Dynamic changes in pyroptosis following spinal cord injury and the identification of crucial molecular signatures through machine learning and single-cell sequencing.

The pathological cascade of spinal cord injury (SCI) is highly intricate. The onset of neuroinflammation can exacerbate the extent of damage. Pyroptosis is a form of inflammation-linked programmed cell death (PCD), the inhibition of pyroptosis can partially mitigate neuroinflammation. It is imperative to delineate the principal cell types susceptible to pyroptosis and concomitantly identify key genes associated with this process. We initially defined the pyroptosis-related genes (PRGs) and analyzed their expression at different time points post SCI. The results demonstrate a substantial upregulation of differentially expressed genes (DEGs) related to pyroptosis on the 7 days post-injury (dpi), these DEGs in the 7 dpi are closely related to the inflammatory response. Subsequently, immune infiltration analysis revealed a predominant presence of inflammatory microglia. Through correlation analysis, we postulated that pyroptosis primarily manifested within the inflammatory microglia. Employing machine learning algorithms, we identified four pyroptosis-related molecular signatures, which were experimentally validated using BV2 cells and spinal cord tissue samples. The robustness of the identified molecular signatures was further confirmed through single-cell sequencing data analysis. Overall, our study elucidates the temporal dynamics of pyroptosis and identifies key molecular signatures following SCI. These findings can provide novel evidence for therapeutic interventions in SCI.

Bulk and single-cell RNA sequencing analyses coupled with multiple machine learning to develop a glycosyltransferase associated signature in colorectal cancer.

BACKGROUND: This study aims to identify key glycosyltransferases (GTs) in colorectal cancer (CRC) and establish a robust prognostic signature derived from GTs. METHODS: Utilizing the AUCell, UCell, singscore, ssgsea, and AddModuleScore algorithms, along with correlation analysis, we redefined genes related to GTs in CRC at the single-cell RNA level. To improve risk model accuracy, univariate Cox and lasso regression were employed to discover a more clinically subset of GTs in CRC. Subsequently, the efficacy of seven machine learning algorithms for CRC prognosis was assessed, focusing on survival outcomes through nested cross-validation. The model was then validated across four independent external cohorts, exploring variations in the tumor microenvironment (TME), response to immunotherapy, mutational profiles, and pathways of each risk group. Importantly, we identified potential therapeutic agents targeting patients categorized into the high-GARS group. RESULTS: In our research, we classified CRC patients into distinct subgroups, each exhibiting variations in prognosis, clinical characteristics, pathway enrichments, immune infiltration, and immune checkpoint genes expression. Additionally, we established a Glycosyltransferase-Associated Risk Signature (GARS) based on machine learning. GARS surpasses traditional clinicopathological features in both prognostic power and survival prediction accuracy, and it correlates with higher malignancy levels, providing valuable insights into CRC patients. Furthermore, we explored the association between the risk score and the efficacy of immunotherapy. CONCLUSION: A prognostic model based on GTs was developed to forecast the response to immunotherapy, offering a novel approach to CRC management.

Single-cell and spatial sequencing identifies senescent and germinal tumor cells in adamantinomatous craniopharyngiomas.

Adamantinomatous craniopharyngioma (ACP) is a clinically aggressive tumor without effective treatment method. Previous studies proposed a paracrine tumorigenesis model, in which oncogenic ß-catenin induces senescence in pituitary stem cells and the senescent cells lead the formation of paracrine tumors through secretion of pro-tumorigenic factors. However, there lacks characterization on senescent cells in ACPs. Here, we profiled 12 ACPs with single-cell RNA and TCR-sequencing to elucidate the cellular atlas in ACPs and 3 of them were also subject to spatial sequencing to localize different subpopulations of the tumor cells. In total, we obtained the transcriptome profiles of 70,682 cells. Tumor cells, which were unambiguously identified through the cellular mutation status of the driver CTNNB1 mutations, were clustered into 6 subsets. The whorl-like cluster (WC) cells show distinct molecular features from the other tumor cells and the palisading epithelium (PE) cells consists of a proliferating subset. Other than typical PE and WC, we identified two novel subpopulations of the tumor cells. In one subpopulation, the cells express a high level of cytokines, e.g., FDCSP and S100A8/A9, and are enriched with the senescence-associated secretory phenotype (SASP) factors. Hematoxylin and eosin staining reveals that these SASP cells lack an ordered structures and their nuclei are elongated. In the other subpopulation, the cell sizes are small and they are tightly packed together with an unusual high density expressing a high level of mitochondrial genes (median 10.9%). These cells are the origin of the tumor developmental trajectories revealed by RNA velocity and pseudo-time analysis. Single-cell RNA and TCR analysis reveals that some ACPs are infiltrated with clonally expanded cytotoxic T cells. We propose a hypothesis that WC and PE are formed via different negative regulation mechanisms of the overactivated WNT/ß-catenin signaling which provides a new understanding on the tumorigenesis of ACPs. The study lays a foundation for future studies on targeting senescent cells in ACPs with senolytic compounds or other therapeutic agents.

The construction and experimental verification of a 6-LncRNA model based on Lactic acid metabolism in the tumor microenvironment of Wilms tumor.

BACKGROUND: Lactic acid (LA) can promote the malignant progression of tumors through the crosstalk with the tumor microenvironment (TME). However, the function of long non-coding RNAs (lncRNAs) related to LA metabolism in Wilms tumor (WT) remains unclear. METHODS: Gene expression data and clinical data of WT patients were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). Through the ESTIMATE algorithm and Pearson correlation analysis, lncRNAs related to tumor immunity and LA metabolism were screened. Subsequently, Cox regression analysis and Lasso Cox regression analysis were used to construct a model. Furthermore, candidate genes were identified and a competitive endogenous RNA (ceRNA) network was conducted to explore the specific mechanism of characteristic genes. Finally, based on the strong clinical relevance of UNC5B-AS1, its expression and function were experimentally verified. RESULTS: The immune score and stromal score were found to be closely related to the prognosis of WT. Eventually, a prognostic model (TME-LA-LM) consisting of 6 lncRNAs was successfully identified. The model demonstrated favorable predictive ability and accuracy, with significant variation in immune infiltration and drug susceptibility observed between risk groups. Additionally, the study revealed the involvement of 2 candidate genes and 5 microRNAs (miRNAs) in the tumor's development. Notably, UNC5B-AS1 was highly expressed and found to promote the proliferation and migration of tumor cells. CONCLUSION: This study, for the first time, elucidated the prognostic signatures of WT using lncRNAs related to TME and LA metabolism. The fundings of this research offer valuable insights for future studies on immunotherapy, personalized chemotherapy and mechanism research.

Single-cell multi-modal chromatin profiles revealing epigenetic regulations of cells in hepatocellular carcinoma.

BACKGROUND: Various epigenetic regulations systematically govern gene expression in cells involving various biological processes. Dysregulation of the epigenome leads to aberrant transcriptional programs and subsequently results in diseases, such as cancer. Therefore, comprehensive profiling epigenomics is essential for exploring the mechanisms underlying gene expression regulation during development and disease. METHODS: In this study, we developed single-cell chromatin proteins and accessibility tagmentation (scCPA-Tag), a multi-modal single-cell epigenetic profile capturing technique based on barcoded Tn5 transposases and a droplet microfluidics platform. scCPA-Tag enables the simultaneous capture of DNA profiles of histone modification and chromatin accessibility in the same cell. RESULTS: By applying scCPA-Tag to K562 cells and a hepatocellular carcinoma (HCC) sample, we found that the silence of several chromatin-accessible genes can be attributed to lysine-27-trimethylation of the histone H3 tail (H3K27me3) modification. We characterized the epigenetic features of the tumour cells and different immune cell types in the HCC tumour tissue by scCPA-Tag. Besides, a tumour cell subtype (C2) with more aggressive features was identified and characterized by high chromatin accessibility and a lower abundance of H3K27me3 on tumour-promoting genes. CONCLUSIONS: Our multi-modal scCPA-Tag provides a comprehensive approach for exploring the epigenetic landscapes of heterogeneous cell types and revealing the mechanisms of gene expression regulation during developmental and pathological processes at the single-cell level. HIGHLIGHTS: scCPA-Tag offers a highly efficient and high throughput technique to simultaneously profile histone modification and chromatin accessibility within a single cell. scCPA-Tag enables to uncover multiple epigenetic modification features of cellular compositions within tumor tissues. scCPA-Tag facilitates the exploration of the epigenetic landscapes of heterogeneous cell types and provides the mechanisms governing gene expression regulation.

A Bioinformatics-Based Study on Methylation Alterations of the FBLN1 Gene in Hippocampal Tissue of Alzheimer's Disease Model DKO and DTG Mice.

Alzheimer's disease (AD) is characterized by progressive cognitive decline and late-stage neurobehavioral issues marked by amyloid-beta plaques and Tau protein tangles. This study aims to investigate Fibulin-1(FBLN1) gene expression in the hippocampal tissue of Presenilin-1/Presenilin-2 conditional double-knockout (DKO) and double-transgenic (DTG) mice, using single-cell sequencing and experimental methods to verify abnormal methylation status and correlation with AD. Genomic DNA from DKO and DTG mice was used for genotyping. Reduced Representation Bisulfite Sequencing (RRBS) identified 10 genes with abnormal methylation changes, with protein-protein interaction (PPI) analysis highlighting five core genes, including FBLN1. Single-cell sequencing, RT-PCR, and Western blotting (WB) were used to analyze FBLN1 mRNA and protein levels in the hippocampal tissues of early-stage and mid-stage AD DKO, DTG, and CBAC57 mice. RRBS identified 10 genes with abnormal methylation, with PPI highlighting five core genes. Single-cell sequencing showed significant FBLN1 expression in AD groups. RT-PCR and WB indicated elevated FBLN1 mRNA and protein levels in mid-stage AD DKO and DTG mice compared to CBAC57 mice, with no differences in early-stage AD DKO and CBAC57 mice. RRBS revealed hypomethylation of the FBLN1 gene in mid-stage AD DKO mice. Elevated FBLN1 expression in AD models suggests an age-dependent neurodegenerative mechanism independent of amyloid-beta deposition. This study enhances our understanding of AD's epigenetic mechanisms, which will aid targeted diagnosis, treatment, and prognosis.

Single-cell landscape revealed immune characteristics associated with disease phases in brucellosis patients.

A comprehensive immune landscape for Brucella infection is crucial for developing new treatments for brucellosis. Here, we utilized single-cell RNA sequencing (scRNA-seq) of 290,369 cells from 35 individuals, including 29 brucellosis patients from acute (n = 10), sub-acute (n = 9), and chronic (n = 10) phases as well as six healthy donors. Enzyme-linked immunosorbent assays were applied for validation within this cohort. Brucella infection caused a significant change in the composition of peripheral immune cells and inflammation was a key feature of brucellosis. Acute patients are characterized by potential cytokine storms resulting from systemic upregulation of S100A8/A9, primarily due to classical monocytes. Cytokine storm may be mediated by activating S100A8/A9-TLR4-MyD88 signaling pathway. Moreover, monocytic myeloid-derived suppressor cells were the probable contributors to immune paralysis in acute patients. Chronic patients are characterized by a dysregulated Th1 response, marked by reduced expression of IFN-γ and Th1 signatures as well as a high exhausted state. Additionally, Brucella infection can suppress apoptosis in myeloid cells (e.g., mDCs, classical monocytes), inhibit antigen presentation in professional antigen-presenting cells (APCs; e.g., mDC) and nonprofessional APCs (e.g., monocytes), and induce exhaustion in CD8+ T/NK cells, potentially resulting in the establishment of chronic infection. Overall, our study systemically deciphered the coordinated immune responses of Brucella at different phases of the infection, which facilitated a full understanding of the immunopathogenesis of brucellosis and may aid the development of new effective therapeutic strategies, especially for those with chronic infection.

Single-Cell Transcriptomic Profiling of Cholangiocyte Organoids Derived from Bile Ducts of Primary Sclerosing Cholangitis Patients.

BACKGROUND AND AIMS: Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disorder without effective medical treatment which is characterized by inflammation and fibrotic structures around the bile ducts. Biliary epithelial cells (cholangiocytes) are the target and potential disease drivers in PSC, yet little is known if cholangiocytes from PSC patients differ from non-PSC controls. To characterize cholangiocytes at early rather than end-stage disease, cholangiocyte organoids (COs) were derived from diseased bile ducts of PSC patients and compared to organoids generated from disease controls. METHODS: Cholangiocytes were obtained during endoscopic retrograde cholangiopancreatography (ERCP) brushing of diseased bile duct areas and expanded as organoids using previously established culture methods. Stable CO lines were analyzed for cell type identity, basic cholangiocyte function, and transcriptomic signature. RESULTS: We demonstrate that cholangiocytes, derived from the damaged area within the bile ducts of PSC patients, can be expanded in culture without displaying functional or genetic disease-related features. We further show that COs from patients who later were diagnosed with dysplasia exhibit higher expression of the cancer-associated genes PGC, FXYD2, MIR4435-2HG, and HES1. CONCLUSIONS: Our results demonstrate that PSC organoids are largely similar to control organoids after culture and highlight the significance of COs as a tool for regenerative medicine approaches as well as their potential for discovering new potential biomarkers for diagnosing cholangiocarcinoma.

Breaking barriers: improving time and space resolution of arbuscular mycorrhizal symbiosis with single-cell sequencing approaches.

The cell and molecular bases of arbuscular mycorrhizal (AM) symbiosis, a crucial plant-fungal interaction for nutrient acquisition, have been extensively investigated by coupling traditional RNA sequencing techniques of roots sampled in bulk, with methods to capture subsets of cells such as laser microdissection. These approaches have revealed central regulators of this complex relationship, yet the requisite level of detail to effectively untangle the intricacies of temporal and spatial development remains elusive.The recent adoption of single-cell RNA sequencing (scRNA-seq) techniques in plant research is revolutionizing our ability to dissect the intricate transcriptional profiles of plant-microbe interactions, offering unparalleled insights into the diversity and dynamics of individual cells during symbiosis. The isolation of plant cells is particularly challenging due to the presence of cell walls, leading plant researchers to widely adopt nuclei isolation methods. Despite the increased resolution that single-cell analyses offer, it also comes at the cost of spatial perspective, hence, it is necessary the integration of these approaches with spatial transcriptomics to obtain a comprehensive overview.To date, few single-cell studies on plant-microbe interactions have been published, most of which provide high-resolution cell atlases that will become crucial for fully deciphering symbiotic interactions and addressing future questions. In AM symbiosis research, key processes such as the mutual recognition of partners during arbuscule development within cortical cells, or arbuscule senescence and degeneration, remain poorly understood, and these advancements are expected to shed light on these processes and contribute to a deeper understanding of this plant-fungal interaction.

TooManyCellsInteractive: A visualization tool for dynamic exploration of single-cell data.

BACKGROUND: As single-cell sequencing technologies continue to advance, the growing volume and complexity of the ensuing data present new analytical challenges. Large cellular populations from single-cell atlases are more difficult to visualize and require extensive processing to identify biologically relevant subpopulations. Managing these workflows is also laborious for technical users and unintuitive for nontechnical users. RESULTS: We present TooManyCellsInteractive (TMCI), a browser-based JavaScript application for interactive exploration of cell populations. TMCI provides an intuitive interface to visualize and manipulate a radial tree representation of hierarchical cell subpopulations and allows users to easily overlay, filter, and compare biological features at multiple resolutions. Here we describe the software architecture and demonstrate how we used TMCI in a pan-cancer analysis to identify unique survival pathways among drug-tolerant persister cells. CONCLUSIONS: TMCI will facilitate exploration and visualization of large-scale sequencing data in a user-friendly way. TMCI is freely available at https://github.com/schwartzlab-methods/too-many-cells-interactive. An example tree from data within this article is available at https://tmci.schwartzlab.ca/.

Single-cell sequencing analysis revealed that WDR72 was a novel cancer stem cells related gene in gastric cancer.

Background: Cancer stem cells (CSCs) are pivotal in tumor resistance to chemotherapy and gastric cancer's rapid proliferation and metastasis. We aimed to explore the CSCs-related genes in gastric cancer epithelial cells. Methods: The mRNA expression profile and single-cell sequencing data of gastric cancer were downloaded from the public database. Results: We identified WDR72 as a CSCs-related gene in gastric cancer epithelial cells. WDR72 was highly expressed in gastric cancer tissues, and high expression of WDR72 was associated with inferior prognosis of patients. WDR72 expression had a significant negative correlation with the infiltration of CD8 + T cells and activated memory CD4 + T cells. PD-L1 expression was significantly reduced in gastric cancer patients with high WDR72 expression. WDR72 was correlated with IC50 of multiple small-molecule drugs. Conclusion: We identified a novel CSCs-related gene in gastric cancer epithelial cells, WDR72, which was highly expressed in patients with high stemness scores.

Application of single cell sequencing technology in ovarian cancer research (review).

Ovarian cancer is a malignant tumor of ovary. It has the characteristics of difficult early diagnosis, poor late curative effect and high recurrence rate. It is the biggest disease that seriously threatens women's health. Single cell sequencing technology refers to sequencing the genetic information carried by it at the single cell level to obtain the gene sequence, transcript, protein and epigenetic expression profile information of a certain cell type and conduct integrated analysis. It has unique advantages in the study of tumor occurrence and evolution, and can provide new methods for the study of ovarian cancer. This paper reviews the single cell sequencing technology and its application in ovarian cancer.

l-Theanine Alleviates Ulcerative Colitis by Regulating Colon Immunity via the Gut Microbiota in an MHC-II-Dependent Manner.

Alterations to the gut microbiota are associated with ulcerative colitis (UC), whereas restoration of normobiosis can effectively alleviate UC. l-Theanine has been shown to reshape the gut microbiota and regulate gut immunity. To investigate the mechanisms by which l-theanine alleviates UC, we used l-theanine and l-theanine fecal microbiota solution to treat UC mice. In this study, we used l-theanine and l-theanine fecal microbiota solution to treat UC mice to explore the mechanism by which l-theanine alleviates UC. By reducing inflammation in the colon, we demonstrated that l-theanine alleviates symptoms of UC. Meanwhile, l-theanine can improve the abundance of microbiota related to short-chain fatty acid, bile acid, and tryptophan production. Single-cell sequencing results indicated that l-theanine-mediated suppression of UC was associated with immune cell changes, especially regarding macrophages and T and B cells, and validated the immune cell responses to the gut microbiota. Further, flow cytometry results showed that the ability of dendritic cells, macrophages, and monocytes to present microbiota antigens to colonic T cells in an MHC-II-dependent manner was reduced after treating normal mouse fecal donors with l-theanine. These results demonstrate that l-theanine modulates colon adaptive and innate immunity by regulating the gut microbiota in an MHC-II-dependent manner, thereby alleviating UC.

Combination anti-PD-1 and anti-CTLA-4 therapy generates waves of clonal responses that include progenitor-exhausted CD8+ T cells.

Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. Here, we perform paired single-cell RNA and T cell receptor (TCR) sequencing across time in 36 patients with stage IV melanoma treated with anti-PD-1, anti-CTLA-4, or combination therapy. We develop the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induces waves of clonal T cell responses that peak at distinct time points. Combination therapy results in greater magnitude of clonal responses at 6 and 9 weeks compared to single-agent therapies, including melanoma-specific CD8+ T cells and exhausted CD8+ T cell (TEX) clones. Focused analyses of TEX identify that anti-CTLA-4 induces robust expansion and proliferation of progenitor TEX, which synergizes with anti-PD-1 to reinvigorate TEX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies.

Single-cell RNA sequencing of OSCC primary tumors and lymph nodes reveals distinct origin and phenotype of fibroblasts.

Desmoplasia in fibroblasts within metastatic lymph nodes (MLNs) serves as an indicator of extranodal extension (ENE), which led mortality in oral squamous cell carcinoma (OSCC). However, systematic studies on fibroblasts in MLNs are lacking. Therefore, this study characterized the differences in phenotype, function, and origin of fibroblasts between primary tumors (PTs) and lymph nodes (LNs) in OSCC. We generated single-cell maps of PTs and paired MLNs and draining LNs from three OSCC patients. The transcriptomic atlas, pseudotime analysis, intercellular communication networks and enrichment analysis of the single cells were characterized. Phenotype and function heterogeneity of fibroblast cells between PTs and MLNs were further verified in vitro. Among 44,052 fibroblasts, we identified two distinct subpopulations of cancer-associated myofibroblastic cells (mCAFs): RGS4+ mCAF1 and COMP + mCAF2. Notably, they exhibited distinct distributions, with mCAF1 predominantly localized in the PTs and mCAF2 in the MLNs. Moreover, pseudotime analysis revealed their distinct origins: mCAF1 originated from inherent normal myofibroblastic cells in the PT, whereas mCAF2 originated from fibroblastic reticular cells in the LNs. Further functional experiments using primary fibroblasts revealed that, compared to mCAF1, mCAF2 in MLNs exhibited weaker crosstalk with immune cells but enhanced extracellular matrix activity, which is closely linked to ENE formation in OSCC. Additionally, we identified two fibroblast subgroups in a transforming state, indicating a potential epithelial-mesenchymal transition. Our research offers profound insights into the heterogeneity of fibroblasts between the PT and MLN in OSCC, serving as an essential resource for future drug discovery endeavors.

Enhancing insight into ferroptosis mechanisms in sepsis: A genomic and pharmacological approach integrating single-cell sequencing and Mendelian randomization.

This research investigated the intricate relationship between ferroptosis and sepsis by utilizing advanced genomic and pharmacological methodologies. Specifically, we obtained expression quantitative trait loci (eQTLs) for 435 genes associated with ferroptosis from the eQTLGen Consortium and detected notable cis-eQTLs for 281 of these genes. Next, we conducted a detailed analysis to assess the impact of these eQTLs on susceptibility to sepsis using Mendelian randomization (MR) with data from a cohort of 10,154 sepsis patients and 452,764 controls sourced from the UK Biobank. MR analysis revealed 16 ferroptosis-related genes that exhibited significant associations with sepsis outcomes. To bolster the robustness of these findings, sensitivity analyses were performed to assess pleiotropy and heterogeneity, thus confirming the reliability of the causal inferences. Furthermore, single-cell RNA sequencing data from sepsis patients offered a detailed examination of gene expression profiles, demonstrating varying levels of ferroptosis marker expression across different cell types. Pathway enrichment analysis utilizing gene set enrichment analysis (GSEA) further revealed the key biological pathways involved in the progression of sepsis. Additionally, the use of computational molecular docking facilitated the prediction of interactions between identified genes and potential therapeutic compounds, highlighting novel drug targets. In conclusion, our integrated approach combining genomics and pharmacology offers valuable insights into the involvement of ferroptosis in sepsis, laying the groundwork for potential therapeutic strategies targeting this cell death pathway to enhance sepsis management.

Identification of the novel exhausted T cell CD8 + markers in breast cancer.

Cancer is one of the most concerning public health issues and breast cancer is one of the most common cancers in the world. The immune cells within the tumor microenvironment regulate cancer development. In this study, single immune cell data sets were used to identify marker gene sets for exhausted CD8 + T cells (CD8Tex) in breast cancer. Machine learning methods were used to cluster subtypes and establish the prognostic models with breast cancer bulk data using the gene sets to evaluate the impacts of CD8Tex. We analyzed breast cancer overexpressing and survival-associated marker genes and identified CD8Tex hub genes in the protein-protein-interaction network. The relevance of the hub genes for CD8 + T-cells in breast cancer was evaluated. The clinical associations of the hub genes were analyzed using bulk sequencing data and spatial sequencing data. The pan-cancer expression, survival, and immune association of the hub genes were analyzed. We identified biomarker gene sets for CD8Tex in breast cancer. CD8Tex-based subtyping systems and prognostic models performed well in the separation of patients with different immune relevance and survival. CRTAM, CLEC2D, and KLRB1 were identified as CD8Tex hub genes and were demonstrated to have potential clinical relevance and immune therapy impact. This study provides a unique view of the critical CD8Tex hub genes for cancer immune therapy.

6-Phosphogluconate dehydrogenase promotes glycolysis and fatty acid synthesis by inhibiting the AMPK pathway in lung adenocarcinoma cells.

Abnormal metabolism has emerged as a prominent hallmark of cancer and plays a pivotal role in carcinogenesis and progression of lung adenocarcinoma (LUAD). In this study, single-cell sequencing revealed that the metabolic enzyme 6-phosphogluconate dehydrogenase (PGD), which is a critical regulator of the pentose phosphate pathway (PPP), is significantly upregulated in the malignant epithelial cell subpopulation during malignant progression. However, the precise functional significance of PGD in LUAD and its underlying mechanisms remain elusive. Through the integration of TCGA database analysis and LUAD tissue microarray data, it was found that PGD expression was significantly upregulated in LUAD and closely correlated with a poor prognosis in LUAD patients. Moreover, in vitro and in vivo analyses demonstrated that PGD knockout and inhibition of its activity mitigated the proliferation, migration, and invasion of LUAD cells. Mechanistically, immunoprecipitation-mass spectrometry (IP-MS) revealed for the first time that IQGAP1 is a robust novel interacting protein of PGD. PGD decreased p-AMPK levels by competitively interacting with the IQ domain of the known AMPKα binding partner IQGAP1, which promoted glycolysis and fatty acid synthesis in LUAD cells. Furthermore, we demonstrated that the combination of Physcion (a PGD-specific inhibitor) and metformin (an AMPK agonist) could inhibit tumor growth more effectively both in vivo and in vitro. Collectively, these findings suggest that PGD is a potential prognostic biomarker and therapeutic target for LUAD.

Soma-germline communication drives sex maintenance in the Drosophila testis.

In adult gonads, disruption of somatic sexual identity leads to defective gametogenesis and infertility. However, the underlying mechanisms by which somatic signals regulate germline cells to achieve proper gametogenesis remain unclear. In our previous study, we introduced the chinmoSex Transformation (chinmoST ) mutant Drosophila testis phenotype as a valuable model for investigating the mechanisms underlying sex maintenance. In chinmoST testes, depletion of the Janus Kinase-Signal Transducer and Activator of Transcription downstream effector Chinmo from somatic cyst stem cells (CySCs) feminizes somatic cyst cells and arrests germline differentiation. Here, we use single-cell RNA sequencing to uncover chinmoST -specific cell populations and their transcriptomic changes during sex transformation. Comparative analysis of intercellular communication networks between wild-type and chinmoST testes revealed disruptions in several soma-germline signaling pathways in chinmoST testes. Notably, the insulin signaling pathway exhibited significant enhancement in germline stem cells (GSCs). Chinmo cleavage under targets and tagmentation (CUT&Tag) assay revealed that Chinmo directly regulates two male sex determination factors, doublesex (dsx) and fruitless (fru), as well as Ecdysone-inducible gene L2 (ImpL2), a negative regulator of the insulin signaling pathway. Further genetic manipulations confirmed that the impaired gametogenesis observed in chinmoST testes was partly contributed by dysregulation of the insulin signaling pathway. In summary, our study demonstrates that somatic sex maintenance promotes normal spermatogenesis through Chinmo-mediated conserved sex determination and the insulin signaling pathway. Our work offers new insights into the complex mechanisms of somatic stem cell sex maintenance and soma-germline communication at the single-cell level. Additionally, our discoveries highlight the potential significance of stem cell sex instability as a novel mechanism contributing to testicular tumorigenesis.

A single-cell atlas of the miracidium larva of Schistosoma mansoni reveals cell types, developmental pathways, and tissue architecture.

Schistosoma mansoni is a parasitic flatworm that causes the major neglected tropical disease schistosomiasis. The miracidium is the first larval stage of the life cycle. It swims and infects a freshwater snail, transforms into a mother sporocyst, where its stem cells generate daughter sporocysts that give rise to human-infective cercariae larvae. To understand the miracidium at cellular and molecular levels, we created a whole-body atlas of its ~365 cells. Single-cell RNA sequencing identified 19 transcriptionally distinct cell clusters. In situ hybridisation of tissue-specific genes revealed that 93% of the cells in the larva are somatic (57% neural, 19% muscle, 13% epidermal or tegument, 2% parenchyma, and 2% protonephridia) and 7% are stem. Whereas neurons represent the most diverse somatic cell types, trajectory analysis of the two main stem cell populations indicates that one of them is the origin of the tegument lineage and the other likely contains pluripotent cells. Furthermore, unlike the somatic cells, each of these stem populations shows sex-biased transcriptional signatures suggesting a cell-type-specific gene dosage compensation for sex chromosome-linked loci. The miracidium represents a simple developmental stage with which to gain a fundamental understanding of the molecular biology and spatial architecture of schistosome cells.