Search

BACKGROUND: The effect of vaccination on the epigenome remains poorly characterized. In previous research, we identified an association between seroprotection against influenza and DNA methylation at sites associated with the RIG-1 signaling pathway, which recognizes viral double-stranded RNA and leads to a type I interferon response. However, these studies did not fully account for confounding factors including age, gender, and BMI, along with changes in cell-type composition. RESULTS: Here, we studied the influenza vaccine response in a longitudinal cohort vaccinated over two consecutive years (2019-2020 and 2020-2021), using peripheral blood mononuclear cells and a targeted DNA methylation approach. To address the effects of multiple factors on the epigenome, we designed a multivariate multiple regression model that included seroprotection levels as quantified by the hemagglutination-inhibition (HAI) assay test. CONCLUSIONS: Our findings indicate that 179 methylation sites can be combined as potential signatures to predict seroprotection. These sites were not only enriched for genes involved in the regulation of the RIG-I signaling pathway, as found previously, but also enriched for other genes associated with innate immunity to viruses and the transcription factor binding sites of BRD4, which is known to impact T cell memory. We propose a model to suggest that the RIG-I pathway and BRD4 could potentially be modulated to improve immunization strategies.

Cell atlases serve as vital references for automating cell labeling in new samples, yet existing classification algorithms struggle with accuracy. Here we introduce SIMS (scalable, interpretable machine learning for single cell), a low-code data-efficient pipeline for single-cell RNA classification. We benchmark SIMS against datasets from different tissues and species. We demonstrate SIMS's efficacy in classifying cells in the brain, achieving high accuracy even with small training sets (

BACKGROUND: Disruptions in vascular formation attributable to chemical insults is a pivotal risk factor or potential etiology of developmental defects and various disease settings. Among the thousands of chemicals threatening human health, the highly concerning groups prevalent in the environment and detected in biological monitoring in the general population ought to be prioritized because of their high exposure risks. However, the impacts of a large number of environmental chemicals on vasculature are far from understood. The angioarchitecture complexity and technical limitations make it challenging to analyze the entire vasculature efficiently and identify subtle changes through a high-throughput in vivo assay. OBJECTIVES: We aimed to develop an automated morphometric approach for the vascular profile and assess the vascular morphology of healthconcerning environmental chemicals. METHODS: High-resolution images of the entire vasculature in Tg(fli1a:eGFP) zebrafish were collected using a high-content imaging platform. We established a deep learning-based quantitative framework, ECA-ResXUnet, combined with MATLAB to segment the vascular networks and extract features. Vessel scores based on the rates of morphological changes were calculated to rank vascular toxicity. Potential biomarkers were identified by vessel--endothelium-gene--disease integrative analysis. RESULTS: Whole-trunk blood vessels and the cerebral vasculature in larvae exposed to 150 representative chemicals were automatically segmented as comparable to human-level accuracy, with sensitivity and specificity of 95.56% and 95.81%, respectively. Chemical treatments led to heterogeneous vascular patterns manifested by 31 architecture indexes, and the common cardinal vein (CCV) was the most affected vessel. The antipsychotic medicine haloperidol, flame retardant 2,2-bis(chloromethyl)trimethylenebis[bis(2-chloroethyl) phosphate], and tert-butylphenyl diphenyl phosphate ranked as the top three in vessel scores. Pesticides accounted for the largest group, with a vessel score of [greater than or equal to]1, characterized by a remarkable inhibition of subintestinal venous plexus and delayed development of CCV. Multiple-concentration evaluation of nine per- and polyfluoroalkyl substances (PFAS) indicated a low-concentration effect on vascular impairment and a positive association between carbon chain length and benchmark concentration. Target vessel-directed single-cell RNA sequencing of fli1a (+) cells from larvae treated with k-cyhalothrin, perfluorohexanesulfonic acid, or benzylbutyl phthalate, along with vessel--endothelium-gene--disease integrative analysis, uncovered potential associations with vascular disorders and identified biomarker candidates. DISCUSSION: This study provides a novel paradigm for phenotype-driven screenings of vascular-disrupting chemicals by converging morphological and transcriptomic profiles at a high-resolution level, serving as a powerful tool for large-scale toxicity tests. Our approach and the high-quality morphometric data facilitate the precise evaluation of vascular effects caused by environmental chemicals. https://doi.org/10.1289/EHP13214, Introduction Vascular formation is one of the earliest essential steps during embryogenesis, and intact vasculature plays a crucial role in tissue homeostasis and disease throughout life. (1) Disturbance of vascular [...]

* Context.--In 2021 the World Health Organization distributed a new classification of central nervous system tumors that incorporated modern testing modalities in the diagnosis. Although universally accepted as a scientifically superior system, this schema has created controversy because its deployment globally is challenging in the best of circumstances and impossible in resource-poor health care ecosystems. Compounding this problem is the significant challenge that neuropathologists with expertise in central nervous system tumors are rare. Objective.--To demonstrate diagnostic use of simple unsupervised machine learning techniques using publicly available data sets. I also discuss some potential solutions to the deployment of neuropathology classification in health care ecosystems burdened by this classification schema. Data Sources.--The Cancer Genome Atlas RNA sequencing data from low-grade and high-grade gliomas. Conclusions.--Methylation-based classification will be unable to solve all diagnostic problems in neuropathology. Information theory quantifications generate focused workflows in pathology, resulting in prevention of ordering unnecessary tests and identifying biomarkers that facilitate diagnosis., The neuropathology community has overtly failed to deliver a 'deployable by general surgical pathologist' workflow for the diagnosis of primary central nervous system (CNS) tumors. This outcome is obviously unintentional, [...]

* Context.--Next-generation sequencing-based approaches using RNA have increasingly been used by clinical laboratories for the detection of fusion genes, intragenic rearrangements, and exon-skipping events. Correspondingly, the College of American Pathologists (CAP) has advanced RNA sequencing proficiency testing (PT) to ensure optimal performance of these assays. Objective.--To report on laboratory performance and practices of RNA sequencing for the detection of fusion genes, intragenic rearrangements, and exon-skipping events using CAP PT data from 8 mailings (2018-A through 2021-B). Design.--CAP PT RNA sequencing program results from 153 laboratories across 24 proficiency test specimens, interrogating 22 distinct engineered fusion transcripts, were analyzed for correct identification of the fusion event, associated performance variables, and laboratory practices. Results.--Overall, the 4-year program detection rate (sensitivity) was 95.5% (1486 of 1556 results). False-negative rates were 3.6% (53 of 1463) and 18.3% (17 of 93) for fusion gene and intragenic rearrangement/exon-skipping events, respectively. Only 19 false-positive results were reported among the 8 PT mailings, and most were likely the result of preanalytical or postanalytical errors. There were no practice characteristics (eg, instrumentation, sequencing method) significantly associated with the fusion detection results. Conclusions.--These data reveal a high overall sensitivity and specificity for fusion gene detection by participating laboratories using clinical RNA sequencing. Performance was comparable across all laboratories, regardless of methodology. The fraction of false-negative results for intragenic rearrangement/exon-skipping events was greater than that for the chimeric fusion genes. False-negative results could not be attributed to any specific practice characteristics., Fusion oncogenes represent valuable biomarkers that aid in diagnostic classification and prognostication, as well as serving as therapeutic targets. (1-4) Examples of structural alterations/mutations that may lead to chimeric fusions [...]

LncRNA-based control affects cardiac pathophysiologies like myocardial infarction, coronary artery disease, hypertrophy, and myotonic muscular dystrophy. This study used a gene-break transposon (GBT) to screen zebrafish (Danio rerio) for insertional mutagenesis. We identified three insertional mutants where the GBT captured a cardiac gene. One of the adult viable GBT mutants had bradycardia (heart arrhythmia) and enlarged cardiac chambers or hypertrophy; we named it bigheart. Bigheart mutant insertion maps to grin2bb or N-methyl D-aspartate receptor (NMDAR2B) gene intron 2 in reverse orientation. Rapid amplification of adjacent cDNA ends analysis suggested a new insertion site transcript in the intron 2 of grin2bb. Analysis of the RNA sequencing of wild-type zebrafish heart chambers revealed a possible new transcript at the insertion site. As this putative lncRNA transcript satisfies the canonical signatures, we called this transcript grin2bb associated RNA transcript (grin2bbART). Using in situ hybridization, we confirmed localized grin2bbART expression in the heart, central nervous system, and muscles in the developing embryos and wild-type adult zebrafish atrium and bulbus arteriosus. The bigheart mutant had reduced Grin2bbART expression. We showed that bigheart gene trap insertion excision reversed cardiac-specific arrhythmia and atrial hypertrophy and restored grin2bbART expression. Morpholino-mediated antisense downregulation of grin2bbART in wild-type zebrafish embryos mimicked bigheart mutants; this suggests grin2bbART is linked to bigheart. Cardiovascular tissues use Grin2bb as a calcium-permeable ion channel. Calcium imaging experiments performed on bigheart mutants indicated calcium mishandling in the heart. The bigheart cardiac transcriptome showed differential expression of calcium homeostasis, cardiac remodeling, and contraction genes. Western blot analysis highlighted Camk2d1 and Hdac1 overexpression. We propose that altered calcium activity due to disruption of grin2bbART, a putative lncRNA in bigheart, altered the Camk2d-Hdac pathway, causing heart arrhythmia and hypertrophy in zebrafish.

Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support. [Display omitted] • Astrocyte Gi-GPCR signaling corrects repetitive and anxiety behaviors in Sapap3 KO mice • Gi-GPCR signaling restores astrocyte morphology via the actin cytoskeleton • Astrocyte Gi-GPCR signaling rescues medium spiny neuron excitability • Improved phenotypes are accompanied with restored astrocyte homeostatic functions Soto et al. used multiple approaches to assess whether and how function is affected following astrocyte Gi-DREADD activation in a mouse model of compulsive and anxiety-related behaviors that are accompanied by astrocyte dysfunction. They found that Gi-DREADD activation restored decreased astrocyte morphology and homeostatic functions and led to the correction of compulsive and anxiety behaviors in obsessive-compulsive disorder (OCD)-related Sapap3 knockout mice. Soto et al. provide an astrocyte-specific pharmacological strategy for normalizing detrimental compulsive phenotypes related to OCD and potentially other disorders in which astrocyte morphology and homeostatic support are compromised. [ABSTRACT FROM AUTHOR]

Background: Botrytis cinerea is a broad-host-range pathogen causing gray mold disease and significant yield losses of numerous crops. However, the mechanisms underlying its rapid invasion and efficient killing of plant cells remain unclear. Results: In this study, we elucidated the dynamics of B. cinerea infection in Arabidopsis thaliana by live cell imaging and dual RNA sequencing. We found extensive transcriptional reprogramming events in both the pathogen and the host, which involved metabolic pathways, signaling cascades, and transcriptional regulation. For the pathogen, we identified 591 candidate effector proteins (CEPs) and comprehensively analyzed their co-expression, sequence similarity, and structural conservation. The results revealed temporal co-regulation patterns of these CEPs, indicating coordinated deployment of effectors during B. cinerea infection. Through functional screening of 48 selected CEPs in Nicotiana benthamiana, we identified 11 cell death-inducing proteins (CDIPs) in B. cinerea. Conclusions: The findings provide important insights into the transcriptional dynamics and effector biology driving B. cinerea pathogenesis. The rapid infection of this pathogen involves the temporal co-regulation of CEPs and the prominent role of CDIPs in host cell death. This work highlights significant changes in gene expression associated with gray mold disease, underscoring the importance of a diverse repertoire of effectors crucial for successful infection. [ABSTRACT FROM AUTHOR]

Background: Monocyte Chemotactic Protein 1-Induced Protein 1 (MCPIP1, also called Regnase-1) is a negative modulator of inflammation with tumor-suppressive properties. Mice with keratinocyte-specific deletion of the Zc3h12a gene, encoding MCPIP1, (Mcpip1eKO mice) are more susceptible to the development of epidermal papillomas initiated by 7,12-dimethylbenz[a]-anthracene (DMBA) and promoted by 2-O-tetradecanoylphorbol-13-acetate (TPA). Methods: The aim of this study was to investigate the MCPIP1 RNase-dependent microRNA (miRNA)‒mRNA regulatory network in chemically induced squamous cell carcinoma (SCC)-like skin papillomas. Next-generation sequencing (NGS) coupled with bioinformatic analysis was used to shortlist the MCPIP1-dependent changes in protein-coding genes and miRNAs. The expression levels of the selected miRNAs were analyzed by quantitative PCR in human keratinocytes with MCPIP1 silencing. Functional studies were performed in human keratinocytes transfected with appropriate miRNA mimics. The DIANA-microT-CDS algorithm and DIANA-TarBase v7 database were used to predict potential target genes and identify the experimentally validated targets of differentially expressed (DE) miRNAs. Results: RNA sequencing (RNA-Seq) analysis of control and Mcpip1eKO DMBA/TPA-induced papillomas revealed transcriptome changes, with 2400 DE protein-coding genes and 33 DE miRNAs. The expression of miR-223-3p, miR-376c-3p, and miR-139-5p was confirmed to be dependent on MCPIP1 activity in both murine and human models. We showed that MCPIP1 directly regulates the expression of miR-376c-3p via direct cleavage of the corresponding precursor miRNA. The pro-proliferative activity of miR-223-3p, miR-376c-3p, and miR-139-5p was experimentally confirmed in SCC-like keratinocytes. Bioinformatic prediction of the mRNA targets of the DE-miRNAs revealed 416 genes as putative targets of the 18 upregulated miRNAs and 425 genes as putative targets of the 15 downregulated miRNAs. Further analyses revealed the murine interactions that are conserved in humans. Functional analysis indicated that during the development of cutaneous SCC, the most important pathways/processes mediated by the miRNA‒mRNA MCPIP1-dependent network are the regulation of inflammatory processes, epithelial cell proliferation, Wnt signaling, and miRNA transcription. Conclusions: Loss of MCPIP1 modulates the expression profiles of 33 miRNAs in chemically induced Mcpip1eKO papillomas, and these changes directly affect the miRNA‒mRNA network and the modulation of pathways and processes related to carcinogenesis. [ABSTRACT FROM AUTHOR]

Spinal cord injury (SCI) triggers a cascade of intricate molecular and cellular changes that determine the outcome. In this study, we resolve the spatiotemporal organization of the injured mouse spinal cord and quantitatively assess in situ cell-cell communication following SCI. By analyzing existing single-cell RNA sequencing datasets alongside our spatial data, we delineate a subpopulation of Igfbp2 -expressing astrocytes that migrate from the white matter (WM) to gray matter (GM) and become reactive upon SCI, termed Astro-GMii. Further, Igfbp2 upregulation promotes astrocyte migration, proliferation, and reactivity, and the secreted IGFBP2 protein fosters neurite outgrowth. Finally, we show that IGFBP2 significantly reduces neuronal loss and remarkably improves the functional recovery in a mouse model of SCI in vivo. Together, this study not only provides a comprehensive molecular atlas of SCI but also exemplifies how this rich resource can be applied to endow cells and genes with functional insight and therapeutic potential. [Display omitted] • A comprehensive and dynamic molecular atlas of the spinal cord after acute injury • In situ depiction of cell-cell communication following spinal cord injury (SCI) • Spatial delineation of SCI-induced, WM-to-GM migrating Igfbp2 + -astrocytes • IGFBP2 treatment improves neuronal survival and functional outcomes of SCI in vivo Wang et al. conducted a comprehensive spatial transcriptomic analysis that provides a rich resource and a useful bioinformatic toolkit for disentangling the spatiotemporal re-organization of the injured spinal cord. They further spatially identified a subpopulation of injury-induced migrating astrocytes that secrete IGFBP2, which exhibits potent neuroprotection following SCI. [ABSTRACT FROM AUTHOR]

Background: MicroRNA host gene (MIRHG) lncRNA is a particular lncRNA subclass that can perform both typical and atypical lncRNA functions. The biological function of MIRHG lncRNA MIR194-2HG in cancer is poorly understood. Methods: Loss-of-function studies were performed in vivo and in vitro to reveal the biological function of MIR194-2HG in GC. MicroRNA PCR array, northern blotting, RNA sequencing, chromatin immunoprecipitation, and rescue assays were conducted to uncover the molecular mechanism of MIR194-2HG. Results: In this study, we reported an atypical lncRNA function of MIR194-2HG in GC. MIR194-2HG downregulation was clinically associated with malignant progression and poor prognosis in GC. Functional assays confirmed that MIR194-2HG knockdown significantly promoted GC proliferation and metastasis in vitro and in vivo. Mechanismically, MIR194-2HG was required for the biogenesis of miR-194 and miR-192, which were reported to be tumor-suppressor genes in GC. Moreover, hepatocyte nuclear factor HNF4A directly activated the transcription of MIR194-2HG and its derived miR-194 and miR-192. Meanwhile, BTF3L4 was proved to be a common target gene of miR-192 and miR-194. Rescue assay further confirmed that MIR194-2HG knockdown promotes GC progression through maintaining BTF3L4 overexpression in a miR-194/192-dependent manner. Conclusion: The dysregulated MIR194-2HG/BTF3L4 axis is responsible for GC progression. Targeting HNF4A to inhibit miR-192/194 expression may be a promising strategy for overcoming GC. [ABSTRACT FROM AUTHOR]

This study aims to investigate the interplay between tumor-associated endothelial cells (TECs) and immune cells within the tumor microenvironment (TME) and its impact on tumor prognosis. We conducted single-cell RNA sequencing (scRNA-seq) of tumor, normal, and lymph node tissues obtained from intrahepatic cholangiocarcinoma (ICC) patients to reveal the role of TECs in tumor angiogenesis and their significant heterogeneity. Meanwhile, we identified genes highly expressed in TECs and constructed TEC signatures (TEC.Sig). Next, we calculated TEC scores of samples based on TEC.Sig. Patients with higher TEC scores exhibited a higher frequency of KRAS mutations, which was associated with increased infiltration of neutrophils and immature dendritic cells (iDCs), and decreased numbers of natural killer (NK), CD4 + T, and CD8 + T effector memory (Tem) cells, indicating an inflammation-dominated immunosuppressive phenotype. In contrast, BAP1 mutations and CXCL12 overexpression showed a contrasting trend. Spatial transcriptomics analysis and histological experiments further confirmed that TECs interacted with various tumor-killing immune cells through the CXCL12/CXCR4 axis. Multiple tumor immunotherapy datasets confirmed that the TEC.Sig could predict patient responses to immunotherapy. The TEC score is a promising and reliable biomarker for predicting genetic mutations and prognosis in ICC patients. Enhancing the regulation of the CXCL12/CXCR4 signaling pathway may represent a potential novel therapeutic target for ICC treatment. [ABSTRACT FROM AUTHOR]

The MECOM locus is a gene frequently amplified in high-grade serous ovarian carcinoma (HGSOC). Nevertheless, the body of research examining the associations among MECOM transcripts, patient prognosis, and their role in modulating the tumor immune microenvironment (TIME) remains sparse, particularly in large cohorts. This study assessed the expression of MECOM transcripts in 352 HGSOC patients and 88 normal ovarian tissues from the combined GTEx/TCGA database. Using resources such as the UCSC Genome Browser, Ensembl, and NextProt, two transcripts corresponding to classical protein isoforms from MECOM were identified. Cox proportional hazards regression analysis, Kaplan-Meier survival curves, and a comprehensive TIME evaluation algorithm were employed to elucidate the connections between the expression levels of these transcripts and both patient prognosis and TIME status. Chromatin Immunoprecipitation sequencing (ChIP-seq) data for the two protein isoforms, as well as RNA sequencing data post-targeted silencing, were analyzed to identify potential regulatory targets of the different transcription factors. Elevated expression of the MECOM isoform transcripts was correlated with poorer survival in HGSOC patients, potentially through the modulation of cancer-associated fibroblasts (CAFs) and immunosuppressive cell populations. In contrast, higher levels of EVI1 isoform transcripts were linked to enhanced survival, possibly due to the regulation of CD8+ T cells, macrophages, and a reduction in the expression of JUN protein, or its DNA-binding activity on downstream genes. Diverse protein isoforms derived from MECOM were found to differentially affect the survival and tumor development in ovarian cancer patients through specific mechanisms. Investigating the molecular mechanisms underlying disease pathogenesis and identifying potential drug target proteins at the level of splice variant isoforms were deemed crucial. [ABSTRACT FROM AUTHOR]

ABSTRACT Canine mucosal melanoma (CMM) is the most common oral malignancy in dogs and is significantly more aggressive than its cutaneous counterpart (CCM), yet the reasons for this disparity remain unclear. Cancer‐associated stroma (CAS) plays a crucial role in tumour progression, but a detailed understanding of CAS in canine melanoma is missing. To assess stromal reprogramming, we analysed CAS from 21 CMM, 14 CCM and normal stroma from 10 skin and 9 oral mucosa samples by laser‐capture microdissection followed by RNA sequencing. Results were assessed in relation to subtypes, prognostic factors including mitotic count (MC), ulceration, necrosis, pigmentation and immune cell infiltration (CD3, CD20 and CD68), scored using immunohistochemistry and RNA in situ hybridisation. Stromal reprogramming was evident in both subtypes but significantly more pronounced in CMM. Immune‐excluded tumours exhibited higher MC than desert/cold ones. MC strongly correlated with genes associated with B‐cells, T‐helper cells and CTLA4 in CCM, suggesting CAS reprogramming to depend on tumour malignancy. Finally, we identify an immune‐suppressive stromal signature in a subset of CMM characterised by the downregulation of key immune checkpoints and pathways. Together, these findings provide a solid foundation for understanding the role of CAS in canine melanoma, specific to cutaneous and mucosal subtypes. [ABSTRACT FROM AUTHOR]

Tay-Sachs disease (TSD) is a rare genetic disorder with diverse clinical manifestations, often leading to underdiagnosis due to symptom similarities with other neurological conditions. In this study, we aimed to identify the genetic mutations underlying late-onset TSD in a 27-year-old patient with progressive neurological symptoms. Whole-exome sequencing revealed two hexA gene mutations associated with TSD: a previously known variant, c.805G > A (p.Gly269Ser), and a novel splice-site mutation, c.346 + 2dupT. Through clinical assessments, genetic analysis, and functional investigations--including RNA sequencing and enzymatic activity assays--we confirmed the pathogenicity of the novel mutation. Our findings highlight the efficacy of advanced genomic technologies in diagnosing intricate genetic disorders and emphasize the significance of functional validation to confirm the effects of mutations. Identifying compound heterozygous mutations in the hexA gene also provides insight into Mendelian inheritance patterns. This case highlights the diagnostic challenges posed by overlapping clinical phenotypes and emphasizes the need for increased genetic awareness among clinicians. Accurate diagnosis of TSD has significant implications for patients and their families, allowing for informed genetic counseling and guiding clinical management decisions. While current treatment options are limited, timely and accurate diagnosis holds promise for future research and therapeutic interventions. This study highlights the value of a multidisciplinary approach in exploring the molecular basis of complex genetic diseases and informing clinical decisions. [ABSTRACT FROM AUTHOR]

Introduction: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by persistent synovitis, systemic inflammation, and autoantibody production. This study aims to explore the role of lactylation in plasma cells and its impact on RA pathogenesis. Methods: We utilized single-cell RNA sequencing (scRNA-seq) data and applied bioinformatics and machine learning techniques. A total of 10,163 cells were retained for analysis after quality control. Clustering analysis identified 13 cell clusters, with plasma cells displaying the highest lactylation scores. We performed pathway enrichment analysis to examine metabolic activity, such as oxidative phosphorylation and glycolysis, in highly lactylated plasma cells. Additionally, we employed 134 machine learning algorithms to identify seven core lactylation-promoting genes and constructed a diagnostic model with an average AUC of 0.918. Results: The RA lactylation score (RAlac_score) was significantly elevated in RA patients and positively correlated with immune cell infiltration and immune checkpoint molecule expression. Differential expression analysis between two plasma cell clusters revealed distinct metabolic and immunological profiles, with cluster 2 demonstrating increased immune activity and extracellular matrix interactions. qRT-PCR validation confirmed that NDUFB3, NGLY1, and SLC25A4 are highly expressed in RA. Conclusion: This study highlights the critical role of lactylation in plasma cells for RA pathogenesis and identifies potential biomarkers and therapeutic targets, which may offer insights for future therapeutic strategies. [ABSTRACT FROM AUTHOR]

Formalin-fixed paraffin-embedded (FFPE) samples are valuable but underutilized in single-cell omics research due to their low RNA quality. In this study, leveraging a recent advance in single-cell genomic technology, we introduce snPATHO-seq, a versatile method to derive high-quality single-nucleus transcriptomic data from FFPE samples. We benchmarked the performance of the snPATHO-seq workflow against existing 10x 3' and Flex assays designed for frozen or fresh samples and highlighted the consistency in snRNA-seq data produced by all workflows. The snPATHO-seq workflow also demonstrated high robustness when tested across a wide range of healthy and diseased FFPE tissue samples. When combined with FFPE spatial transcriptomic technologies such as FFPE Visium, the snPATHO-seq provides a multi-modal sampling approach for FFPE samples, allowing more comprehensive transcriptomic characterization. A combination of an FFPE nuclei preparation protocol and a probe-based transcriptomic profiling technique enables snRNA-seq characterization of archival human FFPE tissues, holding promise for retrospective studies involving aged clinical cohorts. [ABSTRACT FROM AUTHOR]

Background: The potential of microglia as a target for Alzheimer's disease (AD) treatment is promising, yet the clinical and pathological diversity within microglia, driven by genetic factors, poses a significant challenge. Subtyping AD is imperative to enable precise and effective treatment strategies. However, existing subtyping methods fail to comprehensively address the intricate complexities of AD pathogenesis, particularly concerning genetic risk factors. To address this gap, we have employed systems biology approaches for AD subtyping and identified potential therapeutic targets. Methods: We constructed patient-specific microglial molecular regulatory network models by utilizing existing literature and single-cell RNA sequencing data. The combination of large-scale computer simulations and dynamic network analysis enabled us to subtype AD patients according to their distinct molecular regulatory mechanisms. For each identified subtype, we suggested optimal targets for effective AD treatment. Results: To investigate heterogeneity in AD and identify potential therapeutic targets, we constructed a microglia molecular regulatory network model. The network model incorporated 20 known risk factors and crucial signaling pathways associated with microglial functionality, such as inflammation, anti-inflammation, phagocytosis, and autophagy. Probabilistic simulations with patient-specific genomic data and subsequent dynamics analysis revealed nine distinct AD subtypes characterized by core feedback mechanisms involving SPI1, CASS4, and MEF2C. Moreover, we identified PICALM, MEF2C, and LAT2 as common therapeutic targets among several subtypes. Furthermore, we clarified the reasons for the previous contradictory experimental results that suggested both the activation and inhibition of AKT or INPP5D could activate AD through dynamic analysis. This highlights the multifaceted nature of microglial network regulation. Conclusions: These results offer a means to classify AD patients by their genetic risk factors, clarify inconsistent experimental findings, and advance the development of treatments tailored to individual genotypes for AD. [ABSTRACT FROM AUTHOR]

The article "Whole Blood Transcriptomics Identifies Differences in Innate Immune Pathway Expression in Infants at Risk for Peanut Allergy" explores the differential gene expression in infants at risk for peanut allergy. The study aims to analyze the transcriptomic signature of infants at risk for peanut allergy using whole blood RNA sequencing. Results suggest that disruption of the innate immune response may play a role in peanut allergy development, highlighting the importance of understanding immune developmental defects associated with food allergies. Further investigation into the innate immune response in infants at high risk of peanut allergy may have implications for the development of clinical reactivity in sensitized individuals. [Extracted from the article]

The development of acute respiratory distress syndrome (ARDS) in sepsis is associated with substantial morbidity and mortality. However, the molecular pathogenesis underlying sepsis‐induced ARDS remains elusive. Neutrophil heterogeneity and dysfunction contribute to uncontrolled inflammation in patients with ARDS. A specific subset of neutrophils undergoing reverse transendothelial migration (rTEM), which is characterized by an activated phenotype, is implicated in the systemic dissemination of inflammation. Using single‐cell RNA sequencing (scRNA‐seq), it identified functionally activated neutrophils exhibiting the rTEM phenotype in the lung of a sepsis mouse model using cecal ligation and puncture. The prevalence of neutrophils with the rTEM phenotype is elevated in the blood of patients with sepsis‐associated ARDS and is positively correlated with disease severity. Mechanically, scRNA‐seq and proteomic analys revealed that inflamed endothelial cell (EC) released extracellular vesicles (EVs) enriched in karyopherin subunit beta‐1 (KPNB1), promoting abluminal‐to‐luminal neutrophil rTEM. Additionally, EC‐derived EVs are elevated and positively correlated with the proportion of rTEM neutrophils in clinical sepsis. Collectively, EC‐derived EV is identified as a critical regulator of neutrophil rTEM, providing insights into the contribution of rTEM neutrophils to sepsis‐associated lung injury. [ABSTRACT FROM AUTHOR]

The transition from acute kidney injury (AKI) to chronic kidney disease (CKD) is a critical clinical issue. Although previous studies have suggested macrophages as a key player in promoting inflammation and fibrosis during this transition, the heterogeneity and dynamic characterization of macrophages are still poorly understood. Here, we used integrated single‐cell RNA sequencing and spatial transcriptomic to characterize the spatiotemporal heterogeneity of macrophages in murine AKI‐to‐CKD model of unilateral ischemia‐reperfusion injury. A marked increase in macrophage infiltration at day 1 was followed by a second peak at day 14 post AKI. Spatiotemporal profiling revealed that injured tubules and macrophages co‐localized early after AKI, whereas in late chronic stages had spatial proximity to fibroblasts. Further pseudotime analysis revealed two distinct lineages of macrophages in this transition: renal resident macrophages differentiated into the pro‐repair subsets, whereas infiltrating monocyte‐derived macrophages contributed to chronic inflammation and fibrosis. A novel macrophage subset, extracellular matrix remodeling‐associated macrophages (EAMs) originating from monocytes, linked to renal fibrogenesis and communicated with fibroblasts via insulin‐like growth factors (IGF) signalling. In sum, our study identified the spatiotemporal dynamics of macrophage heterogeneity with a unique subset of EAMs in AKI‐to‐CKD transition, which could be a potential therapeutic target for preventing CKD development. [ABSTRACT FROM AUTHOR]

NK2 Homeobox 1 (NKX2‐1) is a well‐characterized pathological marker that delineates lung adenocarcinoma (LUAD) progression. The advancement of LUAD is influenced by the immune tumor microenvironment through paracrine signaling. However, the involvement of NKX2‐1 in modeling the tumor immune microenvironment is still unclear. Here, the downregulation of NKX2‐1 is observed in high‐grade LUAD. Meanwhile, single‐cell RNA sequencing and Visium in situ capturing profiling revealed the recruitment and infiltration of neutrophils in orthotopic syngeneic tumors exhibiting strong cell‐cell communication through the activation of CXCLs/CXCR2 signaling. The depletion of NKX2‐1 triggered the expression and secretion of CXCL1, CXCL2, CXCL3, and CXCL5 in LUAD cells. Chemokine secretion is analyzed by chemokine array and validated by qRT‐PCR. ATAC‐seq revealed the restrictive regulation of NKX2‐1 on the promoters of CXCL1, CXCL2, and CXCL5 genes. This phenomenon led to increased tumor growth, and conversely, tumor growth decreased when inhibited by the CXCR2 antagonist SB225002. This study unveils how NKX2‐1 modulates the infiltration of tumor‐promoting neutrophils by inhibiting CXCLs/CXCR2‐dependent mechanisms. Hence, targeting CXCR2 in NKX2‐1‐low tumors is a potential antitumor therapy that may improve LUAD patient outcomes. [ABSTRACT FROM AUTHOR]

The continental bedrock contains groundwater-bearing fractures that are home to microbial populations that are vital in mediating the Earth's biogeochemical cycles. However, their diversity is poorly understood due to the difficulty of obtaining samples from this environment. Here, a groundwater-bearing fracture at 975 m depth was isolated by employing packers in order to characterize the microbial community via metagenomes combined with prokaryotic and eukaryotic marker genes (16S and 18S ribosomal RNA gene). Genome-resolved analyses revealed a community dominated by sulfate-reducing Bacillota, predominantly represented by Candidatus Desulforudis audaxviator and with Wood-Ljungdahl as the most prevalent pathway for inorganic carbon fixation. Moreover, the eukaryotic community had a considerable diversity and was comprised of mainly flatworms, chlorophytes, crustaceans, ochrophytes, and fungi. These findings support the important role of the Bacillota, with the sulfate reducer Candidatus Desulforudis audaxviator as its main representative, as primary producers in the often energy-limited groundwaters of the continental subsurface. Metagenomics combined with 16S and 18S ribosomal RNA sequencing on groundwater retrieved from a packer-isolated borehole provide insights into the microbial community at 975 m depth. [ABSTRACT FROM AUTHOR]

Background: Lack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act. Methods: We applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC). Results: Methylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation. Conclusions: We did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third most common malignancy, with increasing prevalence and mortality. How the ethoxy-erianin phosphate (EBTP) mediates CRC development remains unclear. Therefore, the current study evaluated the effects of EBTP on the proliferation, migration, and angiogenesis of CRC cells using CCK-8, Wound-healing, Transwell, and Tube formation assays. RNA sequencing and molecular docking techniques helped predict that EBTP could inhibit angiogenesis by regulating PIK3R2 expression while clarifying the mechanism behind EBTP-mediated CRC angiogenesis. Subsequently, several in vitro experiments indicated that PIK3R2 overexpression significantly improved the proliferation, migration, and angiogenesis of CRC cells while knocking down PIK3R2 expression inhibited their proliferation, migration, and angiogenesis. Simultaneously, PIK3R2 expression in CRC cells gradually decreased with increased EBTP concentration and action duration. Moreover, PIK3R2 overexpression in CRC cells could reverse the inhibitory EBTP effect in angiogenesis. Mouse experiments also depicted that EBTP inhibited CRC angiogenesis by down-regulating PIK3R2 expression. In addition, EBTP could inhibit PI3K/AKT pathway activity and indirectly control PIK3R2 expression through the lncRNA TMPO-AS1/miR-126-3p axis. Our findings highlighted that EBTP could inhibit CRC angiogenesis using the TMPO-AS1/miR-126-3p/PIK3R2/PI3k/AKT axis, providing a novel strategy for anti-angiogenic therapy in CRC. [ABSTRACT FROM AUTHOR]

Triple‐negative breast cancer (TNBC), a highly aggressive form of breast cancer, currently lacks targeted therapies and is associated with high recurrence rates in medical practice. In this study, the effectiveness of a specially developed magnetic nanodrug, namely doxorubicin (DOX)‐loaded Fe3O4 vortex magnetic nanoannulars coated with a cancer cell membrane (DOX‐VMAs@CM), against cancer under a varying low‐frequency magnetic field (MF), is investigated. This advanced nanodrug can specifically target and accumulate in tumors. Additionally, it considerably improves tumor suppression compared with that of DOX chemotherapy alone, indicating the possibility of homologous targeting of the cancer cell membrane and the vibrating effect of the MF. The DOX‐VMAs@CM nanoparticles (NPs) exhibit exceptional safety characteristics because of their ability to specifically target tumors. The results of RNA sequencing suggest that the potential mechanisms may involve cellular respiration, cell cycle, and ferroptosis regulation. Altogether, chemotherapy is successfully optimized by implementing a delivery system and MF intervention, and the developed DOX‐VMAs@CM NPs hold significant potential for use in the clinical treatment of TNBC. [ABSTRACT FROM AUTHOR]

Tumor-infiltrating B cells play a significant role in tumor development, progression, and prognosis, yet a comprehensive classification system is lacking. To address this gap, we present a pan-cancer single-cell RNA sequencing (scRNA-seq) atlas of tumor-infiltrating B and plasma cells across a large sample cohort. We identify key B cell subset signatures, revealing distinct subpopulations and highlighting the heterogeneity and functional diversity of these cells in the tumor microenvironment. We explore associations between B cell subsets and checkpoint inhibitor therapy responses, finding subset-specific effects on overall response. Additionally, we examine B and T cell crosstalk, identifying unique ligand-receptor pairs for specific B cell subsets, spatially validated. This comprehensive dataset serves as a valuable resource, providing a detailed atlas that enhances the understanding of B cell complexity in tumors and opens new avenues for research and therapeutic strategies. [Display omitted] • A scRNA-seq atlas identifies ten clusters of intratumoral B and plasma cells • Specific B cell subsets are associated with checkpoint inhibitor therapy responses • Atlas-derived B cell gene signatures validated in spatial analyses • Publicly available Shiny tool enables users to interactively explore the atlas Fitzsimons et al. present a comprehensive pan-cancer single-cell RNA-seq atlas of intratumoral B cells, identifying ten distinct B and plasma cell clusters. They uncover associations with checkpoint inhibitor therapy response, explore B-T cell crosstalk, validate findings with spatial data, and offer an interactive tool for further research. [ABSTRACT FROM AUTHOR]

Aims: Porocarcinoma is a malignant sweat gland tumour differentiated toward the upper part of the sweat duct and may arise from the transformation of a preexisting benign poroma. In 2019, Sekine et al. demonstrated the presence of YAP1::MAML2 and YAP1::NUTM1 fusions in most poromas and porocarcinomas. Recently, our group identified PAK2‐fusions in a subset of benign poromas. Herein we report a series of 12 porocarcinoma cases harbouring PAK1/2/3 fusions. Methods and Results: Five patients were male and the median age was 79 years (ranges: 59–95). Tumours were located on the trunk (n = 7), on the thigh (n = 3), neck (n = 1), or groin area (n = 1). Four patients developed distant metastases. Microscopically, seven cases harboured a benign poroma component and a malignant invasive part. Ductal formations were observed in all, while infundibular/horn cysts and cells with vacuolated cytoplasm were detected in seven and six tumours, respectively. In three cases, the invasive component consisted of a proliferation of elongated cells, some of which formed pseudovascular spaces, whereas the others harboured a predominant solid or trabecular growth pattern. Immunohistochemical staining for CEA and EMA confirmed the presence of ducts. Focal androgen receptor expression was detected in three specimens. Whole RNA sequencing evidenced LAMTOR1::PAK1 (n = 2), ZDHHC5::PAK1 (n = 2), DLG1::PAK2, CTDSP1::PAK1, CTNND1::PAK1, SSR1::PAK3, CTNNA1::PAK2, RNF13::PAK2, ROBO1::PAK2, and CD47::PAK2. Activating mutation of HRAS (G13V, n = 3, G13R, n = 1, Q61L, n = 2) was present in six cases. Conclusion: Our study suggests that PAK1/2/3 fusions is the oncogenic driver of a subset of porocarcinomas lacking YAP1 rearrangement. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening organ malfunction induced by an imbalanced immunological reaction to infection in the host. Many studies have utilized traditional RNA sequencing (RNA-seq) data to identify important biological targets to predict sepsis prognosis. However, alterations in core cells and functional status cannot be effectively detected in sepsis patients. The goal of this study was to identify key cells through single-cell RNA-seq (scRNA-seq), and combine bulk RNA-seq data and multiple algorithm analysis to construct a stable prognostic model for sepsis. The scRNA-seq and bulk RNA-seq data from sepsis patients were collected from the Gene Expression Omnibus (GEO) database. The R package "Seurat" was used to process the scRNA-seq data. Cell communication was investigated using the R package "CellChat". The pseudo-time of the cells was calculated using the R package "monocle". The R package "limma" was used to identify differentially expressed genes (DEGs) between the sepsis group and the control group. Weighted gene correlation network analysis (WGCNA) was used to identify critical modules. Eight kinds of machine learning and 90 algorithm combinations were used to construct the prognostic model for sepsis. Quantitative real-time PCR (qRT‒PCR) was performed to determine the expression of key genes in the cecal ligation and puncture (CLP)-induced sepsis mouse model. The immunological status and related properties of DEGs were then investigated in the high- and low-risk groups delineated by the model. By combining the scRNA-seq data from nine samples, 13 clusters and 9 cell types were identified. CellChat analysis revealed that the number and strength of interactions between platelets and a variety of cells increased. We identified key platelet genes from the scRNA-seq data and combined these genes and the results of differential analysis and WGCNA of the bulk RNA-seq data. After univariate Cox regression analysis, we calculated the Cindex of the model constructed by the combination of 90 algorithms, and we finally determined the "CoxBoost + Lasso" combination. Multivariate Cox regression was used to construct the final prognostic model. The qRT-PCR results revealed significant differences in five key prognostic genes between the CLP and sham groups. The data was classified into high- and low-risk groups based on the model score. The high-risk group had a poorer survival rate and less immune infiltration. We identified the importance of platelets in sepsis patients through scRNA-seq, and established prognostic models with key genes that were identified via scRNA-seq combined with bulk RNA-seq analysis. The results of this model were closely associated with patient survival rates and immunological status and this model is useful for the prognostic management of sepsis. [ABSTRACT FROM AUTHOR]

Introduction: The balanced fatty acid profile of olive oil not only enhances its stability but also contributes to its positive effects on health, making it a valuable dietary choice. Olive oil's high content of unsaturated fatty acids and low content of saturated fatty acids contribute to its beneficial effects on cardiovascular diseases and cancer. The quantities of these fatty acids in olive oil may fluctuate due to various factors, with genotype being a crucial determinant of the oil's quality. Methods: This study investigated the genetic basis of oil quality by comparing the transcriptome of two Iranian cultivars with contrasting oil profiles: Mari, known for its high oleic acid content, and Shengeh, characterized by high linoleic acid at Jaén index four. Results and discussion: Gas chromatography confirmed a significant difference in fatty acid composition between the two cultivars. Mari exhibited significantly higher oleic acid content (78.48%) compared to Shengeh (48.05%), while linoleic acid content was significantly lower in Mari (4.76%) than in Shengeh (26.69%). Using RNA sequencing at Jaén index four, we analyzed genes involved in fatty acid biosynthesis. Differential expression analysis identified 2775 genes showing statistically significant differences between the cultivars. Investigating these genes across nine fundamental pathways involved in oil quality led to the identification of 25 effective genes. Further analysis revealed 78 transcription factors and 95 transcription binding sites involved in oil quality, with BPC6 and RGA emerging as unique factors. This research provides a comprehensive understanding of the genetic and molecular mechanisms underlying oil quality in olive cultivars. The findings have practical implications for olive breeders and producers, potentially streamlining cultivar selection processes and contributing to the production of high-quality olive oil. [ABSTRACT FROM AUTHOR]

Introduction: Gliomas are the most common and aggressive type of primary brain tumor, with a poor prognosis despite current treatment approaches. Understanding the molecular mechanisms underlying glioma development and progression is critical for improving therapies and patient outcomes. Methods: The current study comprehensively analyzed large-scale single-cell RNA sequencing and bulk RNA sequencing of glioma samples. By utilizing a series of advanced computational methods, this integrative approach identified the gene UPP1 (Uridine Phosphorylase 1) as a novel driver of glioma tumorigenesis and immune evasion. Results: High levels of UPP1 were linked to poor survival rates in patients. Functional experiments demonstrated that UPP1 promotes tumor cell proliferation and invasion and suppresses anti-tumor immune responses. Moreover, UPP1 was found to be an effective predictor of mutation patterns, drug response, immunotherapy effectiveness, and immune characteristics. Conclusions: These findings highlight the power of combining diverse machine learning methods to identify valuable clinical markers involved in glioma pathogenesis. Identifying UPP1 as a tumor growth and immune escape driver may be a promising therapeutic target for this devastating disease. [ABSTRACT FROM AUTHOR]

Introduction: Development of an effective vaccine against tuberculosis is a critical step towards reducing the global burden of disease. A therapeutic vaccine might also reduce the high rate of TB recurrence and help address the challenges of drug-resistant strains. ID93+GLA-SE is a candidate subunit vaccine that will soon be evaluated in a phase 2b efficacy trial for prevention of recurrent TB among patients undergoing TB treatment. ID93+GLA-SE vaccination was shown to elicit robust CD4+ T cell and IgG antibody responses among recently treated TB patients in the TBVPX-203 Phase 2a study (NCT02465216), but the mechanisms underlying these responses are not well understood. Methods: In this study we used specimens from TBVPX-203 participants to describe the changes in peripheral blood gene expression that occur after ID93 +GLA-SE vaccination. Results: Analyses revealed several distinct modules of co-varying genes that were either up-or down-regulated after vaccination, including genes associated with innate immune pathways at 3 days post-vaccination and genes associated with lymphocyte expansion and B cell activation at 7 days post-vaccination. Notably, the regulation of these gene modules was affected by the dose schedule and by participant sex, and early innate gene signatures were correlated with the ID93-specific CD4+ T cell response. vDiscussion: The results provide insight into the complex interplay of the innate and adaptive arms of the immune system in developing responses to vaccination with ID93+GLA-SE and demonstrate how dosing and schedule can affect vaccine responses. [ABSTRACT FROM AUTHOR]

How immune cells are spatiotemporally coordinated in the lung to effectively monitor, respond to, and resolve infection and inflammation in primed form needs to be fully illustrated. Here we apply immunocartography, a high-resolution technique that integrates spatial and single-cell RNA sequencing (scRNA-seq) through deconvolution and co-localization analyses, to the SARS-CoV-2-infected Syrian hamster model. We generate a comprehensive transcriptome map of the whole process of pulmonary infection from physiological condition, infection initiation, severe pneumonia to natural recovery at organ scale and single-cell resolution, with 142,965 cells and 45 lung lobes from 25 hamsters at 5 time points. Integrative analysis identifies that alveolar dendritic cell–T cell immunity hubs, where Ccr7+Ido1+ dendritic cells, Cd160+Cd8+ T cells, and Tnfrsf4+Cd4+ T cells physiologically co-localize, rapidly expand during SARS-CoV-2 infection, eliminate SARS-CoV-2 with the aid of Slamf9+ macrophages, and then restore to physiological levels after viral clearance. We verify the presence of these cell subpopulations in the immunity hubs in normal and SARS-CoV-2-infected hACE2 mouse models, as well as in publicly available human scRNA-seq datasets, demonstrating the potential broad relevance of our findings in lung immunity. [ABSTRACT FROM AUTHOR]

Introduction: Nitraria tangutorum Bobr., a prominent xerophytic shrub, exhibits remarkable adaptability to harsh environment and plays a significant part in preventing desertification in northwest China owing to its exceptional drought and salinity tolerance. Methods: To investigate the drought-resistant mechanism underlying N. tangutorum, we treated 8-week-old seedlings with polyethylene glycol (PEG)- 6000 (20%, m/m) to induce drought stress. 27 samples from different tissues (leaves, roots and stems) of N. tangutorum at 0, 6 and 24 h after drought stress treatment were sequenced using PacBio single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing to obtain a comprehensive transcriptome. Results: The PacBio SMRT sequencing generated 44,829 non-redundant transcripts and provided valuable reference gene information. In leaves, roots and stems, we identified 1162, 2024 and 232 differentially expressed genes (DEGs), respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that plant hormone signaling and mitogen-activated protein kinase (MAPK) cascade played a pivotal role in transmitting stress signals throughout the whole N. tangutorum plant following drought stress. The interconversion of starch and sucrose, as well as the biosynthesis of amino acid and lignin, may represent adaptive strategies employed by N. tangutorum to effectively cope with drought. Transcription factor analysis showed that AP2/ERF-ERF, WRKY, bHLH, NAC and MYB families were mainly involved in the regulation of drought response genes. Furthermore, eight physiological indexes, including content of proline, hydrogen peroxide (H2O2), malondialdehyde (MDA), total amino acid and soluble sugar, and activities of three antioxidant enzymes were all investigate after PEG treatment, elucidating the drought tolerance mechanism from physiological perspective. The weighted gene co-expression network analysis (WGCNA) identified several hub genes serve as key regulator in response to drought through hormone participation, ROS cleavage, glycolysis, TF regulation in N. tangutorum. Discussion: These findings enlarge genomic resources and facilitate research in the discovery of novel genes research in N. tangutorum, thereby establishing a foundation for investigating the drought resistance mechanism of xerophyte. [ABSTRACT FROM AUTHOR]

Introduction: The advent of RNA sequencing (RNA-Seq) has significantly advanced our understanding of the transcriptomic landscape, revealing intricate gene expression patterns across biological states and conditions. However, the complexity and volume of RNA-Seq data pose challenges in identifying differentially expressed genes (DEGs), critical for understanding the molecular basis of diseases like cancer. Methods: We introduce a novel Machine Learning-Enhanced Genomic Data Analysis Pipeline (ML-GAP) that incorporates autoencoders and innovative data augmentation strategies, notably the MixUp method, to overcome these challenges. By creating synthetic training examples through a linear combination of input pairs and their labels, MixUp significantly enhances the model's ability to generalize from the training data to unseen examples. Results: Our results demonstrate the ML-GAP's superiority in accuracy, efficiency, and insights, particularly crediting the MixUp method for its substantial contribution to the pipeline's effectiveness, advancing greatly genomic data analysis and setting a new standard in the field. Discussion: This, in turn, suggests that ML-GAP has the potential to perform more accurate detection of DEGs but also offers new avenues for therapeutic intervention and research. By integrating explainable artificial intelligence (XAI) techniques, ML-GAP ensures a transparent and interpretable analysis, highlighting the significance of identified genetic markers. [ABSTRACT FROM AUTHOR]

Background: Exhaustion is a key factor that influences the efficacy of chimeric antigen receptor T (CAR-T) cells. Our previous study demonstrated that a bromodomain protein 4 (BRD4) inhibitor can revise the phenotype and function of exhausted T cells from leukemia patients. This study aims to elucidate the mechanism by which a BRD4 inhibitor reduces CAR-T cell exhaustion using single-cell RNA sequencing (scRNA-Seq). Methods: Exhausted CD123-specific CAR-T cells were prepared by co-culture with CD123 antigen-positive MV411 cells. After elimination of MV411 cells and upregulation of inhibitory receptors on the surface, exhausted CAR-T cells were treated with a BRD4 inhibitor (JQ1) for 72 h. The CAR-T cells were subsequently isolated, and scRNA-Seq was conducted to characterize phenotypic and functional changes in JQ1-treated cells. Results: Both the proportion of exhausted CD8+ CAR-T cells and the exhausted score of CAR-T cells decreased in JQ1-treated compared with control-treated cells. Moreover, JQ1 treatment led to a higher proportion of naïve, memory, and progenitor exhausted CD8+ CAR-T cells as opposed to terminal exhausted CD8+ CAR-T cells accompanied by enhanced proliferation, differentiation, and activation capacities. Additionally, with JQ1 treatment, BATF activity and expression in naïve, memory, and progenitor exhausted CD8+ CAR-T cells decreased, whereas EGR1 activity and expression increased. Interestingly, AML patients with higher EGR1 and EGR1 target gene ssGSEA scores, coupled with lower BATF and BATF target gene ssGSEA scores, had the best prognosis. Conclusions: Our study reveals that a BRD4 inhibitor can reduce CAR-T cell exhaustion and block exhausted T cell terminal differentiation by downregulating BATF activity and expression together with upregulating EGR1 activity and expression, presenting an approach for improving the effectiveness of CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

Introduction: Illicit drug use, particularly the synthetic opioid fentanyl, presents a significant global health challenge. Previous studies have shown that fentanyl enhances viral replication; yet, the mechanisms by which it affects HIV pathogenesis remain unclear. This study investigated the impact of fentanyl on HIV replication in CD4+ T lymphocytes. Methods: CD4+ T lymphocytes from HIV-negative donors were activated, infected with HIVNL4-3, and treated with fentanyl. HIV proviral DNA and p24 antigen expression were quantified using real-time PCR and ELISA, respectively. Single-cell RNA libraries were analyzed to identify differentially expressed genes. Results: Results indicated that fentanyl treatment increased HIV p24 expression and proviral DNA levels, and naltrexone mitigated these effects. Single-cell RNAseq analysis identified significantly altered gene expression in CD4+ T lymphocytes. Discussion: The results of our findings suggest that fentanyl promotes HIV replication ex vivo, emphasizing the need for a deeper understanding of opioid-virus interactions to develop better treatment strategies for individuals with HIV and opioid use disorder. [ABSTRACT FROM AUTHOR]

Background: Glioma is the predominant malignant brain tumor that lacks effective treatment options due to its shielding by the blood-brain barrier (BBB). Astrocytes play a role in the development of glioma, yet the diverse cellular composition of astrocytoma has not been thoroughly researched. Methods: We examined the internal diversity of seven distinct astrocytoma subgroups through single-cell RNA sequencing (scRNA-seq), pinpointed crucial subgroups using CytoTRACE, monocle2 pseudotime analysis, and slingshot pseudotime analysis, employed various techniques to identify critical subgroups, and delved into cellular communication analysis. Then, we combined the clinical information of GBM patients and used bulk RNA sequencing (bulk RNA-seq) to analyze the prognostic impact of the relevant molecules on GBM patients, and we performed in vitro experiments for validation. Results: The analysis of the current study revealed that C0 IGFBP7+ Glioma cells were a noteworthy subpopulation of astrocytoma, influencing the differentiation and progression of astrocytoma. A predictive model was developed to categorize patients into high- and low-scoring groups based on the IGFBP7 Risk Score (IGRS), with survival analysis revealing a poorer prognosis for the high-IGRS group. Analysis of immune cell infiltration, identification of genes with differential expression, various enrichment analyses, assessment of copy number variations, and evaluation of drug susceptibility were conducted, all of which highlighted their significant influence on the prognosis of astrocytoma. Conclusion: This research enhances comprehension of the diverse cell composition of astrocytoma, delves into the various factors impacting the prognosis of astrocytoma, and offers fresh perspectives on treating glioma. [ABSTRACT FROM AUTHOR]

The efficacy and side effects of radiotherapy (RT) depend on parameters like dose and the volume of irradiated tissue. RT induces modulations of the tumor immune microenvironment (TIME) that are dependent on the dose. Low dose RT (LDRT, i.e., single doses of 0.5–2 Gy) has been shown to promote immune infiltration into the tumor. Here we hypothesize that partial tumor irradiation combining the immunostimulatory/non-lethal properties of LDRT with cell killing/shrinkage properties of high dose RT (HDRT) within the same tumor mass could enhance anti-tumor responses when combined with immunomodulators. In models of colorectal and breast cancer in immunocompetent female mice, partial irradiation (PI) with millimetric precision to deliver LDRT (2 Gy) and HDRT (16 Gy) within the same tumor induces substantial tumor control when combined with anti-PD1. Using flow cytometry, cytokine profiling and single-cell RNA sequencing, we identify a crosstalk between the TIME of the differentially irradiated tumor volumes. PI reshapes tumor-infiltrating CD8+ T cells into more cytotoxic and interferon-activated phenotypes but also increases the infiltration of pro-tumor neutrophils driven by CXCR2. The combination of the CXCR2 antagonist SB225002 with PD1 blockade and PI improves tumor control and mouse survival. Our results suggest a strategy to reduce RT toxicity and improve the therapeutic index of RT and immune checkpoint combinations. Low dose-radiation therapy (LDRT) can promote anti-tumor immune responses. Here the authors propose to combine the immunostimulatory properties of LDRT with the cell killing/shrinkage properties of high dose RT within the same tumor mass as a strategy to optimize RT-induced anti-tumor immune responses. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) remains the primary cause of mortality among gynecological malignancies, and the identification of reliable molecular biomarkers to prognosticate OC outcomes is yet to be achieved. The gene palmitoyl protein thioesterase 2 (PPT2), which has been sparsely studied in OC, was closely associated with metabolism. This study aimed to determine the association between PPT2 expression, prognosis, immune infiltration, and potential molecular mechanisms in OC. We obtained the RNA-seq and clinical data from The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx) and Gene Expression Omnibus (GEO) databases, then Kaplan-Meier analysis, univariate Cox regression, multivariate Cox regression, nomogram, and calibration were conducted to assess and verify the role of PPT2. Gene set enrichment analysis (GSEA) was used to figure out the closely correlated pathways with PPT2. Overexpression experiment was performed to explore the function of PPT2. Our findings showed that PPT2 mRNA expression was apparent down-regulation in OC tissue compared to normal ovarian tissues in TCGA, GTEx datasets, and GEO datasets. This differential expression was also confirmed in our in-house datasets at both the mRNA and protein levels. Decreased PPT2 expression correlated with lower survival rates in TCGA, several GEO datasets, and our in-house datasets. Multivariate analysis revealed that PPT2 was an independent factor in predicting better outcomes for OC patients in TCGA and GEO. A negative correlation was revealed between immune infiltration and PPT2 expression through Single-sample GSEA (ssGSEA). Additionally, PPT2 was negatively correlated with an up-regulated immune score, stromal score, and estimate score, suggesting that patients with low PPT2 expression might benefit more from immunotherapy. Numerous chemical agents showed lower IC50 in patients with high PPT2 expression. In single-cell RNA sequencing (scRNA-seq) analysis of several OC datasets, we found PPT2 was mainly expressed in endothelial cells. Furthermore, we found that PPT2 inhibited OC cell proliferation in vitro. Our results demonstrated that PPT2 was considered a favorable prognostic biomarker for OC and may be vital in predicting response to immunotherapy and chemotherapy. Further research was needed to fully understand the relationship between PPT2 and immunotherapy efficacy in OC patients. [ABSTRACT FROM AUTHOR]

The incidence of central nervous system (CNS) disease has persistently increased over the last several years. There is an urgent need for effective methods to improve the cure rates of CNS disease. However, the precise molecular basis underlying the development and progression of major CNS diseases remains elusive. A complete molecular map will contribute to research on CNS disease treatment strategies. Emerging technologies such as single-cell RNA sequencing (scRNA-seq) and Spatial Transcriptomics (ST) are potent tools for exploring the molecular complexity, cell heterogeneity, and functional specificity of CNS disease. scRNA-seq and ST can provide insights into the disease at cellular and spatial transcription levels. This review presents a survey of scRNA-seq and ST studies on CNS diseases, such as chronic neurodegenerative diseases, acute CNS injuries, and others. These studies offer novel perspectives in treating and diagnosing CNS diseases by discovering new cell types or subtypes associated with the disease, proposing new pathophysiological mechanisms, uncovering novel therapeutic targets, and identifying putative biomarkers. Implications of scRNA-seq and ST for CNS diseases. A comprehensive understanding of cellular information and spatial context using scRNA-seq and ST, helping to elucidate the biological functions, the intricate connection between crucial regulatory pathways, and cell communication after CNS diseases. [ABSTRACT FROM AUTHOR]

Chemical modifications to mRNA respond dynamically to environmental cues and are important modulators of gene expression. Nanopore direct RNA sequencing has been applied for assessing the presence of pseudouridine (ψ) modifications through basecalling errors and signal analysis. These approaches strongly depend on the sequence context around the modification, and the occupancies derived from these measurements are not quantitative. In this work, we combine direct RNA sequencing of synthetic RNAs bearing site-specific modifications and supervised machine learning models (ModQuant) to achieve near-analytical, site-specific ψ quantification. Our models demonstrate that the ionic current signal features important for accurate ψ classification are sequence dependent and encompass information extending beyond n + 2 and n − 2 nucleotides from the ψ site. This is contradictory to current models, which assume that accurate ψ classification can be achieved with signal information confined to the 5-nucleotide k-mer window (n + 2 and n − 2 nucleotides from the ψ site). We applied our models to quantitatively profile ψ occupancy in five mRNA sites in datasets from seven human cell lines, demonstrating conserved and variable sites. Our study motivates a wider pipeline that uses ground-truth RNA control sets with site-specific modifications for quantitative profiling of RNA modifications. The ModQuant pipeline and guide are freely available at https://github.com/wanunulab/ModQuant. [ABSTRACT FROM AUTHOR]

Oncolytic virotherapy is emerging as a promising therapeutic avenue for cancer treatment, harnessing both innate and tumor-specific immune responses for targeted tumor elimination. In this study, we present a novel oncolytic virus (oHSV1-IL15B) derived from herpes simplex virus-1 (HSV-1), armed with IL-15/IL-15Rα complex, with a focus on treating colon cancer combined with oncolytic HSV-1 expressing anti-PD-1 antibody (oHSV1-aPD1). Results from our study reveal that recombinant oHSV-1 virus equipped with IL-15/IL-15Rα complex exhibited significant anti-tumor effects in a murine CT26 colon adenocarcinoma model. Notably, oHSV1-IL15B combined with oHSV-1-aPD1 demonstrates superior tumor inhibition and prolonged overall survival compared to oHSV1-mock and monotherapy groups. Further exploration highlights the impact of oHSV1-IL15B, oHSV-1-aPD1 and combined group on antitumor capacity, revealing a substantial increase in CD8+ T and CD4+ T cell proportions of CT26-bearing BALB/c mice and promoting apoptosis in tumor tissue. The study emphasizes the pivotal role of cytotoxic CD8+T cells in oncolytic virotherapy, demonstrating that recombinant oHSV1-IL15B combined with oncolytic HSV-1-aPD1 induces a robust tumor-specific T cell response. RNA sequence analysis highlighted oHSV1-IL15B combined with oHSV1-aPD1 improved tumors immune microenvironment on immune response, antiviral response-related genes and apoptosis-related genes, which contributed to anti-tumor immunotherapy. The findings underscore the promising antitumor activity achieved through the combination of IL-15/IL-15Rα complex and anti-PD-1 antibody with oHSV-1. This research opens avenues for diverse therapeutic strategies, suggesting the potential of synergistically utilizing cytokines and anti-PD-1 antibody with oncolytic viruses to enhance immunotherapy for cancer management. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sncRNAs) derived from tRNAs are known as tRNA-derived small RNAs (tsRNAs). These tsRNAs are further categorized into tRNA-derived fragments (tRFs) and tRNA halves (tiRNAs), which play significant roles in the various molecular mechanisms underlying certain human diseases. However, the generation of tsRNAs and their potential roles during Dengue virus (DENV) infection is not yet known. Here, we performed small RNA sequencing to identify the generation and alterations in tsRNAs expression profiles of DENV-infected Huh7 cells. Upon DENV infection, tRNA fragmentation was found to be increased. We identified a significant number of differentially expressed tsRNAs during DENV infection. Interestingly, the 3′tRF population showed upregulation, while the i-tRF population exhibited downregulation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to analyze the impact of differentially expressed tsRNAs on DENV pathogenesis. Our results suggest that differentially expressed tsRNAs are involved in transcriptional regulation via RNA polymerase II promoter and metabolic pathways. Overall, our study contributes significantly to our understanding of the roles played by tsRNAs in the complex dynamics of DENV infection. [ABSTRACT FROM AUTHOR]

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas. [ABSTRACT FROM AUTHOR]

Metabolic RNA labeling‐based time‐resolved single‐cell RNA sequencing (scRNA‐seq) has provided unprecedented tools to dissect the temporal dynamics and the complex gene regulatory networks of gene expression. However, this technology fails to reveal the spatial organization of cells in tissues, which also regulates the gene expression by intercellular communication. Herein, it is demonstrated that integrating time‐resolved scRNA‐seq with spatial transcriptomics is a new paradigm for spatiotemporal analysis. Metabolic RNA labeling‐based time‐resolved Well‐TEMP‐seq is first applied to profile the transcriptional dynamics of glioblastoma (GBM) cells and discover two potential pathways of EZH2‐mediated mesenchymal transition in GBM. With spatial transcriptomics, it is further revealed that the crosstalk between CCL2+ malignant cells and IL10+ tumor‐associated macrophages in the tumor microenvironment through an EZH2‐FOSL2‐CCL2 axis contributes to the mesenchymal transition in GBM. These discoveries show the power of integrative spatiotemporal scRNA‐seq to elucidate the complex gene regulatory mechanism and advance the understanding of cellular processes in disease. [ABSTRACT FROM AUTHOR]

Background: Flowers can be a source of essential oils used in the manufacture of substances with high economic value. The ethylene response factor (ERF) gene family plays a key role in regulating secondary metabolite biosynthesis in plants. However, until now, little has been known about the involvement of ERF transcription factors (TFs) in floral terpenoid biosynthesis. Results: In this study, an aromatic plant, Primula forbesii Franch., was used as research material to explore the key regulatory effects of PfERF106 on the biosynthesis of terpenoids. PfERF106, which encodes an IXb group ERF transcription factor, exhibited a consistent expression trend in the flowers of P. forbesii and was transcriptionally induced by exogenous ethylene. Transient silencing of PfERF106 in P. forbesii significantly decreased the relative contents of key floral terpenes, including (z)-β-ocimene, sabinene, β-pinene, γ-terpinene, linalool, eremophilene, α-ionone, and α-terpineol. In contrast, constitutive overexpression of PfERF106 in transgenic tobacco significantly increased the relative contents of key floral terpenes, including cis-3-hexen-1-ol, linalool, caryophyllene, cembrene, and sclareol. RNA sequencing of petals of PfERF106-silenced plants and empty-vector control plants revealed 52,711 expressed unigenes and 9,060 differentially expressed genes (DEGs). KEGG annotation analysis revealed that the DEGs were enriched for involvement in secondary metabolic biosynthetic pathways, including monoterpene and diterpene synthesis. Notably, 10 downregulated DEGs were determined to be the downstream target genes of PfERF106 affecting the biosynthesis of terpenoids in P. forbesii. Conclusion: This study characterized the key positive regulatory effects of PfERF106 on the biosynthesis of terpenoids, indicating high-quality genetic resources for aroma improvement in P. forbesii. Thus, this study advances the artificial and precise directional regulation of metabolic engineering of aromatic substances. [ABSTRACT FROM AUTHOR]

Tenosynovial giant cell tumor (TGCT) is a rare type of tumor that originates from the synovium of joints and tendon sheaths. It is characterized by recurring genetic abnormalities, often involving the CSF1 gene. Common symptoms include pain and swelling, which are not specific to TGCT, so MRI and a pathological biopsy are needed for an accurate diagnosis. We report the case of a 45-year-old man who experienced painful swelling in his right hip for six months. Initially, this was diagnosed as Erdheim-Chester disease. However, whole exome sequencing (WES) and RNA-Sequencing revealed a CSF1::GAPDHP64 fusion, leading to a revised diagnosis of TGCT. The patient was treated with pegylated interferon and imatinib, which resulted in stable disease after three months. Single-cell transcriptome analysis identified seven distinct cell clusters, revealing that neoplastic cells expressing CSF1 attract macrophages. Analysis of ligand-receptor interactions showed significant communication between neoplastic cells and macrophages mediated by CSF1 and CSF1R. Our findings emphasize the importance of comprehensive molecular analysis in diagnosing and treating rare malignancies like TGCT. [ABSTRACT FROM AUTHOR]

The sequencing of a kidney sample (KW2013002) from a stranded Megaptera novaeangliae (Humpback whale) calf is the first chromosome-level reference genome for this species1. The calf, a 457 cm and 2,500 lbs male, was found stranded in Hawai'i Kai, HI, in 2013 and was marked as abandoned/orphaned. In 2023, 1 g of kidney was sequenced with PacBio long-read DNA sequencing, chromatin conformation capture (Hi-C), RNA sequencing, and mitochondrial sequencing to comprehensively characterize the genome and transcriptome of M. novaeangliae. Data validation includes a synteny analysis, mitochondrial annotation, and a comparison of BUSCO scores (scaffold v. reference genome and Balaenoptera musculus (Blue whale) v. M. novaeangliae). BUSCO analysis was performed on an M. novaeangliae scaffold-level assembly to determine genomic completeness of the reference genome, with a scaffold BUSCO score of 91.2% versus a score of 95.4%. Synteny analysis was performed using the B. musculus genome as comparison to determine chromosome-level coverage and structure. Further, a time-based phylogenetic tree was constructed using the sequenced data and publicly available genomes. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) exhibits significant genetic and epigenetic diversity, evolving into sub-clonal populations with varied metastatic potentials and treatment responses. Predicting metastatic disease in CRC patients remains challenging, underscoring the need for reliable biomarkers. While most research on therapeutic targets and biomarkers has focused on proteins, non-coding RNAs such as long non-coding RNAs (lncRNAs) comprise most of the transcriptome and demonstrate superior tissue- and cancer-specific expression. We utilised spatial transcriptomics to investigate lncRNAs in CRC tumours, offering more precise cell-type-specific expression data compared to bulk RNA sequencing. Our analysis identified 301 lncRNAs linked to malignant CRC regions, which we validated with public data. Further validation using RNA–FISH revealed three lncRNAs (LINC01978, PLAC4, and LINC01303) that are detectable in stage II tumours but not in normal epithelium and are upregulated in metastatic tissues. These lncRNAs hold potential as biomarkers for early risk assessment of metastatic disease. [ABSTRACT FROM AUTHOR]