subtypes may promote MC-proliferation and differentiation, and that MCs may regulate IC phenotype and autoimmunity. Our findings suggest that bidirectional regulatory interactions between ICs and MCs may be important to enthesis mineralization, and suggest that progenitor MCs have a unique ability to limit autoimmunity during development., (© 2024. The Author(s).)

Background: Metabolic distress is often associated with heart failure with preserved ejection fraction (HFpEF) and represents a therapeutic challenge. Metabolism-induced systemic inflammation links comorbidities with HFpEF. How metabolic changes affect myocardial inflammation in the context of HFpEF is not known., Methods: We found that ApoE knockout mice fed a Western diet recapitulate many features of HFpEF. Single-cell RNA sequencing was used for expression analysis of CD45 + cardiac cells to evaluate the involvement of inflammation in diastolic dysfunction. We focused bioinformatics analysis on macrophages, obtaining high-resolution identification of subsets of these cells in the heart, enabling us to study the outcomes of metabolic distress on the cardiac macrophage infiltrate and to identify a macrophage-to-cardiomyocyte regulatory axis. To test whether a clinically relevant sodium glucose cotransporter-2 inhibitor could ameliorate the cardiac immune infiltrate profile in our model, mice were randomized to receive the sodium glucose cotransporter-2 inhibitor dapagliflozin or vehicle for 8 weeks., Results: ApoE knockout mice fed a Western diet presented with reduced diastolic function, reduced exercise tolerance, and increased pulmonary congestion associated with cardiac lipid overload and reduced polyunsaturated fatty acids. The main immune cell types infiltrating the heart included 4 subpopulations of resident and monocyte-derived macrophages, determining a proinflammatory profile exclusively in ApoE knockout-Western diet mice. Lipid overload had a direct effect on inflammatory gene activation in macrophages, mediated through endoplasmic reticulum stress pathways. Investigation of the macrophage-to-cardiomyocyte regulatory axis revealed the potential effects on cardiomyocytes of multiple inflammatory cytokines secreted by macrophages, affecting pathways such as hypertrophy, fibrosis, and autophagy. Finally, we describe an anti-inflammatory effect of sodium glucose cotransporter-2 inhibition in this model., Conclusions: Using single-cell RNA sequencing in a model of diastolic dysfunction driven by hyperlipidemia, we have determined the effects of metabolic distress on cardiac inflammatory cells, in particular on macrophages, and suggest sodium glucose cotransporter-2 inhibitors as potential therapeutic agents for the targeting of a specific phenotype of HFpEF., Competing Interests: None.

Diabetic foot ulcer (DFU) is a chronic and serious complication of diabetes mellitus. It is mainly caused by hyperglycaemia, diabetic peripheral vasculopathy and diabetic peripheral neuropathy. These conditions result in ulceration of foot tissues and chronic wounds. If left untreated, DFU can lead to amputation or even endanger the patient's life. Single-cell RNA sequencing (scRNA-seq) is a technique used to identify and characterise transcriptional subpopulations at the single-cell level. It provides insight into cellular function and the molecular drivers of disease. The objective of this paper is to examine the subpopulations, genes and molecules of cells associated with chronic wounds of diabetic foot by using scRNA-seq. The paper aims to explore the wound-healing mechanism of DFU from three aspects: inflammation, angiogenesis and extracellular matrix remodelling. The goal is to gain a better understanding of the mechanism of DFU wound healing and identify possible DFU therapeutic targets, providing new insights for the application of DFU personalised therapy., (© 2024 The Author(s). Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.)

The occurrence of poisoning incidents caused by cyanobacterial blooms has aroused wide public concern. Microcystin-leucine arginine (MC-LR) is a well-established toxin produced by cyanobacterial blooms, which is widely distributed in eutrophic waters. MC-LR is not only hazardous to the water environment but also exerts multiple toxic effects including liver toxicity in both humans and animals. However, the underlying mechanisms of MC-LR-induced liver toxicity are unclear. Herein, we used advanced single-cell RNA sequencing technology to characterize MC-LR-induced liver injury in mice. We established the first single-cell atlas of mouse livers in response to MC-LR. Our results showed that the differentially expressed genes and pathways in diverse cell types of liver tissues of mice treated with MC-LR are highly heterogeneous. Deep analysis showed that MC-LR induced an increase in a subpopulation of hepatocytes that highly express Gstm3, which potentially contributed to hepatocyte apoptosis in response to MC-LR. Moreover, MC-LR increased the proportion and multiple subtypes of Kupffer cells with M1 phenotypes and highly expressed proinflammatory genes. Furthermore, the MC-LR increased several subtypes of CD8 + T cells with highly expressed multiple cytokines and chemokines. Overall, apart from directly inducing hepatocytes apoptosis, MC-LR activated proinflammatory Kupffer cell and CD8 + T cells, and their interaction may constitute a hostile microenvironment that contributes to liver injury. Our findings not only present novel insight into underlying molecular mechanisms but also provide a valuable resource and foundation for additional discovery of MC-LR-induced liver toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2023. Published by Elsevier B.V.)

Although next-generation sequencing has enabled diagnoses for many patients with Mendelian disorders, the majority remain undiagnosed. Here, we present a sibling pair who were clinically diagnosed with Escobar syndrome, however targeted gene testing was negative. Exome sequencing (ES), and later genome sequencing (GS), revealed compound heterozygous TTN variants in both siblings, a maternally inherited frameshift variant [(NM&#95;133378.4):c.36812del; p.(Asp12271Valfs*10)], and a paternally inherited missense variant [(NM&#95;133378.4):c.12322G > A; p.(Asp4108Asn)]. This result was considered nondiagnostic due to poor clinical fit and limited pathogenicity evidence for the missense variant of uncertain significance (VUS). Following initial nondiagnostic RNA sequencing (RNAseq) on muscle and further pursuit of other variants detected on the ES/GS, a reanalysis of noncanonical splice sites in the muscle transcriptome identified an out-of-frame exon retraction in TTN, near the known VUS. Interim literature included reports of patients with similar TTN variants who had phenotypic concordance with the siblings, and a diagnosis of a congenital titinopathy was given 4 years after the TTN variants had been initially reported. This report highlights the value of reanalysis of RNAseq with a different approach, expands the phenotypic spectrum of congenital titinopathy and also illustrates how a perceived phenotypic mismatch, and failure to consider known variants, can result in a prolongation of the diagnostic journey., (© 2024 Wiley Periodicals LLC.)

Women typically live longer than men, and constitute the majority of centenarians. We applied RNA-sequencing (RNA-seq) of blood-derived lymphoblastoid cell lines (LCLs) from women aged 60-80 years and centenarians (100-105 years), validated the RNA-seq findings by real-time PCR, and additionally measured the differentially expressed genes in LCLs from young women aged 20-35 years. Top RNA-seq genes with differential expression between the age groups included three selenoproteins (GPX1, SELENOW, SELENOH) and three heat shock proteins (HSPA6, HSPA1A, HSPA1B), with the highest expression in LCLs from young women, indicating that young women are better protected from oxidative stress. The expression of two additional genes, BID encoding BH3-interacting domain death agonist and CD99 encoding CD99 antigen, showed unique age dependence, with similar expression levels in young and centenarian women while exhibiting higher and lower expression levels, respectively, in LCLs from women aged 60-80 years compared with the two other age groups. This age-related differential expression of BID and CD99 suggests elevated inflammation susceptibility in middle-aged women compared with either young or centenarian women. Our findings, once validated with human peripheral blood mononuclear cells and further cell types, may lead to novel healthy aging diagnostics and therapeutics., (© 2024 The Author(s). Drug Development Research published by Wiley Periodicals LLC.)

Objective: Synovial fluid (SF)-derived T cells are frequently studied as a proxy for investigating the synovial tissue (ST) T cell infiltrate in inflammatory arthritis. However, because ST is the primary site of inflammatory activity, there is debate as to whether SF provides a true reflection of the ST T cell population., Methods: In this study, we used single-cell RNA sequencing paired with single-cell T cell receptor (TCR) sequencing to directly compare memory T cells from paired samples of SF and ST from six patients with inflammatory arthritis to investigate their similarity in terms of TCR repertoire and T cell subset composition., Results: The TCR repertoires of SF and ST T cells were strikingly similar, particularly for CD8+ T cells. A median of 49% of the total CD8+ TCR repertoire in SF was shared with ST, compared with 20% shared with blood. Similarly, 47% of the ST CD8+ TCR repertoire was shared with SF compared to 25% with blood. Furthermore, once the effect of collagenase digestion on gene expression by ST T cells had been accounted for, the frequencies of specific CD8+ and CD4+ T cell subsets were, in general, similar in SF and ST and were distinct from blood., Conclusion: Our results suggest that T cells migrate and equilibrate between the SF and ST and maintain similar phenotypes in both sites. We conclude that SF is an appropriate proxy for investigating the T cell infiltrate in inflamed synovium, particularly in terms of investigating the TCR repertoire., (© 2024 The Author(s). Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

Introduction: Kinesin family member 20A (KIF20A) is essential for cell proliferation and is implicated in promoting tumor progression, but its role in hepatocellular carcinoma (HCC) remains poorly studied., Methods: Through the analysis of bulk RNA-sequencing (bulk RNA-seq) and single-cell RNA sequencing (scRNA-seq) data, the expression of KIF20A and its relationship with diagnosis, prognosis, and the immune microenvironment were examined. The association between KIF20A and the malignant progression and metastasis of HCC was confirmed through in vitro and in vivo experiments. Furthermore, patient re-staging was performed using Recursive Partitioning Analysis (RPA) to enhance clinical benefit., Results: In this study, we firstly found KIF20A was overexprerssed in HCC both by bulk RNA-seq and scRNA-seq, and then the overexpression of KIF20A significantly promoted the proliferation, invasion, and metastasis in vitro. In vivo, the overexpression of KIF20A promoted the growth and lung metastasis of HCC. Furthermore, gene set variation analysis of bulk RNA-seq and scRNA-seq revealed that KIF20A might be associated with cell cycle related signaling pathways of E2F and G2M, and overexpression of KIF20A inhibited the activity of p21 and bax, as well as shortened G2 phase. Importantly, we found that KIF20A could induce T cell exhaustion via the SPP1-CD44 axe using scRNA-seq. Additionally, KIF20A was also correlated with the expression of immune checkpoint inhibitors (ICIs), and KIF20Ahigh subgroup might be benefited from the ICIs therapy., Conclusion: KIF20A emerges as a pivotal driver of HCC progression, intricately regulating cell cycle pathways and modulating immune responses, which position KIF20A as a promising target for HCC management., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Su, Zhong, He, You, Xin, Wang and Liu.)

The pathogenesis of sepsis, with a high mortality rate and often poor prognosis, has not been fully elucidated. Therefore, an in-depth study on the pathogenesis of sepsis at the molecular level is essential to identify key sepsis-related genes. The aim of this study was to explore the key genes and potential molecular mechanisms of sepsis using a bioinformatics approach. In addition, key genes with miRNA network correlation analysis and immune infiltration correlation analysis were investigated. The scRNA dataset (GSE167363) and RNA-seq dataset (GSE65682, GSE134347) from GEO database were used for screening out differentially expressed genes using single-cell sequencing and transcriptome sequencing. The analysis of immune infiltration was evaluated by the CIBERSORT method. Key genes and possible mechanisms were identified by WGCNA analysis, GSVA analysis, GSEA enrichment analysis and regulatory network analysis, and miRNA networks associated with key genes were constructed. Nine key genes associated with the development of sepsis, namely IL7R, CD3D, IL32, GPR183, HLA-DPB1, CD81, PEBP1, NCL, and ETS1 were screened, and the specific signaling mechanisms associated with the key genes causing sepsis were predicted. Immune profiling showed immune heterogeneity between control and sepsis samples. A regulatory network of 82 miRNAs, 266 pairs of mRNA-miRNA relationship pairs was also constructed. These nine key genes have the potential to become biomarkers for the diagnosis of sepsis and provide new targets and research directions for the treatment of sepsis.

Purpose: Breast cancer is a common malignancy in women, and its metastasis is a leading cause of cancer-related deaths. Single-cell RNA sequencing (scRNA-seq) can distinguish the molecular characteristics of metastasis and identify predictor genes for patient prognosis. This article explores gene expression in primary breast cancer tumor tissue against metastatic cells in the lymph node and liver using scRNA-seq., Methods: Breast cancer scRNA-seq data from the Gene Expression Omnibus were used. The data were processed using R and the Seurat package. The cells were clustered and identified using Metascape. InferCNV is used to analyze the variation in copy number. Differential expression analysis was conducted for the cancer cells using Seurat and was enriched using Metascape., Results: We identified 18 distinct cell clusters, 6 of which were epithelial. CNV analysis identified significant alterations with duplication of chromosomes 1, 8, and 19. Differential gene analysis resulted in 17 upregulated and 171 downregulated genes for the primary tumor in the primary tumor vs. liver metastasis comparison and 43 upregulated and 4 downregulated genes in the primary tumor in the primary tumor vs lymph node metastasis comparison. Several enriched terms include Ribosome biogenesis, NTP synthesis, Epithelial dedifferentiation, Autophagy, and genes associated with epithelial-to-mesenchymal transitions., Conclusion: No single gene or pathway can clearly explain the mechanisms behind tumor metastasis. Several mechanisms contribute to lymph node and liver metastasis, such as the loss of differentiation, epithelial-to-mesenchymal transition, and autophagy. These findings necessitate further study of metastatic tissue for effective drug development., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Drought is a significant global environmental stress. Biostimulants offer a sustainable solution to enhance crop tolerance and mitigate productivity losses. This study assessed the impact of foliar application of ERANTHIS®, a biostimulant derived from the algae Ascophyllum nodosum and Laminaria digitata and yeast extracts, on tomato plants under mild water stress. Evaluations were conducted at 5 and 24 hours after the third treatment. Under optimal water conditions, the biostimulant showed a priming effect, with an early increase of stress markers and a timing-specific modulation of ROS non enzymatic and enzymatic ROS scavenging activities. Under drought stress, the biostimulant later decreased stress markers, by aligning the majority of analyzed ROS scavengers closer to levels in well-irrigated plants. Transcriptome analysis using RNA-seq data revealed differentially expressed genes (DEGs) and multivariate data highlighted groups of co-regulated genes (k-means clustering). Genes involved in water channel activity, transcription regulator activity, and oxidoreductase activity were significantly modulated. Cluster analysis identified distinct gene clusters influenced by the biostimulant under optimal conditions, including early responses (cell wall modification, hormone signaling) and late responses (RNA modification, nutrient uptake process). Under water stress, early responses involved actin filament organization and MAPK signaling, while late responses were related to plasma membrane components and cell wall organization. This study, integrating biochemical and transcriptomic data, provides a comprehensive understanding of how a biostimulant primes plants under optimal conditions and mitigates water stress effects, offering valuable insights for sustainable agriculture., (© 2024 Green Has Italia S.P.A and The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Tri (2-Ethylhexyl) phosphate (TEHP), widely used as a fire retardant and plasticizer, has been commonly found in the environment. Its potential health-related risks, especially reproductive toxicity, have aroused concern. However, the potential cellular mechanisms remain unexplored. In this study, we aimed to investigate the molecular mechanisms underlying TEHP-caused cell damage in Sertoli cells, which play a crucial role in supporting spermatogenesis. Our findings indicate that TEHP induces apoptosis in 15P-1 mouse Sertoli cells. Subsequently, we conducted RNA sequencing analyses, which suggested that ER stress, autophagy, and MAPK-related pathways may participate in TEHP-induced cytotoxicity. Furthermore, we demonstrated that TEHP triggers ER stress, activates p38 MAPK, and inhibits autophagy flux. Then, we showed that the inhibition of ER stress or p38 MAPK activation attenuates TEHP-induced apoptosis, while the inhibition of autophagy flux is responsible for TEHP-induced apoptosis. These results collectively reveal that TEHP induces ER stress, activates p38, and inhibits autophagy flux, ultimately leading to apoptosis in Sertoli cells. These shed light on the molecular mechanisms underlying TEHP-associated testicular toxicity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Idiopathic carpal tunnel syndrome is the most common entrapment neuropathy in hand surgery, and it is characterized by Noninflammatory fibrosis of subsynovial connective tissues. The prevalence and incidence differ between male and female individuals, and the mechanism underlying this difference remains largely unclear. In the present study, we collected subsynovial connective tissues from six male and six female patients diagnosed with idiopathic carpal tunnel syndrome during surgery. We performed a comprehensive gene expression analysis using RNA sequencing to compare the gene expression profiles between male and female patients with idiopathic carpal tunnel syndrome. We identified 26 genes with significantly different expressions between male and female patients, in which POSTN, COL1A1, and COL3A1, which are involved in extracellular matrix organization, and IGF1, an important fibrotic factor, were significantly upregulated in male patients. Immunohistochemistry confirmed the expression of proteins encoded by these genes in tissues, and male patients tended to show increased POSTN expression. Our results indicate that fibrosis of subsynovial connective tissues is induced by different mechanisms in male and female patients, and genes involved in extracellular matrix organization, especially POSTN, might be important factors in male patients. This study provides insight into the pathogenesis of idiopathic carpal syndrome and might contribute to the development of new treatment strategies., (© 2024 Orthopaedic Research Society.)

Techniques for triggering neural differentiation of embryonic and induced pluripotent stem cells into neural stem cells and neurons have been established. However, neural induction of mesenchymal stem cells, including dental pulp stem cells (DPSCs), has been assessed primarily based on neural-related gene regulation, and detailed studies into the characteristics and differentiation status of cells are lacking. Therefore, this study was aimed at evaluating the cellular components and differentiation pathways of neural lineage cells obtained via neural induction of human DPSCs. Human DPSCs were induced to neural cells in monolayer culture and examined for gene expression and mechanisms underlying differentiation using microarray-based ingenuity pathway analysis. In addition, the neural lineage cells were subjected to single-cell RNA sequencing (scRNA-seq) to classify cell populations based on gene expression profiles and to elucidate their differentiation pathways. Ingenuity pathway analysis revealed that genes exhibiting marked overexpression, post-neuronal induction, such as FABP7 and ZIC1, were associated with neurogenesis. Furthermore, in canonical pathway analysis, axon guidance signals demonstrated maximum activation. The scRNA-seq and cell type annotations revealed the presence of neural progenitor cells, astrocytes, neurons, and a small number of non-neural lineage cells. Moreover, trajectory and pseudotime analyses demonstrated that the neural progenitor cells initially engendered neurons, which subsequently differentiated into astrocytes. This result indicates that the aforementioned neural induction strategy generated neural stem/progenitor cells from DPSCs, which might differentiate and proliferate to constitute neural lineage cells. Therefore, neural induction of DPSCs may present an alternative approach to pluripotent stem cell-based therapeutic interventions for nervous system disorders., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

This study aimed to evaluate the efficacy and therapeutic mechanism of parthenolide (PTL) in breast cancer (BC) through a comprehensive strategy integrating network pharmacology, single-cell RNA sequencing (scRNA-seq) and metabolomics. In network pharmacology, 70 therapeutic targets were identified, of which 16 core targets were filtered out through seven classical algorithms of Cytohubba plugin. Additionally, the hub module of PPI network was extracted using MCODE plugin. Molecular docking and molecular dynamics simulation showed a potent binding affinity between PTL and JNK, subsequently validated by MST and SPR assays. Further, Mendelian randomization analysis indicated that JNK was causally associated with BC. GO and KEGG enrichment analyses revealed that PTL counteracted BC via promoting ROS generation, inducing apoptosis and suppressing proliferation, which potentially involved the coordinated regulation of MAPK and FoxO1 pathways. Moreover, ssGSEA and scRNA-seq analysis suggested that PTL may act on T cell immune microenvironment of BC. Subsequently, these bioinformatics-based predictions were experimentally validated using in-vitro and in-vivo models. Finally, metabolome profiling unveiled that PTL remodeled the glycine, serine and threonine metabolism as well as biosynthesis of unsaturated fatty acids, and thereby contributed to BC inhibition. From molecular, immune and metabolic perspectives, this study not only provided a unique insight into the mechanistic details of PTL against BC, but also proposed a novel promising therapeutic strategy for BC., Competing Interests: Declaration of competing interest The authors have no competing interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although metastatic disease is the leading cause of cancer-related deaths, its tumor microenvironment remains poorly characterized due to technical and biospecimen limitations. In this study, we assembled a multi-modal spatial and cellular map of 67 tumor biopsies from 60 patients with metastatic breast cancer across diverse clinicopathological features and nine anatomic sites with detailed clinical annotations. We combined single-cell or single-nucleus RNA sequencing for all biopsies with a panel of four spatial expression assays (Slide-seq, MERFISH, ExSeq and CODEX) and H&E staining of consecutive serial sections from up to 15 of these biopsies. We leveraged the coupled measurements to provide reference points for the utility and integration of different experimental techniques and used them to assess variability in cell type composition and expression as well as emerging spatial expression characteristics across clinicopathological and methodological diversity. Finally, we assessed spatial expression and co-localization features of macrophage populations, characterized three distinct spatial phenotypes of epithelial-to-mesenchymal transition and identified expression programs associated with local T cell infiltration versus exclusion, showcasing the potential of clinically relevant discovery in such maps., Competing Interests: Competing interests A. Regev, J.K. and D.L.A. are co-inventors on patent application number 17/156,392, filed by the Broad Institute, for inventions relating to work in this manuscript. A. Regev is a co-founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas and, until 31 July 2020, a scientific advisory board (SAB) member of Thermo Fisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics and Asimov. From 1 August 2020, A. Regev is an employee of Genentech and has equity in Roche. A. Rotem is an employee of AstraZeneca since September 2020 and has equity in AstraZeneca. E.S.B. co-founded a company that is exploring commercial applications of expansion microscopy. G.N. holds equity in and consults for Akoya Biosciences. J.G.T.Z. owns stocks in the biotechnology exchange-traded funds CNCR, IDNA, IBB and XBI, owns stock in Novo Nordisk and owns stock in Adaptive Biotechnologies, 2seventy bio and bluebird bio. J.J-V. is a contractor at Genentech since 2021. M.S. is a contractor at Genentech since November 2020. O.R.-R. is a co-inventor on patent applications filed by the Broad Institute for inventions related to single-cell genomics. She has given numerous lectures on the subject of single-cell genomics to a wide variety of audiences and, in some cases, has received remuneration to cover time and costs. O.R.-R. is an employee of Genentech since 19 October 2020 and has equity in Roche. S.J.R. is a member of the SAB of Immunitas Therapeutics and receives research funding from Bristol Myers Squibb and Kite/Gilead. X.Z. is a co-founder of and consultant for Vizgen. The other authors declare no competing interests., (© 2024. The Author(s).)

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively., Competing Interests: Declaration of Competing Interest All authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The intestinal helminth Ascaris lumbricoides infects over 800 million people. Infections are often chronic and immunity is not sterilizing due to host-immune modulation, therefore reinfection is common after antihelmintic treatment. We have previously demonstrated a role for Ascaris spp. extracellular vesicles (EVs) in host immune modulation but whether EVs are recognized by the adaptive immune system and are present systemically in the host remains unknown. Therefore, we employed a well-established trickle infection model in pigs to mimic natural Ascaris infection in humans. EVs were isolated from adult Ascaris suum followed by immunoblotting of EV and EV-depleted secretory fractions using plasma from infected and uninfected pigs. Next, EVs were isolated from pig plasma at day 56 post first infection and subjected to deep small RNAseq analysis. RNAs were aligned to A. suum and Sus scrofa miRNA complements to detect A. suum EVs and elucidate the host EV micro RNA (miRNA) response to infection, respectively. Infection generates robust antibody responses against A. suum EVs that is distinct from EV-depleted fractions. However, A. suum miRNAs were not detectable in EVs from the peripheral blood. Notably, host plasma-derived EV miRNA profiles showed significant changes between infected and uninfected pigs, indicating that Ascaris infection drives systemic changes in host EV composition., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Aim: This study was to explore role of immune landscape and the immune cells in acute lymphoblastic leukemia (ALL) progression., Background: The most prevalent genetic alteration in childhood ALL is the ETV6-RUNX1 fusion. The increased proliferation of B progenitor cells could expedite the disease's progression due to irregularities in the cell cycle. Nevertheless, the mechanisms by which particular cell clusters influence the cell cycle and promote the advancement of ALL are still not well understood., Objective: This study was to explore role of immune landscape and the immune cells in ALL progression., Methods: Single-cell RNA sequencing (scRNA-seq) data of ETV6-RUNX1 and healthy pediatric samples obtained from GSE132509 were clustered and annotated using the Seurat package, and differentially highly expressed genes identified in each cluster were analyzed using DAVID for pathway annotation. Chromosome amplification and deletion were analyzed using the inferCNV package. SCENIC evaluated the regulation of transcription factors and target gene formation in cells. cellphoneDB and CellChat were served to infer ligand-receptor pairs that mediate interactions between subpopulations. The role of the target gene in regulating ALL progression was assessed using RT-qPCR, Transwell and scratch healing assays., Results: The bone marrow mononuclear cells (BMMCs) from ETV6-RUNX1 and healthy pediatric samples in GSE132509 were divided into 11 clusters, and B cell cluster 1 was identified as B progenitor cell, which was amplified on chromosome 6p. B progenitor cells were divided into seven clusters. Expression levels of amplified genes in chromosome 6p of B progenitor cell cluster 5 were the highest, and its specific highly expressed genes were annotated to pathways promoting cell cycle progression. Regulons formed in B progenitor cell cluster 5 were all involved in promoting cell cycle progression, so it was regarded as the B progenitor cell cluster that drives cell cycle progression. The key regulator of the B progenitor cell is E2F1, which promotes the migration and invasion ability of the cell line HAP1. The major ligand-receptor pairs that mediate the communication of B progenitor cell cluster 5 with cytotoxic NK/T cells or naive T cells included FAM3C-CLEC2D, CD47-SIRPG, HLAE-KLRC2, and CD47-KLRC2. HLAE-KLRC1 and TGFB1-(TGFBR1+TGFBR2)., Conclusion: This study outlined the immune cell landscape of ETV6-RUNX1 ALL and identified chromosome 6p amplification in B progenitor cells, described the major B progenitor cell cluster driving cell cycle progression and its potential regulatory mechanisms on NK cells and T cells, providing cellular and molecular insights into ETV6-RUNX1 ALL., Competing Interests: The authors declare that they have no competing interests., (© 2024 Qu et al.)

The growth of the ovarian antral follicle is a complex process that is difficult to study, especially in human and nonhuman primates. Understanding the antral stage of development is key to new approaches to regulating reproduction. This study analyzed cohorts of three sizes of developing antral follicles obtained from adult rhesus macaque females using RNA sequencing of oocytes and cumulus and granulosa cells. The overall objective of this study was to identify key developmental changes in gene expression in oocytes, granulosa, and cumulus cells, as nonhuman primate antral stage follicles transition through progressively larger sizes in the absence of exogenous hormonal stimulation. Only a relatively small number of genes displayed altered mRNA expression levels in any of the three cell types during this period. Most of the identified differentially expressed genes (DEGs) decreased in the granulosa cells or increased in the cumulus cells. Although the number of DEGs observed was small, these DEGs indicate predicted effects on distinct upstream regulators in the cumulus and granulosa cells. This study is particularly important because it shows for the first time the gene expression changes during antral follicle growth in a medically relevant model. NEW & NOTEWORTHY Changes in gene expression in oocytes, granulosa, and cumulus cells were determined in nonhuman primate antral stage ovarian follicles transitioning through progressively larger sizes without exogenous hormonal stimulation. Only a small number of genes displayed altered mRNA expression levels in any of the three cell types. Most of the differentially expressed genes (DEGs) decreased in granulosa cells or increased in cumulus cells. These results identified upstream regulators of antral follicle development.

Factors that determine nonresponse to immune checkpoint inhibitor (ICI) remain unclear. The protumor activities of cancer-associated fibroblasts (CAFs) suggest that they are potential therapeutic targets for cancer treatment. There is, however, a lack of CAF-related signature in predicting response to immunotherapy in gastric cancer (GC). Single-cell RNA sequencing (scRNA-seq) and RNA sequencing (RNA-seq) data of GC immunotherapy were downloaded from the Gene Expression Omnibus database. Bulk RNA-seq data were obtained from The Cancer Genome Atlas. The R package 'Seurat' was used for scRNA-seq data processing. Cellular infiltration, receptor-ligand interactions, and evolutionary trajectory analysis were further explored. Differentially expressed genes affecting overall survival were obtained using the limma package. Weighted Gene Correlation Network Analysis was used to identify key modules of immunotherapy nonresponder. Prognostic model was constructed by univariate Cox and least absolute contraction and selection operator analysis using the intersection of activated fibroblast genes (AFGs) with key module genes. The differences in clinicopathological features, immune microenvironment, immunotherapy prediction, and sensitivity to small molecule agents between the high- and low-risk groups were further investigated. Based on scRNA-seq, we finally identified 20 AFGs associations with the prognosis of GC patients. AFGs' high expression levels were correlated with both poor prognosis and tumor progression. Three genes ( FRZB , SPARC , and FKBP10 ) were identified as immunotherapy nonresponse-related fibroblast genes and used to construct the prognostic signature. This signature is an independent significant risk factor affecting the clinical outcomes of GC patients. Remarkably, there were more CD4 memory T cells, resting mast cells, and M2 macrophages infiltrating in the high-risk group, which was characterized by higher tumor immune exclusion. Moreover, patients with higher risk scores were more prone to not respond to immunotherapy but were more sensitive to various small molecule agents, such as memantine. In conclusion, this study constructed a fibroblast-associated ICI nonresponse gene signature, which could predict the response to immunotherapy. This study potentially revealed a novel way to overcome immune resistance in GC., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Recent studies have suggested that cell death may be involved in bone loss or the resolution of inflammation in periodontitis. Immunogenic cell death (ICD), a recently identified cell death pathway, may be involved in the development of this disease., Methods: By analyzing single-cell RNA sequencing (scRNA-seq) for periodontitis and scoring gene set activity, we identified cell populations associated with ICD, which were further verified by qPCR, enzyme linked immunosorbent assay (ELISA) and immunofluorescence (IF) staining. By combining the bulk transcriptome and applying machine learning methods, we identified several potential ICD-related hub genes, which were then used to build diagnostic models. Subsequently, consensus clustering analysis was performed to identify ICD-associated subtypes, and multiple bioinformatics algorithms were used to investigate differences in immune cells and pathways between subtypes. Finally, qPCR and immunohistochemical staining were performed to validate the accuracy of the models., Results: Single-cell gene set activity analysis found that in non-immune cells, fibroblasts had a higher ICD activity score, and KEGG results showed that fibroblasts were enriched in a variety of ICD-related pathways. qPCR, Elisa and IF further verified the accuracy of the results. From the bulk transcriptome, we identified 11 differentially expressed genes (DEGs) associated with ICD, and machine learning methods further identified 5 hub genes associated with ICD. Consensus cluster analysis based on these 5 genes showed that there were differences in immune cells and immune functions among subtypes associated with ICD. Finally, qPCR and immunohistochemistry confirmed the ability of these five genes as biomarkers for the diagnosis of periodontitis., Conclusion: Fibroblasts may be the main cell source of ICD in periodontitis. Adaptive immune responses driven by ICD may be one of the pathogenesis of periodontitis. Five key genes associated with ICD (ENTPD1, TLR4, LY96, PRF1 and P2RX7) may be diagnostic biomarkers of periodontitis and future therapeutic targets., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wu, Yin, Liang, Zhou, Hu, Yuan, Gu, Zhao, Gao, Cheng, Yang, Zhang, Wang, Sun and Shao.)