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BACKGROUND: Preeclampsia is a serious disease of pregnancy that lacks early diagnosis methods or effective treatment, except delivery. Dysregulated uterine immune cells and spiral arteries are implicated in preeclampsia, but the mechanistic link remains unclear. METHODS: Single-cell RNA sequencing and spatial transcriptomics were used to identify immune cell subsets associated with preeclampsia. Cell-based studies and animal models including conditional knockout mice and a new preeclampsia mouse model induced by recombinant mouse galectin-9 were applied to validate the pathogenic role of a CD11chigh subpopulation of decidual macrophages (dMφ) and to determine its underlying regulatory mechanisms in preeclampsia. A retrospective preeclampsia cohort study was performed to determine the value of circulating galectin-9 in predicting preeclampsia. RESULTS: We discovered a distinct CD11chigh dMφ subset that inhibits spiral artery remodeling in preeclampsia. The proinflammatory CD11chigh dMφ exhibits perivascular enrichment in the decidua from patients with preeclampsia. We also showed that trophoblast-derived galectin-9 activates CD11chigh dMφ by means of CD44 binding to suppress spiral artery remodeling. In 3 independent preeclampsia mouse models, placental and plasma galectin-9 levels were elevated. Galectin-9 administration in mice induces preeclampsia-like phenotypes with increased CD11chigh dMφ and defective spiral arteries, whereas galectin-9 blockade or macrophage-specific CD44 deletion prevents such phenotypes. In pregnant women, increased circulating galectin-9 levels in the first trimester and at 16 to 20 gestational weeks can predict subsequent preeclampsia onset. CONCLUSIONS: These findings highlight a key role of a distinct perivascular inflammatory CD11chigh dMφ subpopulation in the pathogenesis of preeclampsia. CD11chigh dMφ activated by increased galectin-9 from trophoblasts suppresses uterine spiral artery remodeling, contributing to preeclampsia. Increased circulating galectin-9 may be a biomarker for preeclampsia prediction and intervention. [ABSTRACT FROM AUTHOR]

ABSTRACTExtensive research has explored the role of gut microbiota in colorectal cancer (CRC). Nonetheless, metatranscriptomic studies investigating the in situ functional implications of host-microbe interactions in CRC are scarce. Therefore, we characterized the influence of CRC core pathogens and biofilms on the tumor microenvironment (TME) in 40 CRC, paired normal, and healthy tissue biopsies using fluorescence in situ hybridization (FISH) and dual-RNA sequencing. FISH revealed that Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. Dual-RNA sequencing demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors in CRC samples with high bacterial activity. In addition, bacterial activity correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells in CRC samples. Specifically, Bacteroides fragilis and Fusobacterium nucleatum correlated with the infiltration of neutrophils and CD4+ T-cells, respectively. The collective bacterial activity/biomass appeared to exert a more significant influence on the TME than core pathogens, underscoring the intricate interplay between gut microbiota and CRC. These results emphasize how biofilms and core pathogens shape the immune phenotype and TME in CRC while highlighting the need to extend the bacterial scope beyond CRC pathogens to advance our understanding and identify treatment targets.

This article discusses the limitations of using rodent models in heart transplantation research and highlights the shift towards using human specimens for discovery research. The authors focus on a study that uses single-nucleus RNA sequencing to investigate the cellular landscape of transplanted pediatric hearts. The study identifies important candidate mediators of heart transplant failure, such as graft-infiltrating macrophages and natural killer cells. The authors also explore the dynamics of immune cells within the failing transplanted heart and highlight the potential therapeutic targets that could be investigated further. However, the study has limitations due to the small sample size and the difficulty in obtaining healthy heart transplant tissue. Overall, the article emphasizes the potential of single-cell sequencing and other molecular pathology techniques in advancing our understanding of heart transplantation. [Extracted from the article]

Objective To summarize the clinical features and treatment of genetic epilepsy with febrile seizures plus (GEFS+) caused by SCN1A gene mutations, to improve the understanding of SCN1A gene mutations, and to provide a basis for the clinical diagnosis and treatment of this disease. Methods Clinical data were collected from a child with GEFS+ and her family members who attended Department of Neurology in our hospital, and the third-generation gene sequencing technique was used to perform genetic analysis and validation of peripheral blood DNA of the child and her parents and aunt. Results A girl, aged 7 years, was admitted to the hospital due to "paroxysmal seizures lasting for more than 4 years", with loss of consciousness during seizures and more seizures during pyrexia. The seizures increased after the use of oxcarbazepine and were controlled after the admi-nistration of sodium valproate. Her father had a history of seizures, while her aunt had no clinical symptoms. Third-generation gene sequencing showed the same heterozygous mutation, c.4787G>A(p.R1596H), in the SCN1A gene in the child and her father and aunt. Conclusion The onset of GEFS+ is associated with SCN1A gene mutations, but there is heterogeneity in clinical manifestations among patients in one family, and sodium channel blockers as anti-epileptic drugs will aggravate epileptic seizures in GEFS+.

Background: Irritable bowel syndrome (IBS) is a functional bowel disorder (FBD). Objectives: To assess the therapeutic effects of paeoniflorin (PF) on IBS in rats. Method: Sixty male Sprague–Dawley rats were randomly divided into normal, model, positive drug, low-dose PF, medium-dose PF and high-dose PF groups (n = 10). After gavage for 2 consecutive weeks, the effect of PF on abdominal pain symptoms was assessed based on the abdominal withdrawal reflex (AWR) score, fecal water content and pathological changes in colon tissues. D-lactate, interleukin-1β (IL-1β), transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay, and phosphorylated nuclear factor kappa B (p-NF-κB) p65 was detected by Western blotting. The abundance and diversity changes of intestinal flora were explored using 16S ribosomal RNA sequencing. Result: In PF groups, the mucosal morphology of colon tissues was intact, and the glands were arranged neatly and structured clearly, without obvious inflammatory cell infiltration. Compared with the model group, PF groups had significantly elevated pain threshold, and mRNA and protein levels of zonula occludens-1 (ZO-1) and occludin, decreased AWR score at 20 mmHg pressure, fecal water content, mRNA levels of IL-1β, TGF-β, and TNF-α, protein level of p-NF-κB p65 and level of serum D-lactate, and reduced levels of serum IL-1β, TGF-β, and TNF-α (p < 0.05, p < 0.01). PF groups had higher abundance of Lactobacillus, Akkermansia, Alistipes, and Bacteroides, but lower abundance of Desulfovibrio, Parasutterella, and Enterococcus than those of the model group. Conclusions: PF exerts therapeutic effects on IBS in rats probably by regulating the intestinal flora, and then up-regulating the expressions of ZO-1 and occludin in colon tissue while down-regulating the levels of IL-1β, TGF-β, TNF-α, D-lactate and p-NF-κB p65. [ABSTRACT FROM AUTHOR]

Background: Transplant arteriosclerosis is a major complication in long-term survivors of heart transplantation. Increased lymph flow from donor heart to host lymph nodes has been reported to play a role in transplant arteriosclerosis, but how lymphangiogenesis affects this process is unknown. Methods: Vascular allografts were transplanted among various combinations of mice, including wild-type, Lyve1 -CreERT2;R26-tdTomato, CAG -Cre-tdTomato, severe combined immune deficiency, Ccr2 KO, Foxn1 KO, and lghm / lghd KO mice. Whole-mount staining and 3-dimensional reconstruction identified lymphatic vessels within the grafted arteries. Lineage tracing strategies delineated the cellular origin of lymphatic endothelial cells. Adeno-associated viral vectors and a selective inhibitor were used to regulate lymphangiogenesis. Results: Lymphangiogenesis within allograft vessels began at the anastomotic sites and extended from preexisting lymphatic vessels in the host. Tertiary lymphatic organs were identified in transplanted arteries at the anastomotic site and lymphatic vessels expressing CCL21 (chemokine [C-C motif] ligand 21) were associated with these immune structures. Fibroblasts in the vascular allografts released VEGF-C (vascular endothelial growth factor C), which stimulated lymphangiogenesis into the grafts. Inhibition of VEGF-C signaling inhibited lymphangiogenesis, neointima formation, and adventitial fibrosis of vascular allografts. These studies identified VEGF-C released from fibroblasts as a signal stimulating lymphangiogenesis extending from the host into the vascular allografts. Conclusions: Formation of lymphatic vessels plays a key role in the immune response to vascular transplantation. The inhibition of lymphangiogenesis may be a novel approach to prevent transplant arteriosclerosis. [ABSTRACT FROM AUTHOR]

Background: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations.Methods: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection.Results: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival.Conclusions: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions. [ABSTRACT FROM AUTHOR]

Accumulation of alpha-synuclein (α-syn) is a key pathological hallmark of synucleinopathies and has been shown to negatively impact neuronal function and activity. α-syn is an important factor contributing to astrocyte overactivation, though the effect of astrocyte overactivation on neurons remains unclear. Single-cell RNA sequencing data of mouse brain frontal cortex and midbrain from Hua-Syn (A53T) and wild type mice were utilized from the GEO database. Enrichment analysis, protein-protein interaction networks, and cell-cell interaction networks all indicated enhanced communication between astrocytes and neurons, along with the involvement of TNF and inflammation-related signaling pathways. In vitro experiments were performed to further explore the mechanism of neurotoxicity in astrocyte-neuron crosstalk. Astrocytes were treated by α-syn, neuronal TNFR1 receptors were antagonized by R-7050, and the cells were co-cultured after 24 h treatment. ELISA results revealed that cytokines such as TNF-α and IL-6 were significantly upregulated in astrocytes following the endocytosis of α-syn. Immunofluorescence (IF) showed neuronal dendritic reduction, axon elongation and increased co-localisation of TNFR1 receptor expression. Western blot showed up-regulation of PKR, P-eIF2α and ATF4 protein expression. Conversely, after antagonizing neuronal TNFR1 receptors with the R-7050 chemical inhibitor, neuronal synaptic structure was significantly restored and the expression of PKR, P-eIF2α and ATF4 was down-regulated. In summary, TNF-α acts as a signaling molecule mediating the up-regulated astrocyte-neuron crosstalk, providing new insights into the pathogenesis of α-syn-related neurological disorders., 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 B.V. All rights reserved.)

Objective: Osteoporosis (OP) is a disease characterized by decreased bone mass, deteriorated microstructure, and increased fragility and fracture risk. The diagnosis and prevention of OP and its complications have become major public health challenges. Therefore, exploring the complex ecological connections between the immune and skeletal systems may provide new insights for clinical prevention and treatment strategies., Methods: First, we performed single-cell RNA sequencing on human lumbar lamina tissue and conducted clustering and subgroup analysis of quality-controlled single-cell transcriptome data to identify target subgroups. Subsequently, enrichment analysis and pseudotime analysis were performed. In addition, we conducted in-depth studies on the gene regulatory network between different cell subgroups and the communication between bone immune cells., Results: In this study, we identified several cell subgroups that may be involved in the progression of OP. For example, the CCL4 + NKT and CXCL8 + neutrophils subgroups promote OP progression by mediating an inflammatory environment that disrupts bone homeostasis, and the MNDA + Mac subgroup promotes osteoclast differentiation to promote OP. Moreover, the TNFAIP6 + Obl, NR4A2 + B and HMGN2 + erythrocyte subgroups promoted the balance of bone metabolism and suppressed OP. In the cell communication network, Obl closely interacts with immune cell subgroups through the CXCR4-CXCL12, CTGF-ITGB2, and TNFSF14-TNFRSF14 axes., Conclusion: Our research revealed specific subgroups and intercellular interactions that play crucial roles in the pathogenesis of OP, providing potential new insights for more precise therapeutic interventions for OP., 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 B.V. All rights reserved.)

Background and Objectives: Neutrophils, underestimated in multiple sclerosis (MS), are gaining increased attention for their significant functions in patients with MS and the experimental autoimmune encephalomyelitis (EAE) animal model. However, the precise role of neutrophils in cervical lymph nodes (CLNs), the primary CNS-draining lymph nodes where the autoimmune response is initiated during the progression of EAE, remains poorly understood., Methods: Applying single-cell RNA sequencing (scRNA-seq), we constructed a comprehensive immune cell atlas of CLNs during development of EAE. Through this atlas, we concentrated on and uncovered the transcriptional landscape, phenotypic and functional heterogeneity of neutrophils, and their crosstalk with immune cells within CLNs in the neuroinflammatory processes in EAE., Results: Notably, we observed a substantial increase in the neutrophil population in EAE mice, with a particular emphasis on the significant rise within the CLNs. Neutrophils in CLNs were categorized into 3 subtypes, and we explored the specific roles and developmental trajectories of each distinct neutrophil subtype. Neutrophils were found to engage in extensive interactions with other immune cells, playing crucial roles in T-cell activation. Moreover, our findings highlighted the strong migratory ability of neutrophils to CLNs, partly regulated by triggering the receptor expressed on myeloid cells 1 (TREM-1). Inhibiting TREM1 with LR12 prevents neutrophil migration both in vivo and in vitro. In addition, in patients with MS, we confirmed an increase in peripheral neutrophils with an upregulation of TREM-1., Discussion: Our research provides a comprehensive and precise single-cell atlas of CLNs in EAE, highlighting the role of neutrophils in regulating the periphery immune response. In addition, TREM-1 emerged as an essential regulator of neutrophil migration to CLNs, holding promise as a potential therapeutic target in MS.

Aim: Endothelial cells (EDs) play a key role in angiogenesis and are associated with granulomatous lesions in patients with chronic apical periodontitis (CAP). This study aimed to investigate the diversity of EDs using single-cell ribonucleic acid sequencing (scRNA-seq) and to evaluate the regulation of intercellular adhesion molecule 1 (ICAM1) on the ferroptosis-related protein, prostaglandin-endoperoxide synthase 2 (PTGS2), in CAP., Methodology: EDs from the uploaded scRNA-seq data of five CAP samples (GSE181688 and GSE197680) were categorized using distinct marker genes. The interactions between vein EDs (veinEndo) and other cell types were analysed using CellPhoneDB. Differentially expressed proteins in the proteomics of human umbilical vein EDs (HUVECs) and THP-1-derived macrophages infected with Porphyromonas gingivalis were compared with the differentially expressed genes (DEGs) of VeinEndo in scRNA-seq of CAP versus healthy control periodontal tissues. The protein-protein interaction of ICAM1-PTGS2 in macrophages and HUVECs was validated by adding recombinant ICAM1, ICAM1 inhibitor and PTGS2 inhibitor using real-time polymerase chain reaction (PCR), western blotting, and immunofluorescence staining., Results: EDs in patients with CAP were divided into eight subclusters: five vein ED, capillaries, arterials and EC (PLA). There were 29 mutually upregulated DEGs and two mutually downregulated DEGs in vein cells in the scRNA-seq data, as well as differentially expressed proteins in the proteomics of HUVECs. Real-time PCR and immunofluorescence staining showed that ICAM1 and PTGS2 were highly expressed in CAP, infected HUVECs, and macrophages. Recombinant protein ICAM1 may improve PTGS2 expression, reactive oxygen species (ROS), and Fe 2+ levels and decrease glutathione peroxidase 4 (GPX4) and SLC7A11 protein levels. ICAM1 inhibitor may inverse the above changes., Conclusions: scRNA-seq revealed the diversity of EDs in CAP and identified the possible regulation of ICAM1 by the ferroptosis-related protein, PTGS2, in infected HUVECs and macrophages, thus providing a basis for therapeutic approaches that target the inflammatory microenvironment of CAP., (© 2024 British Endodontic Society. Published by John Wiley & Sons Ltd.)

Background: We previously demonstrated that Shaziling and Yorkshire pigs differ in growth rate and meat quality. However, the molecular mechanisms responsible for such phenotypic differences remain unclear. In the present study, we performed a transcriptomic analysis of 36 longissimus dorsi (LM) and 36 soleus (SM) muscle samples from Shaziling and Yorkshire pigs at six postnatal stages (30, 60, 90, 150, 210 and 300 days) to explore the differences in postnatal skeletal muscle of Shaziling and Yorkshire pigs., Results: Muscle morphological changes and the number of differentially expressed genes indicated the two stages of 60-90 days and 150-210 days were critical for the muscle growth and development in Shaziling pigs. Genes such as FLNC, COL1A1, NRAP, SMYD1, TNNI3, CRYAB and PDLIM3 played vital roles in the muscle growth, and genes such as CCDC71L, LPIN1, CPT1A, UCP3, NR4A3 and PDK4 played dominant roles in the lipid metabolism. Additionally, in contrast to the LM, the percentage of slow-twitch muscle fibers in the SM of both breeds consistently decreased from 30 to 150 days of age, but there was a significant rebound at 210 days of age. However, the percentage of slow-twitch muscle fibers in the SM of Shaziling pigs was higher than that in Yorkshire pigs, which may be associated with the calcium signaling pathway and the PPARβ/δ signaling pathway., Conclusion: The present study detected two critical periods and many functional genes for the muscle growth and development of Shaziling pigs, and showed differences in muscle fiber characteristics between Shaziling and Yorkshire pigs. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Background: Phytophthora capsici is a destructive oomycete pathogen, causing huge economic losses for agricultural production. The genus Trichoderma represents one of the most extensively researched categories of biocontrol agents, encompassing a diverse array of effective strains. The commercial biocontrol agent Trichoderma harzianum strain T-22 exhibits pronounced biocontrol effects against many plant pathogens, but its activity against P. capsici is not known., Results: T. harzianum T-22 significantly inhibited the growth of P. capsici mycelia and the culture filtrate of T-22 induced lysis of P. capsici zoospores. Electron microscopic analyses indicated that T-22 significantly modulated the ultrastructural composition of P. capsici, with a severe impact on the cell wall integrity. Dual RNA sequencing revealed multiple biological processes involved in the inhibition during the interaction between these two microorganisms. In particular, a marked upregulation of genes was identified in T. harzianum that are implicated in cell wall degradation or disruption. Concurrently, the presence of T. harzianum appeared to potentiate the susceptibility of P. capsici to cell wall biosynthesis inhibitors such as mandipropamid and dimethomorph. Further investigations showed that mandipropamid and dimethomorph could strongly inhibit the growth and development of P. capsici but had no impact on T. harzianum even at high concentrations, demonstrating the feasibility of combining T. harzianum and these cell wall synthesis inhibitors to combat P. capsici., Conclusion: These findings provided enhanced insights into the biocontrol mechanisms against P. capsici with T. harzianum and evidenced compatibility between specific biological and chemical control strategies. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Ovarian development in animals is a complicated biological process, requiring the simultaneous coordination among various genes and pathways. To understand the dynamic changes and molecular regulatory mechanisms of ovarian development in mud crab (Scylla paramamosain), both histological observation and whole transcriptome sequencing of ovarian tissues at different mating stages were implemented in this study. The histological results revealed that ovarian development was delayed in unmated females (60 days after courtship behavior but not mating), who exhibited an oocyte diameter of 56.38 ± 15.17 μm. Conversely, mated females exhibited accelerated the ovarian maturation process, with females reaching ovarian stage III (proliferative stage) 23 days after mating and attained an average oocyte diameter of 132.19 ± 15.07 μm. Thus, mating process is essential in promoting the rapid ovarian development in mud crab. Based on the whole transcriptome sequencing analysis, a total of 518 mRNAs, 1502 lncRNAs, 18 circRNAs and 151 miRNAs were identified to be differentially expressed between ovarian tissues at different mating stages. Notably, six differentially expressed genes (DEGs) associated with ovarian development were identified, including ovary development-related protein, red pigment concentrating hormone receptor, G2/mitotic-specific cyclin-B3-like, lutropin-chorio gonadotropic hormone receptor, renin receptor, and SoxB2. More importantly, both DEGs and targets of differentially expressed non-coding RNAs (DEncRNAs) were enriched in renin-angiotensin system, TGF-β signaling, cell adhesion molecules, MAPK signaling pathway, and ECM-receptor interaction, suggesting that these pathways may play significant roles in the ovarian development of mud crabs. Moreover, competition endogenous RNA (ceRNA) networks were constructed while mRNAs were differentially expressed between mating stages were involved in Gene Ontology (GO) biological processes such as developmental process, reproduction, and growth. These findings could provide solid foundations for the future development of female mud crab maturation enhancement strategy, and improve the understanding of the ovarian maturation process in crustaceans., 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 Inc. All rights reserved.)

Despite advances in treatment, atherosclerotic cardiovascular disease remains the leading cause of death in patients with diabetes. Even when risk factors are mitigated, the disease progresses, and thus, newer targets need to be identified that directly inhibit the underlying pathobiology of atherosclerosis in diabetes. A single-cell sequencing approach was used to distinguish the proatherogenic transcriptional profile in aortic cells in diabetes using a streptozotocin-induced diabetic Apoe-/- mouse model. Human carotid endarterectomy specimens from individuals with and without diabetes were also evaluated via immunohistochemical analysis. Further mechanistic studies were performed in human aortic endothelial cells (HAECs) and human THP-1-derived macrophages. We then performed a preclinical study using an activator protein-1 (AP-1) inhibitor in a diabetic Apoe-/- mouse model. Single-cell RNA sequencing analysis identified the AP-1 complex as a novel target in diabetes-associated atherosclerosis. AP-1 levels were elevated in carotid endarterectomy specimens from individuals with diabetes compared with those without diabetes. AP-1 was validated as a mechanosensitive transcription factor via immunofluorescence staining for regional heterogeneity of endothelial cells of the aortic region exposed to turbulent blood flow and by performing microfluidics experiments in HAECs. AP-1 inhibition with T-5224 blunted endothelial cell activation as assessed by a monocyte adhesion assay and expression of genes relevant to endothelial function. Furthermore, AP-1 inhibition attenuated foam cell formation. Critically, treatment with T-5224 attenuated atherosclerosis development in diabetic Apoe-/- mice. This study has identified the AP-1 complex as a novel target, the inhibition of which treats the underlying pathobiology of atherosclerosis in diabetes., (© 2024 by the American Diabetes Association.)

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

Lung adenocarcinoma (LUAD), the most common subtype of lung cancer globally, has seen improved prognosis with advancements in diagnostic, surgical, radiotherapy, and molecular therapy techniques, while its 5-year survival rate remains low. Molecular biomarkers provide prognostic value. Oxidative stress factors, such as reactive nitrogen species and ROS, are crucial in various stages of tumor progression, influencing cell transformation, proliferation, angiogenesis, and metastasis. ROS demonstrate dual roles, affecting tumor cells, hypoxia sensitivity, and the microenvironment. Comprehensive analysis of oxidative stress in LUAD has not been conducted to date. Therefore, we systematically investigated the regulatory patterns of oxidative stress in LUAD based on oxidative stress-related genes and correlated these patterns with cellular infiltration characteristics of the tumor immune microenvironment. The model utilizes single-factor Cox analysis to screen key differential genes with prognostic value and employs least absolute shrinkage and selection operator (LASSO) penalized Cox regression analysis to construct a prognostic-related prediction model. Ten candidate genes were selected based on this model. The risk score was constructed using the coefficients and expression levels of these ten genes. Furthermore, the impact of this risk score on overall survival (OS) was determined. Two genes with the most significant differential expression, SFTPB and S100P, were selected through qRT-PCR. Cell experiments including CCK-8, Edu, transwell assays confirmed their effects on lung cancer cells growth, consistent with the results of bioinformatics analysis. These findings suggested that this model held potential clinical value for evaluating the prognosis of lung adenocarcinoma., 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 B.V. All rights reserved.)

Background: Increased oxidative stress (OS) activity following intracerebral hemorrhage (ICH) had significantly impacting patient prognosis. Identifying optimal genes associated with OS could enhance the understanding of OS after ICH., Methods: We employed single-cell RNA sequencing (scRNA-seq) to investigate the heterogeneity of OS across various cellular tiers following ICH, aiming to acquire biological insights into ICH. We utilized AUCell, Ucell, singscore, ssgsea, and AddModuleScore algorithms, along with correlation analysis, to identify hub genes influencing high OS post-ICH. Furthermore, we employed four machine learning algorithms, eXtreme Gradient Boosting, Boruta, Random Forest, and Least Absolute Shrinkage and Selection Operator, to identify the optimal feature genes. To validate the accuracy of our analysis, we conducted validation in ICH animal experiments., Results: After analyzing the scRNA-seq dataset using various algorithms, we found that OS activity exhibited heterogeneity across different cellular layers following ICH, with particularly heightened activity observed in monocytes. Further integration of bulk data and machine learning algorithms revealed that ANXA2 and COTL1 were closely associated with high OS after ICH. Our animal experiments demonstrated an increase in OS expression post-ICH. Additionally, the protein expression of ANXA2 and COTL1 was significantly elevated and co-localized with microglia. Pearson correlation coefficient analysis revealed a significant correlation between ANXA2 and OS, indicating strong consistency (r = 0.84, p < 0.05). Similar results were observed for COTL1 and OS (r = 0.69, p < 0.05)., Conclusions: Following ICH, ANXA2 and COTL1 might penetrate the brain via monocytes, localize within microglia, and enhance OS activity. This might help us better understand OS after ICH., 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 B.V. All rights reserved.)

Background: The occurrence and progression of hepatocellular carcinoma (HCC) are significantly affected by DNA damage response (DDR). Exploring DDR-related biomarkers can help predict the prognosis and immune characteristics of HCC., Methods: First, the single-cell RNA sequencing (scRNA-seq) dataset GSE242889 was processed and performed manual annotation. Then we found the marker genes of DDR-active subgroups based on "AUCell" algorithm. The "Limma" R package was used to identify differentially expressed genes (DEGs) between tumor and normal samples of HCC. The risk prognostic model was constructed by filtering genes using univariate Cox and LASSO regression analyses. Finally, the signatures were analyzed for immune infiltration, gene mutation, and drug sensitivity. Last but not least, KPNA2, which had the largest coefficient in our model was validated by experiments including western blot, MTT, colony formation and γ-H2AX assays., Results: We constructed a prognostic model based on 5 DDR marker genes including KIF2C, CDC20, KPNA2, UBE2S and ADH1B for HCC. We also proved that the model had an excellent performance in both training and validation cohorts. Patients in the high-risk group had a poorer prognosis, different immune features, gene mutation frequency, immunotherapy response and drug sensitivity compared with the low-risk group. Besides, our experimental results proved that KPNA2 was up-regulated in liver cancer cells than in hepatocytes. More importantly, the knockdown of KPNA2 significantly inhibited cell variability, proliferation and promoted DNA damage., Conclusions: We innovatively integrated scRNA-seq and bulk RNA sequencing to construct the DDR-related prognostic model. Our model could effectively predict the prognosis, immune landscape and therapy response of HCC., 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. Published by Elsevier B.V.)

Main Conclusion: Transcriptome analysis in potato varieties revealed genes associated with tuber yield-related traits and developed gene expression markers. This study aimed to identify genes involved in high tuber yield and its component traits in test potato varieties (Kufri Frysona, Kufri Khyati, and Kufri Mohan) compared to control (Kufri Sutlej). The aeroponic evaluation showed significant differences in yield-related traits in the varieties. Total RNA sequencing was performed using tuber and leaf tissues on the Illumina platform. The high-quality reads (QV > 25) mapping with the reference potato genomes revealed statistically significant (P < 0.05) differentially expressed genes (DEGs) into two categories: up-regulated (> 2 Log 2 fold change) and down-regulated (< -2 Log 2 fold change). DEGs were characterized by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Collectively, we identified genes participating in sugar metabolism, stress response, transcription factors, phytohormones, kinase proteins, and other genes greatly affecting tuber yield and its related traits. A few selected genes were UDP-glucose glucosyltransferase, glutathion S-transferase, GDSL esterase/lipase, transcription factors (MYB, WRKY, bHLH63, and BURP), phytohormones (auxin-induced protein X10A, and GA20 oxidase), kinase proteins (Kunitz-type tuber invertase inhibitor, BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1) and laccase. Based on the selected 17 peptide sequences representing 13 genes, a phylogeny tree and motifs were analyzed. Real time-quantitative polymerase chain reaction (RT-qPCR) analysis was used to validate the RNA-seq results. RT-qPCR based gene expression markers were developed for the genes such as 101 kDa heat shock protein, catechol oxidase B chloroplastic, cysteine protease inhibitor 1, Kunitz-type tuber invertase inhibitor, and laccase to identify high yielding potato genotypes. Thus, our study paved the path for potential genes associated with tuber yield traits in potato under aeroponics., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Reproducibility is a major concern in biomedical studies, and existing publication guidelines do not solve the problem. Batch effects and quality imbalances between groups of biological samples are major factors hampering reproducibility. Yet, the latter is rarely considered in the scientific literature., Results: Our analysis uses 40 clinically relevant RNA-seq datasets to quantify the impact of quality imbalance between groups of samples on the reproducibility of gene expression studies. High-quality imbalance is frequent (14 datasets; 35%), and hundreds of quality markers are present in more than 50% of the datasets. Enrichment analysis suggests common stress-driven effects among the low-quality samples and highlights a complementary role of transcription factors and miRNAs to regulate stress response. Preliminary ChIP-seq results show similar trends. Quality imbalance has an impact on the number of differential genes derived by comparing control to disease samples (the higher the imbalance, the higher the number of genes), on the proportion of quality markers in top differential genes (the higher the imbalance, the higher the proportion; up to 22%) and on the proportion of known disease genes in top differential genes (the higher the imbalance, the lower the proportion). We show that removing outliers based on their quality score improves the resulting downstream analysis., Conclusions: Thanks to a stringent selection of well-designed datasets, we demonstrate that quality imbalance between groups of samples can significantly reduce the relevance of differential genes, consequently reducing reproducibility between studies. Appropriate experimental design and analysis methods can substantially reduce the problem., (© 2024. The Author(s).)

Next-generation sequencing (NGS) has revolutionized genomic research by enabling high-throughput, cost-effective genome and transcriptome sequencing accelerating personalized medicine for complex diseases, including cancer. Whole genome/transcriptome sequencing (WGS/WTS) provides comprehensive insights, while targeted sequencing is more cost-effective and sensitive. In comparison to short-read sequencing, which still dominates the field due to high speed and cost-effectiveness, long-read sequencing can overcome alignment limitations and better discriminate similar sequences from alternative transcripts or repetitive regions. Hybrid sequencing combines the best strengths of different technologies for a more comprehensive view of genomic/transcriptomic variations. Understanding each technology's strengths and limitations is critical for translating cutting-edge technologies into clinical applications. In this study, we sequenced DNA and RNA libraries of reference samples using various targeted DNA and RNA panels and the whole transcriptome on both short-read and long-read platforms. This study design enables a comprehensive analysis of sequencing technologies, targeting protocols, and library preparation methods. Our expanded profiling landscape establishes a reference point for assessing current sequencing technologies, facilitating informed decision-making in genomic research and precision medicine., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objectives: The present study used single-cell RNA sequencing (scRNA-seq) to characterize the cellular composition of ovarian carcinosarcoma (OCS) and identify its molecular characteristics., Methods: scRNA-seq was performed in resected primary OCS for an in-depth analysis of tumor cells and the tumor microenvironment. Immunohistochemistry staining was used for validation. The scRNA-seq data of OCS were compared with those of high-grade serous ovarian carcinoma (HGSOC) tumors and other OCS tumors., Results: Both malignant epithelial and malignant mesenchymal cells were observed in the OCS patient of this study. We identified four epithelial cell subclusters with different biological roles. Among them, epithelial subcluster 4 presented high levels of breast cancer type 1 susceptibility protein homolog ( BRCA1 ) and DNA topoisomerase 2-α ( TOP2A ) expression and was related to drug resistance and cell cycle. We analyzed the interaction between epithelial and mesenchymal cells and found that fibroblast growth factor (FGF) and pleiotrophin (PTN) signalings were the main pathways contributing to communication between these cells. Moreover, we compared the malignant epithelial and mesenchymal cells of this OCS tumor with our previous published HGSOC scRNA-seq data and OCS data. All the epithelial subclusters in the OCS tumor could be found in the HGSOC samples. Notably, the mesenchymal subcluster C14 exhibited specific expression patterns in the OCS tumor, characterized by elevated expression of cytochrome P450 family 24 subfamily A member 1 ( CYP24A1 ), collagen type XXIII α1 chain ( COL23A1 ), cholecystokinin ( CCK ), bone morphogenetic protein 7 ( BMP7 ), PTN , Wnt inhibitory factor 1 ( WIF1 ), and insulin-like growth factor 2 ( IGF2 ). Moreover, this subcluster showed distinct characteristics when compared with both another previously published OCS tumor and normal ovarian tissue., Conclusions: This study provides the single-cell transcriptomics signature of human OCS, which constitutes a new resource for elucidating OCS diversity.

Background: Many individuals experience long COVID after SARS-CoV-2 infection. As microbiota can influence health, it may change with COVID-19. This study investigated differences in oral microbiota between COVID-19 patients with and without long COVID., Methods: Based on a prospective follow-up investigation, this nested case-control study evaluated the differences in oral microbiota in individuals with and without long COVID (Symptomatic and Asymptomatic groups), which were assessed by 16S rRNA sequencing on tongue coating samples. A predictive model was established using machine learning based on specific differential microbial communities., Results: One-hundred-and-eight patients were included (n=54 Symptomatic group). The Symptomatic group had higher Alpha diversity indices (observed&#95;otus, Chao1, Shannon, and Simpson indices), differences in microbial composition (Beta diversity), and microbial dysbiosis with increased diversity and relative abundance of pathogenic bacteria. Marker bacteria (c&#95;&#95;Campylobacterota, o&#95;&#95;Coriobacteriales, o&#95;&#95;Pseudomonadales, and o&#95;&#95;Campylobacterales) were associated with long COVID by linear discriminant analysis effect size and receiver operating characteristic curves (AUC 0.821)., Conclusion: There were distinct variations in oral microbiota between COVID-19 patients with and without long COVID. Changes in oral microbiota may indicate long COVID., Competing Interests: Declaration of competing interest All the authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Inferring gene regulatory networks from single-cell RNA-sequencing trajectories has been an active area of research yet methods are still needed to identify regulators governing cell transitions. We developed DREAMIT (Dynamic Regulation of Expression Across Modules in Inferred Trajectories) to annotate transcription-factor activity along single-cell trajectory branches, using ensembles of relations to target genes. Using a benchmark representing several different tissues, as well as external validation with ATAC-Seq and Perturb-Seq data on hematopoietic cells, the method was found to have higher tissue-specific sensitivity and specificity over competing approaches., (© 2024. The Author(s).)

Hematopoiesis is a tightly regulated process that gets skewed toward myelopoiesis. This restrains lymphopoiesis, but the role of lymphocytes in this process is not well defined. To unravel the intricacies of neutrophil responses in COVID-19, we performed bulk RNAseq on neutrophils from healthy controls and COVID-19 patients. Principal component analysis revealed distinguishing neutrophil gene expression alterations in COVID-19 patients. ICU and ward patients displayed substantial transcriptional changes, with ICU patients exhibiting a more pronounced response. Intriguingly, neutrophils from COVID-19 patients, notably ICU patients, exhibited an enrichment of immunoglobulin (Ig) and B cell lineage-associated genes, suggesting potential lineage plasticity. We validated our RNAseq findings in a larger cohort. Moreover, by reanalyzing single-cell RNA sequencing (scRNAseq) data on human bone marrow (BM) granulocytes, we identified the cluster of granulocyte-monocyte progenitors (GMP) enriched with Ig and B cell lineage-associated genes. These cells with lineage plasticity may serve as a resource depending on the host's needs during severe systemic infection. This distinct B cell subset may play a pivotal role in promoting myelopoiesis in response to infection. The scRNAseq analysis of BM neutrophils in infected mice further supported our observations in humans. Finally, our studies using an animal model of acute infection implicate IL-7/GM-CSF in influencing neutrophil and B cell dynamics. Elevated GM-CSF and reduced IL-7 receptor expression in COVID-19 patients imply altered hematopoiesis favoring myeloid cells over B cells. Our findings provide novel insights into the relationship between the B-neutrophil lineages during severe infection, hinting at potential implications for disease pathogenesis., Importance: This study investigates the dynamics of hematopoiesis in COVID-19, focusing on neutrophil responses. Through RNA sequencing of neutrophils from healthy controls and COVID-19 patients, distinct gene expression alterations are identified, particularly in ICU patients. Notably, neutrophils from COVID-19 patients, especially in the ICU, exhibit enrichment of immunoglobulin and B cell lineage-associated genes, suggesting potential lineage plasticity. Validation in a larger patient cohort and single-cell analysis of bone marrow granulocytes support the presence of granulocyte-monocyte progenitors with B cell lineage-associated genes. The findings propose a link between B-neutrophil lineages during severe infection, implicating a potential role for these cells in altered hematopoiesis favoring myeloid cells over B cells. Elevated GM-CSF and reduced IL-7 receptor expression in stress hematopoiesis suggest cytokine involvement in these dynamics, providing novel insights into disease pathogenesis., Competing Interests: The authors declare no conflict of interest.

Peanut (Arachis hypogaea L.) is an important leguminous oil and economic crop that produces flowers aboveground and fruits belowground. Subterranean fruit-pod development, which significantly affects peanut production, involves complex molecular mechanisms that likely require the coordinated regulation of multiple genes in different tissues. To investigate the molecular mechanisms that underlie peanut fruit-pod development, we characterized the anatomical features of early fruit-pod development and integrated single-nucleus RNA-sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) data at the single-cell level. We identified distinct cell types, such as meristem, embryo, vascular tissue, cuticular layer, and stele cells within the shell wall. These specific cell types were used to examine potential molecular changes unique to each cell type during pivotal stages of fruit-pod development. snRNA-seq analyses of differentially expressed genes revealed cell-type-specific insights that were not previously obtainable from transcriptome analyses of bulk RNA. For instance, we identified MADS-box genes that contributes to the formation of parenchyma cells and gravity-related genes that are present in the vascular cells, indicating an essential role for the vascular cells in peg gravitropism. Overall, our single-nucleus analysis provides comprehensive and novel information on specific cell types, gene expression, and chromatin accessibility during the early stages of fruit-pod development. This information will enhance our understanding of the mechanisms that underlie fruit-pod development in peanut and contribute to efforts aimed at improving peanut production., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Pancreatic cancer (PC) poses a substantial global health challenge, ranking as the fourth leading cause of cancer-related deaths due to its high mortality rate. Late-stage diagnoses are common due to the absence of specific symptoms. Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of PC cases. Recent research has suggested a potential link between elevated serum levels of bile acids (BAs) and tumorigenesis of PDAC. This study aims to understand how taurochenodeoxycholic acid (TCDCA), a secondary BA, influences PDAC using RNA sequencing techniques on the Capan-1 cell line. We identified 2,950 differentially expressed genes (DEGs) following TCDCA treatment, with 1,597 upregulated and 1,353 downregulated genes. These DEGs were associated with critical PDAC pathways, including coagulation, angiogenesis, cell migration, and signaling regulation. Furthermore, we reviewed relevant literature highlighting genes like DKK-1, KRT80, UPLA, and SerpinB2, known for their roles in PDAC tumorigenesis and metastasis. Our study sheds light on the complex relationship between BAs and PDAC, offering insights into potential diagnostic markers and therapeutic targets. Further research is needed to unravel these findings' precise mechanisms and clinical implications, potentially improving PDAC diagnosis and treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Zoltan Vereb reports financial support was provided by Hungarian Academy of Sciences. Viktoria Venglovecz reports financial support was provided by National, Research, Development and Innovation Office. Lajos Kemeny reports financial support was provided by Hungarian Centre of Excellence for Molecular Medicine. Zoltan Vereb reports financial support was provided by National, Research, Development and Innovation Office. If there are other authors, they 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 The Authors. Published by Elsevier B.V. All rights reserved.)

During maturation oocytes at the germinal vesicle (GV) stage progress to metaphase II (MII). However, during in vitro maturation a proportion often fail to progress. To understand these processes, we employed RNA sequencing to examine the transcriptome profile of these three groups of oocytes from the pig. We compared our findings with similar public oocyte data from humans. The transcriptomes in oocytes that failed to progress was similar to those that did. We found in both species, the most upregulated genes in MII oocytes were associated with chromosome segregation and cell cycle processes, while the most down regulated genes were relevant to ribosomal and mitochondrial pathways. Moreover, those genes involved in chromosome segregation during GV to MII transition were conserved in pig and human. We also compared MII and GV oocyte transcriptomes at the isoform transcript level in both species. Several thousands of genes (including DTNBP1, MAPK1, RAB35, GOLGA7, ATP1A1 and ATP2B1) identified as not different in expression at a gene transcript level were found to have differences in isoform transcript levels. Many of these genes were involved in ATPase-dependent or GTPase-dependent intracellular transport in pig and human, respectively. In conclusion, our study suggests the failure to progress to MII in vitro may not be regulated at the level of the genome and that many genes are differentially regulated at the isoform level, particular those involved ATPase- or GTPase-dependent intracellular transport., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Tang 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: Xenotransplantation of genetically engineered porcine organs has the potential to address the challenge of organ donor shortage. Two cases of porcine-to-human kidney xenotransplantation were performed, yet the physiological effects on the xenografts and the recipients' immune responses remain largely uncharacterized., Methods: We performed single-cell RNA sequencing (scRNA-seq) and longitudinal RNA-seq analyses of the porcine kidneys to dissect xenotransplantation-associated cellular dynamics and xenograft-recipient interactions. We additionally performed longitudinal scRNA-seq of the peripheral blood mononuclear cells (PBMCs) to detect recipient immune responses across time., Findings: Although no hyperacute rejection signals were detected, scRNA-seq analyses of the xenografts found evidence of endothelial cell and immune response activation, indicating early signs of antibody-mediated rejection. Tracing the cells' species origin, we found human immune cell infiltration in both xenografts. Human transcripts in the longitudinal bulk RNA-seq revealed that human immune cell infiltration and the activation of interferon-gamma-induced chemokine expression occurred by 12 and 48 h post-xenotransplantation, respectively. Concordantly, longitudinal scRNA-seq of PBMCs also revealed two phases of the recipients' immune responses at 12 and 48-53 h. Lastly, we observed global expression signatures of xenotransplantation-associated kidney tissue damage in the xenografts. Surprisingly, we detected a rapid increase of proliferative cells in both xenografts, indicating the activation of the porcine tissue repair program., Conclusions: Longitudinal and single-cell transcriptomic analyses of porcine kidneys and the recipient's PBMCs revealed time-resolved cellular dynamics of xenograft-recipient interactions during xenotransplantation. These cues can be leveraged for designing gene edits and immunosuppression regimens to optimize xenotransplantation outcomes., Funding: This work was supported by NIH RM1HG009491 and DP5OD033430., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Opentrons LabWorks/Neochromosome, Inc.; and serves or served on the scientific advisory boards of the following: CZ Biohub New York, LLC; Logomix, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sangamo, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. R.A.M. is on scientific advisory boards for eGenesis, Sanofi, Regeneron, CareDx, and Hansa Biopharma; is a consultant to Recombinetics; reports consulting fees from Hansa Medical, Regeneron, Thermo Fisher Scientific, Genentech, CareDx, One Lambda, ITB Med, Sanofi, and PPD Development; and reports grant support from Hansa Biopharma, all unrelated to the present work. R.A.M. also reports grant support from United Therapeutics Corporation, PBC. All other authors have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Gene misexpression is the aberrant transcription of a gene in a context where it is usually inactive. Despite its known pathological consequences in specific rare diseases, we have a limited understanding of its wider prevalence and mechanisms in humans. To address this, we analyzed gene misexpression in 4,568 whole-blood bulk RNA sequencing samples from INTERVAL study blood donors. We found that while individual misexpression events occur rarely, in aggregate they were found in almost all samples and a third of inactive protein-coding genes. Using 2,821 paired whole-genome and RNA sequencing samples, we identified that misexpression events are enriched in cis for rare structural variants. We established putative mechanisms through which a subset of SVs lead to gene misexpression, including transcriptional readthrough, transcript fusions, and gene inversion. Overall, we develop misexpression as a type of transcriptomic outlier analysis and extend our understanding of the variety of mechanisms by which genetic variants can influence gene expression., Competing Interests: Declaration of interests The authors declare the following interests: T.V. has received PhD studentship funding from AstraZeneca. J.M. completed this work while employed by the University of Cambridge but is now an employee of Genomics plc. S.P. is a current employee and stockholder of AstraZeneca. D.J.R. is an employee of NHS Blood and Transplant. A.B. is currently an employee of Bayer AG, Research and Early Development Precision Medicine, Research & Development, Pharmaceutical Division, Wuppertal, DE. A.P. is a current employee and stockholder of AstraZeneca. D.S.P. is a current employee and stockholder of AstraZeneca. K.K. is a current employee and stockholder of AstraZeneca. J.D. serves on scientific advisory boards for AstraZeneca, Novartis, and UK Biobank and has received multiple grants from academic, charitable, and industry sources outside of the submitted work. M.I. is a trustee of the Public Health Genomics (PHG) Foundation, is a member of the Scientific Advisory Board of Open Targets, and has a research collaboration with AstraZeneca, which is unrelated to this study., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2024. The Author(s).)

Introduction: Gastric cancer (GC) remains a major global health threat ranking as the fifth most prevalent cancer. Hypoxia, a characteristic feature of solid tumors, significantly contributes to the malignant progression of GC. Mitochondria are the major target of hypoxic injury that promotes mitochondrial dysfunction during the development of cancers including GC. However, the gene signature and prognostic model based on hypoxia- and mitochondrial dysfunction-related genes (HMDRGs) in the prediction of GC prognosis have not yet been established., Methods: The gene expression profile datasets of stomach cancer patients were retrieved from The Cancer Genome Atlas and the Gene Expression Omnibus databases. Prognostic genes were selected using Least Absolute Shrinkage and Selection Operator Cox (LASSO-Cox) regression analysis to construct a prognostic model. Immune infiltration was evaluated through ESTIMATE, CIBERSORT, and ssGSEA analyses. Tumor immune dysfunction and exclusion (TIDE) and immunophenoscore (IPS) were utilized to explore implications for immunotherapy. Furthermore, in vitro experiments were conducted to validate the functional roles of HMDRGs in GC cell malignancy., Results: In this study, five HMDRGs (ZFP36, SERPINE1, DUSP1, CAV1, and AKAP12) were identified for developing a prognostic model in GC. This model stratifies GC patients into high- and low-risk groups based on median risk scores. A nomogram predicting overall survival (OS) was constructed and showed consistent results with observed OS. Immune infiltration analysis indicated that individuals in the high-risk group tend to exhibit increased immune cell infiltration. Additionally, analysis of cancer immunotherapy responses revealed that high-risk group patients exhibit poorer responses to cancer immunotherapy compared to the low-risk group. Immunohistochemistry (IHC) staining indicated that the expression levels of HMDRGs were remarkably correlated with GC, of which, SERPINE1 displayed the most pronounced up-regulation, while ZFP36 exhibited the most notable down-regulation in GC patients. Furthermore, in vitro investigation validated that SERPINE1 and ZFP36 contribute to the malignant processes of GC cells correlated with mitochondrial dysfunction., Conclusions: This study presents a novel and efficient approach to evaluate GC prognosis and immunotherapy efficacy, and also provides insights into understanding the pathogenesis of GC., 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, Cui, Wang, Wang, Yu and Xiong.)

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

This study investigates the cellular origin and tissue heterogeneity in bipolar disorder (BD) by integrating multiomics data. Four distinct datasets were employed, including single-cell RNA sequencing (scRNA-seq) data (embryonic and fetal brain, n = 8, 1,266 cells), BD Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) data (adult brain, n = 210), BD bulk RNA-seq data (adult brain, n = 314), and BD genome-wide association study (GWAS) summary data (n = 413,466). The integration of scRNA-seq data with multiomics data relevant to BD was accomplished using the single-cell disease relevance score (scDRS) algorithm. We have identified a novel brain cell cluster named ADCY1, which exhibits distinct genetic characteristics. From a high-resolution genetic perspective, glial cells emerge as the primary cytopathology associated with BD. Specifically, astrocytes were significantly related to BD at the RNA-seq level, while microglia showed a strong association with BD across multiple panels, including the transcriptome-wide association study (TWAS), ATAC-seq, and RNA-seq. Additionally, oligodendrocyte precursor cells displayed a significant association with BD in both ATAC-seq and RNA-seq panel. Notably, our investigation of brain regions affected by BD revealed significant associations between BD and all three types of glial cells in the dorsolateral prefrontal cortex (DLPFC). Through comprehensive analyses, we identified several BD-associated genes, including CRMP1, SYT4, UCHL1, and ZBTB18. In conclusion, our findings suggest that glial cells, particularly in specific brain regions such as the DLPFC, may play a significant role in the pathogenesis of BD. The integration of multiomics data has provided valuable insights into the etiology of BD, shedding light on potential mechanisms underlying this complex psychiatric disorder., (© 2024. The Author(s).)

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

Multimodal measurement of single cells provides deep insights into the intricate relationships between individual molecular layers and the regulatory mechanisms underlying intercellular variations. Here, we reported DMF-DM-seq, a highly integrated, sensitive, and automated platform for single-cell mRNA and microRNA (miRNA) co-sequencing based on digital microfluidics. This platform first integrates the processes of single-cell isolation, lysis, component separation, and simultaneous sequencing library preparation of mRNA and miRNA within a single DMF device. Compared with the current half-cell measuring strategy, DMF-DM-seq enables complete separation of single-cell mRNA and miRNA via a magnetic field application, resulting in a higher miRNA detection ability. DMF-DM-seq revealed differential expression patterns of single cells of noncancerous breast cells and noninvasive and aggressive breast cancer cells at both mRNA and miRNA levels. The results demonstrated the anticorrelated relationship between miRNA and their mRNA targets. Further, we unravel the tumor growth and metastasis-associated biological processes enriched by miRNA-targeted genes, along with important miRNA-interaction networks involved in significant signaling pathways. We also deconstruct the miRNA regulatory mechanisms underlying different signaling pathways across different breast cell types. In summary, DMF-DM-seq offers a powerful tool for a comprehensive study of the expression heterogeneity of single-cell mRNA and miRNA, which will be widely applied in basic and clinical research.

The role of monocytes in postmenopausal osteoporosis is widely recognized; however, the mechanisms underlying monocyte reprogramming remain unknown. In this study, single-cell RNA sequencing (scRNA-seq) was conducted on CD14+ bone marrow monocytes obtained from 3 postmenopausal women with normal BMD and 3 women with postmenopausal osteoporosis (PMOP). Monocle2 was used to classify the monocytes into 7 distinct clusters. The proportion of cluster 1 significantly decreased in PMOP patients, while the proportion of cluster 7 increased. Further analysis via GSEA, transcription factor activity analysis, and sc-metabolic analysis revealed significant differences between clusters 1 and 7. Cluster 7 exhibited upregulated pathways associated with inflammation, immunity, and osteoclast differentiation, whereas cluster 1 demonstrated the opposite results. Monocle2, TSCAN, VECTOR, and scVelo data indicated that cluster 1 represented the initial subset and that cluster 7 represents one of the terminal subsets. BayesPrism and ssGSEA were employed to analyze the bulk transcriptome data obtained from the GEO database. The observed alterations in the proportions of 1 and 7 were validated and found to have diagnostic significance. CD16 serves as the marker gene for cluster 7, thus leading to an increased proportion of CD16+ monocytes in women with PMOP. Flow cytometry was used to assess the consistency of outcomes with those of the bioinformatic analysis. Subsequently, an additional scRNA-seq analysis was conducted on bone marrow mononuclear cells obtained from 3 patients with PMOP and 3 postmenopausal women with normal BMD. The differential proportions of cluster 1 and cluster 7 were once again confirmed, with the pathological effect of cluster 7 may attribute to cell-cell communication. The scRNA-seq findings suggest that an imbalance in monocyte subsets is a characteristic feature of PMOP. These findings elucidate the limitations of utilizing bulk transcriptome data for detecting alterations in monocytes, which may influence novel research inquiries., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society for Bone and Mineral Research.)

The placenta is crucial for fetal development, yet the impact of environmental stressors such as arsenic exposure remains poorly understood. We apply single-cell RNA sequencing to analyze the response of the mouse placenta to arsenic, revealing cell-type-specific gene expression, function, and pathological changes. Notably, the Prap1 gene, which encodes proline-rich acidic protein 1 (PRAP1), is significantly upregulated in 26 placental cell types including various trophoblast cells. Our study shows a female-biased increase in PRAP1 in response to arsenic and localizes it in the placenta. In vitro and ex vivo experiments confirm PRAP1 upregulation following arsenic treatment and demonstrate that recombinant PRAP1 protein reduces arsenic-induced cytotoxicity and downregulates cell cycle pathways in human trophoblast cells. Moreover, PRAP1 knockdown differentially affects cell cycle processes, proliferation, and cell death depending on the presence of arsenic. Our findings provide insights into the placental response to environmental stress, offering potential preventative and therapeutic approaches for environment-related adverse outcomes in mothers and children., (© 2024. The Author(s).)

Purpose: The purpose of this study was to elucidate the relationship between the tumor microenvironment (TME) and cellular diversity in bladder cancer (BLCA) progression, leveraging single-cell RNA sequencing (scRNA-seq) data to identify potential prognostic biomarkers and construct a prognostic model for BLCA., Methods: We analyzed scRNA-seq data of normal and tumor bladder cells from the Gene Expression Omnibus (GEO) database to uncover crucial markers within the bladder TME. The study compared gene expression in normal versus tumor bladder cells, identifying differentially expressed genes. These genes were subsequently assessed for their prognostic significance using patient follow-up data from The Cancer Genome Atlas. Prognostic models were constructed using Least Absolute Shrinkage and Selection Operator and multivariate Cox regression analyses, focusing on eight genes of interest. The predictive performance of the model was also tested against additional GEO data sets (GSE31684, GSE13507, and GSE32894)., Results: The prognostic model demonstrated reliable prediction of patient outcomes. Validation through gene set enrichment analysis and immune cell infiltration assessment supported the model's efficacy. The results from both the univariate and multivariate analyses suggest that the risk score is an independent prognostic factor with a hazard ratio of 2.97 (95% CI, 2.28 to 3.9, P < .001). In the validation cohort, the AUC at 1, 2, and 3 years is 0.74, 0.74, and 0.72, respectively., Conclusion: Our findings proposed biomarkers with prognostic potential, laying the groundwork for future in vitro validation and therapeutic exploration. This contributes to a deeper understanding of the genes associated with bladder TME and may improve prognostic precision in BLCA management.

Background: CD8+ T cells have been recognized as crucial factors in the prognosis of melanoma. However, there is currently a lack of gene markers that accurately describe their characteristics and functions in acral melanoma (AM), which hinders the development of personalized medicine., Methods: Firstly, we explored the composition differences of immune cells in AM using single-cell RNA sequencing (scRNA-seq) data and comprehensively characterized the immune microenvironment of AM in terms of composition, developmental differentiation, function, and cell communication. Subsequently, we constructed and validated a prognostic risk scoring model based on differentially expressed genes (DEGs) of CD8+ T cells using the TCGA-SKCM cohort through Lasso-Cox method. Lastly, immunofluorescence staining was performed to validate the expression of four genes (ISG20, CCL4, LPAR6, DDIT3) in AM and healthy skin tissues as included in the prognostic model., Results: The scRNA-seq data revealed that memory CD8+ T cells accounted for the highest proportion in the immune microenvironment of AM, reaching 70.5%. Cell-cell communication analysis showed extensive communication relationships among effector CD8+ T cells. Subsequently, we constructed a prognostic scoring model based on DEGs derived from CD8+ T cell sources. Four CD8+ T cell-related genes were included in the construction and validation of the prognostic model. Additionally, immunofluorescence results demonstrated that ISG20 and CCL4 were downregulated, while LPAR6 and DDIT3 were upregulated in AM tissues compared to normal skin tissues., Conclusion: Identifying biomarkers based on the expression levels of CD8+ T cell-related genes may be an effective approach for establishing prognostic models in AM patients. The independently prognostic risk evaluation model we constructed provides new insights and theoretical support for immunotherapy in AM., (© 2024 The Author(s). Skin Research and Technology published by John Wiley & Sons Ltd.)

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

Unstable transcripts have emerged as markers of active enhancers in vertebrates and shown to be involved in many cellular processes and medical disorders. However, their prevalence and role in plants is largely unexplored. Here, we comprehensively captured all actively initiating (nascent) transcripts across diverse crops and other plants using capped small (cs)RNA sequencing. We discovered that unstable transcripts are rare in plants, unlike in vertebrates, and when present, often originate from promoters. In addition, many 'distal' elements in plants initiate tissue-specific stable transcripts and are likely bona fide promoters of as-yet-unannotated genes or non-coding RNAs, cautioning against using reference genome annotations to infer putative enhancer sites. To investigate enhancer function, we integrated data from self-transcribing active regulatory region (STARR) sequencing. We found that annotated promoters and other regions that initiate stable transcripts, but not those marked by unstable or bidirectional unstable transcripts, showed stronger enhancer activity in this assay. Our findings underscore the blurred line between promoters and enhancers and suggest that cis-regulatory elements can encompass diverse structures and mechanisms in eukaryotes, including humans., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous sarcoma characterized by the COL1A1-PDGFB fusion gene. This study utilized single-cell RNA sequencing to dissect the cellular and molecular landscape of primary DFSP. Distinct DFSP cell clusters, exhibiting fibroblast-like traits, revealed variations in pathways associated with proliferation, inflammation and metabolism. Differential gene expression analysis during the differentiation from tumour stem cells to DFSP cells unveiled SMOC2, DCN and TGFBR3 as potential regulators of tumour invasion and immune infiltration through VEGF/TGF-β signalling modulation. Cellular communication analysis highlighted interactions within DFSP cell clusters and with endothelial cells, implicating molecules such as NAMPT, ANGPT2 and PTN in pathogenesis and treatment resistance. These findings offer insights into DFSP intratumour heterogeneity, elucidate molecular mechanisms underlying tumour behaviour, and suggest potential therapeutic targets., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objectives: RNA sequencing (RNA-seq) and bioinformatic analysis were combined and used to explore the anti-inflammatory effects and mechanisms of naringenin (Nar) in lipopolysaccharide (LPS)-stimulated human periodontal ligament stem cells (hPDLSCs)., Methods: Cell counting kit-8, quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA) were adopted to detect the effects of Nar on the proliferation and expression of inflammatory factors in LPS-stimulated hPDLSCs, screening for the optimal anti-inflammatory concentration of Nar. Differentially expressed genes (DEGs) were screened using |log2FC|≥1 and P ≤0.05 as criteria. Volcano plot analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, the String database, and the MCODE module of Cytoscape were utilized to select core genes and enriched pathways. The effects on the nuclear factor κB (NF-κB) signaling pathway were verified using ELISA, qRT-PCR, and Western blot., Results: Appropriate concentrations of Nar could alleviate the expression of inflammatory factors and promote the proliferation of hPDLSCs stimulated by LPS. The best anti-inflammatory effect was achieved with 20 μmol/L Nar. RNA-seq showed significant enrichment of inflammation-related signaling pathways. The anti-inflammatory effect of Nar was mediated by inhibiting the NF-κB signaling pathway, similar to the effect of the NF-κB inhibitor BAY 11-7802., Conclusions: Nar could exert its anti-inflammatory effects by inhibiting the NF-κB signaling pathway, making it a potential therapeutic option for the adjuvant treatment of periodontitis.

Genetic variation that influences gene expression and splicing is a key source of phenotypic diversity 1-5 . Although invaluable, studies investigating these links in humans have been strongly biased towards participants of European ancestries, which constrains generalizability and hinders evolutionary research. Here to address these limitations, we developed MAGE, an open-access RNA sequencing dataset of lymphoblastoid cell lines from 731 individuals from the 1000 Genomes Project 6 , spread across 5 continental groups and 26 populations. Most variation in gene expression (92%) and splicing (95%) was distributed within versus between populations, which mirrored the variation in DNA sequence. We mapped associations between genetic variants and expression and splicing of nearby genes (cis-expression quantitative trait loci (eQTLs) and cis-splicing QTLs (sQTLs), respectively). We identified more than 15,000 putatively causal eQTLs and more than 16,000 putatively causal sQTLs that are enriched for relevant epigenomic signatures. These include 1,310 eQTLs and 1,657 sQTLs that are largely private to underrepresented populations. Our data further indicate that the magnitude and direction of causal eQTL effects are highly consistent across populations. Moreover, the apparent 'population-specific' effects observed in previous studies were largely driven by low resolution or additional independent eQTLs of the same genes that were not detected. Together, our study expands our understanding of human gene expression diversity and provides an inclusive resource for studying the evolution and function of human genomes., (© 2024. The Author(s).)

Background: Abdominal obesity increases the risk for non-alcoholic fatty liver disease (NAFLD), now known as metabolic dysfunction-associated steatotic liver disease (MASLD)., Methods: To elucidate the directional cell-type level biological mechanisms underlying the association between abdominal obesity and MASLD, we integrated adipose and liver single nucleus RNA-sequencing and bulk cis-expression quantitative trait locus (eQTL) data with the UK Biobank genome-wide association study (GWAS) data using colocalization. Then we used colocalized cis-eQTL variants as instrumental variables in Mendelian randomization (MR) analyses, followed by functional validation experiments on the target genes of the cis-eQTL variants., Findings: We identified 17 colocalized abdominal obesity GWAS variants, regulating 17 adipose cell-type marker genes. Incorporating these 17 variants into MR discovers a putative tissue-of-origin, cell-type-aware causal effect of abdominal obesity on MASLD consistently with multiple MR methods without significant evidence for pleiotropy or heterogeneity. Single cell data confirm the adipocyte-enriched mean expression of the 17 genes. Our cellular experiments across human adipogenesis identify risk variant -specific epigenetic and transcriptional mechanisms. Knocking down two of the 17 genes, PPP2R5A and SH3PXD2B, shows a marked decrease in adipocyte lipidation and significantly alters adipocyte function and adipogenesis regulator genes, including DGAT2, LPL, ADIPOQ, PPARG, and SREBF1. Furthermore, the 17 genes capture a characteristic MASLD expression signature in subcutaneous adipose tissue., Interpretation: Overall, we discover a significant cell-type level effect of abdominal obesity on MASLD and trace its biological effect to adipogenesis., Funding: NIH grants R01HG010505, R01DK132775, and R01HL170604; the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant No. 802825), Academy of Finland (Grants Nos. 333021), the Finnish Foundation for Cardiovascular Research the Sigrid Jusélius Foundation and the Jane and Aatos Erkko Foundation; American Association for the Study of Liver Diseases (AASLD) Advanced Transplant Hepatology award and NIH/NIDDK (P30DK41301) Pilot and Feasibility award; NIH/NIEHS F32 award (F32ES034668); Finnish Diabetes Research Foundation, Kuopio University Hospital Project grant (EVO/VTR grants 2005-2021), the Academy of Finland grant (Contract no. 138006); Academy of Finland (Grant Nos 335443, 314383, 272376 and 266286), Sigrid Jusélius Foundation, Finnish Medical Foundation, Finnish Diabetes Research Foundation, Novo Nordisk Foundation (#NNF20OC0060547, NNF17OC0027232, NNF10OC1013354) and Government Research Funds to Helsinki University Hospital; Orion Research Foundation, Maud Kuistila Foundation, Finish Medical Foundation, and University of Helsinki., Competing Interests: Declaration of interests M.L. was funded by the Sigrid Jusélius Foundation during the last 36 months. The other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Neoadjuvant immunotherapy has evolved as an effective option to treat non-small cell lung cancer (NSCLC). B cells play essential roles in the immune system as well as cancer progression. However, the repertoire of B cells and its association with clinical outcomes remains unclear in NSCLC patients receiving neoadjuvant immunotherapy., Methods: Single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing data for LUAD samples were accessed from the TCGA and GEO databases. LUAD-related B cell marker genes were confirmed based on comprehensive analysis of scRNA-seq data. We then constructed the B cell marker gene signature (BCMGS) and validated it. In addition, we evaluated the association of BCGMS with tumor immune microenvironment (TIME) characteristics. Furthermore, we validated the efficacy of BCGMS in a cohort of NSCLC patients receiving neoadjuvant immunotherapy., Results: A BCMGS was constructed based on the TCGA cohort and further validated in three independent GSE cohorts. In addition, the BCMGS was proven to be significantly associated with TIME characteristics. Moreover, a relatively higher risk score indicated poor clinical outcomes and a worse immune response among NSCLC patients receiving neoadjuvant immunotherapy., Conclusions: We constructed an 18-gene prognostic signature derived from B cell marker genes based on scRNA-seq data, which had the potential to predict the prognosis and immune response of NSCLC patients receiving neoadjuvant immunotherapy., (© 2024. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

The epitranscriptome encompasses a diverse array of dynamic and reversible RNA modifications, affecting both coding and noncoding RNAs. Over 170 types of RNA chemical modifications have been identified, underscoring the need for innovative detection methods to deepen our understanding of RNA modification roles and mechanisms. In particular, the base resolution and quantitative information on RNA modifications are critical for understanding the regulation and functions of RNA modifications. Based on detection throughput and principles, existing quantitative RNA modification detection methods can be categorized into two groups, including next-generation sequencing and nanopore direct RNA sequencing. In this review, we focus on methodologies for elucidating the base resolution and stoichiometric information of RNA modifications. In addition, we further discuss the challenges and the potential prospects of the quantitative RNA modification detection methods., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest in this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)