DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development.

Orofacial clefts (OFCs) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the "casual" variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson correlation coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50-0.83. Specifically, our model revealed that SNPs significantly associated with OFCs tended to exhibit higher SAD scores across various variant categories compared to less related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFCs. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFCs, and the model can be extended to study other complex diseases or traits.

Long-Term Erythromycin Treatment Alters the Airway and Gut Microbiota: Data from Chronic Obstructive Pulmonary Disease Patients and Mice with Emphysema.

INTRODUCTION: Although long-term macrolide antibiotics could reduce the recurrent exacerbation of chronic obstructive pulmonary disease (COPD), the side effect of bacterial resistance and the impact on the microbiota remain concerning. We investigated the influence of long-term erythromycin treatment on the airway and gut microbiota in mice with emphysema and patients with COPD. METHODS: We conducted 16S rRNA gene sequencing to explore the effect of erythromycin treatment on the lung and gut microbiota in mice with emphysema. Liquid chromatography-mass spectrometry was used for lung metabolomics. A randomized controlled trial was performed to investigate the effect of 48-week erythromycin treatment on the airway and gut microbiota in COPD patients. RESULTS: The mouse lung and gut microbiota were disrupted after cigarette smoke exposure. Erythromycin treatment depleted harmful bacteria and altered lung metabolism. Erythromycin treatment did not alter airway or gut microbial diversity in COPD patients. It reduced the abundance of pathogens, such as Burkholderia, in the airway of COPD patients and increased levels of symbiotic bacteria, such as Prevotella and Veillonella. The proportions of Blautia, Ruminococcus, and Lachnospiraceae in the gut were increased in COPD patients after erythromycin treatment. The time to the first exacerbation following treatment was significantly longer in the erythromycin treatment group than in the COPD group. CONCLUSION: Long-term erythromycin treatment reduces airway and gut microbe abundance in COPD patients but does not affect microbial diversity and restores microbiota balance in COPD patients by reducing the abundance of pathogenic bacteria.

Development and external validation of a nomogram for predicting short-term prognosis in patients with acute pulmonary embolism.

BACKGROUND: Accurate assessment and timely intervention play a crucial role in ameliorating poor short-term prognosis of acute pulmonary embolism (APE) patients. The currently employed scoring models exhibit a degree of complexity, and some models may not comprehensively incorporate relevant indicators, thereby imposing limitations on the evaluative efficacy. Our study aimed to construct and externally validate a nomogram that predicts 30-day all-cause mortality risk in APE patients. METHODS: Clinical data from APE patients in Intensive Care-IV database was included as a training cohort. Additionally, we utilized our hospital's APE database as an external validation cohort. The nomogram was developed, and its predictive ability was evaluated using receiver operating characteristic (ROC) curves, calibration plots and decision curve analysis. RESULTS: A collective of 1332 patients and 336 patients were respectively enrolled as the training cohort and the validation cohort in this study. Five variables including age, malignancy, oxygen saturation, blood glucose, and the use of vasopressor, were identified based on the results of the multivariate Cox regression model. The ROC value for the nomogram in the training cohort yielded 0.765, whereas in the validation group, it reached 0.907. Notably, these values surpassed the corresponding ROC values for the Pulmonary Embolism Severity Index, which were 0.713 in the training cohort and 0.754 in the validation cohort. CONCLUSIONS: The nomogram including five indicators had a good performance in predicting short-term prognosis in patients with APE, which was easier to apply and provided better recommendations for clinical decision-making.

Genome-wide CRISPR screen reveals the synthetic lethality between BCL2L1 inhibition and radiotherapy.

Radiation therapy (RT) is one of the most commonly used anticancer therapies. However, the landscape of cellular response to irradiation, especially to a single high-dose irradiation, remains largely unknown. In this study, we performed a whole-genome CRISPR loss-of-function screen and revealed temporal inherent and acquired responses to RT. Specifically, we found that loss of the IL1R1 pathway led to cellular resistance to RT. This is in part because of the involvement of radiation-induced IL1R1-dependent transcriptional regulation, which relies on the NF-κB pathway. Moreover, the mitochondrial anti-apoptotic pathway, particularly the BCL2L1 gene, is crucially important for cell survival after radiation. BCL2L1 inhibition combined with RT dramatically impeded tumor growth in several breast cancer cell lines and syngeneic models. Taken together, our results suggest that the combination of an apoptosis inhibitor such as a BCL2L1 inhibitor with RT may represent a promising anticancer strategy for solid cancers including breast cancer.

An atlas of epithelial cell states and plasticity in lung adenocarcinoma.

Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies1. Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8+ alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8+ cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Single-cell multiomics decodes regulatory programs for mouse secondary palate development.

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Evolution of immune and stromal cell states and ecotypes during gastric adenocarcinoma progression.

Understanding tumor microenvironment (TME) reprogramming in gastric adenocarcinoma (GAC) progression may uncover novel therapeutic targets. Here, we performed single-cell profiling of precancerous lesions, localized and metastatic GACs, identifying alterations in TME cell states and compositions as GAC progresses. Abundant IgA+ plasma cells exist in the premalignant microenvironment, whereas immunosuppressive myeloid and stromal subsets dominate late-stage GACs. We identified six TME ecotypes (EC1-6). EC1 is exclusive to blood, while EC4, EC5, and EC2 are highly enriched in uninvolved tissues, premalignant lesions, and metastases, respectively. EC3 and EC6, two distinct ecotypes in primary GACs, associate with histopathological and genomic characteristics, and survival outcomes. Extensive stromal remodeling occurs in GAC progression. High SDC2 expression in cancer-associated fibroblasts (CAFs) is linked to aggressive phenotypes and poor survival, and SDC2 overexpression in CAFs contributes to tumor growth. Our study provides a high-resolution GAC TME atlas and underscores potential targets for further investigation.

Pan-cancer T cell atlas links a cellular stress response state to immunotherapy resistance.

Tumor-infiltrating T cells offer a promising avenue for cancer treatment, yet their states remain to be fully characterized. Here we present a single-cell atlas of T cells from 308,048 transcriptomes across 16 cancer types, uncovering previously undescribed T cell states and heterogeneous subpopulations of follicular helper, regulatory and proliferative T cells. We identified a unique stress response state, TSTR, characterized by heat shock gene expression. TSTR cells are detectable in situ in the tumor microenvironment across various cancer types, mostly within lymphocyte aggregates or potential tertiary lymphoid structures in tumor beds or surrounding tumor edges. T cell states/compositions correlated with genomic, pathological and clinical features in 375 patients from 23 cohorts, including 171 patients who received immune checkpoint blockade therapy. We also found significantly upregulated heat shock gene expression in intratumoral CD4/CD8+ cells following immune checkpoint blockade treatment, particularly in nonresponsive tumors, suggesting a potential role of TSTR cells in immunotherapy resistance. Our well-annotated T cell reference maps, web portal and automatic alignment/annotation tool could provide valuable resources for T cell therapy optimization and biomarker discovery.

oHSV-P10 reduces glioma stem cell enrichment after oncolytic HSV therapy.

Longstanding evidence implicate glioma stem-like cells as the main drivers contributing toward glioblastoma (GBM) therapy resistance and tumor recurrence. Although oncolytic herpes simplex virus (oHSV) viral therapy is a promising biological therapy recently approved for melanoma (in the United States and Europe) and GBM (in Japan); however, the impact of this therapy on GBM stem-like cells (GSCs) is understudied. Here we show that post-oHSV virotherapy activated AKT signaling results in an enrichment of GSC signatures in glioma, which mimics the enrichment in GSC observed after radiation treatment. We also uncovered that a second-generation oncolytic virus armed with PTEN-L (oHSV-P10) decreases this by moderating IL6/JAK/STAT3 signaling. This ability was retained in the presence of radiation treatment and oHSV-P10-sensitized intracranial GBM to radiotherapy. Collectively, our findings uncover potential mechanisms to overcome GSC-mediated radiation resistance via oHSV-P10.

PKR induces TGF-ß and limits oncolytic immune therapy.

BACKGROUND: Mammalian cells have developed multiple intracellular mechanisms to defend against viral infections. These include RNA-activated protein kinase (PKR), cyclic GMP-AMP synthase and stimulation of interferon genes (cGAS-STING) and toll-like receptor-myeloid differentiation primary response 88 (TLR-MyD88). Among these, we identified that PKR presents the most formidable barrier to oncolytic herpes simplex virus (oHSV) replication in vitro. METHODS: To elucidate the impact of PKR on host responses to oncolytic therapy, we generated a novel oncolytic virus (oHSV-shPKR) which disables tumor intrinsic PKR signaling in infected tumor cells. RESULTS: As anticipated, oHSV-shPKR resulted in suppression of innate antiviral immunity and improves virus spread and tumor cell lysis both in vitro and in vivo. Single cell RNA sequencing combined with cell-cell communication analysis uncovered a strong correlation between PKR activation and transforming growth factor beta (TGF-ß) immune suppressive signaling in both human and preclinical models. Using a murine PKR targeting oHSV, we found that in immune-competent mice this virus could rewire the tumor immune microenvironment to increase the activation of antigen presentation and enhance tumor antigen-specific CD8 T cell expansion and activity. Further, a single intratumoral injection of oHSV-shPKR significantly improved the survival of mice bearing orthotopic glioblastoma. To our knowledge, this is the first report to identify dual and opposing roles of PKR wherein PKR activates antivirus innate immunity and induces TGF-ß signaling to inhibit antitumor adaptive immune responses. CONCLUSIONS: Thus, PKR represents the Achilles heel of oHSV therapy, restricting both viral replication and antitumor immunity, and an oncolytic virus that can target this pathway significantly improves response to virotherapy.

deCS: A Tool for Systematic Cell Type Annotations of Single-cell RNA Sequencing Data among Human Tissues.

Single-cell RNA sequencing (scRNA-seq) is revolutionizing the study of complex and dynamic cellular mechanisms. However, cell type annotation remains a main challenge as it largely relies on a priori knowledge and manual curation, which is cumbersome and subjective. The increasing number of scRNA-seq datasets, as well as numerous published genetic studies, has motivated us to build a comprehensive human cell type reference atlas.Here, we present decoding Cell type Specificity (deCS), an automatic cell type annotation method augmented by a comprehensive collection of human cell type expression profiles and marker genes. We used deCS to annotate scRNA-seq data from various tissue types and systematically evaluated the annotation accuracy under different conditions, including reference panels, sequencing depth, and feature selection strategies. Our results demonstrate that expanding the references is critical for improving annotation accuracy. Compared to many existing state-of-the-art annotation tools, deCS significantly reduced computation time and increased accuracy. deCS can be integrated into the standard scRNA-seq analytical pipeline to enhance cell type annotation. Finally, we demonstrated the broad utility of deCS to identify trait-cell type associations in 51 human complex traits, providing deep insights into the cellular mechanisms underlying disease pathogenesis. All documents for deCS, including source code, user manual, demo data, and tutorials, are freely available at https://github.com/bsml320/deCS.

Integrated analysis of racial disparities in genomic architecture identifies a trans-ancestry prognostic subtype in bladder cancer.

The incidence of bladder cancer and patient survival vary greatly among different populations, but the influence of the associated molecular features and evolutionary processes on its clinical treatment and prognostication remains unknown. Here, we analyze the genomic architectures of 505 bladder cancer patients from Asian/Black/White populations. We identify a previously unknown association between AHNAK mutations and activity of the APOBEC-a mutational signature, the activity of which varied substantially across populations. All significantly mutated genes but only half of arm-level somatic copy number alterations (SCNAs) are enriched with clonal events, indicating large-scale SCNAs as rich sources of bladder cancer clonal diversities. The prevalence of TP53 and ATM clonal mutations as well as the associated burden of SCNAs is significantly higher in Whites/Blacks than in Asians. We identify a trans-ancestry prognostic subtype of bladder cancer characterized by enrichment of non-muscle-invasive patients and muscle-invasive patients with good prognosis, increased CREBBP/FGFR3/HRAS/NFE2L2 mutations, decreased intra-tumor heterogeneity and genome instability, and an activated tumor microenvironment.

Innately expressed estrogen-related receptors in the skeletal muscle are indispensable for exercise fitness.

Transcriptional determinants in the skeletal muscle that govern exercise capacity, while poorly defined, could provide molecular insights into how exercise improves fitness. Here, we have elucidated the role of nuclear receptors, estrogen-related receptor alpha and gamma (ERRα/γ) in regulating myofibrillar composition, contractility, and exercise capacity in skeletal muscle. We used muscle-specific single or double (DKO) ERRα/γ knockout mice to investigate the effect of ERRα/γ deletion on muscle and exercise parameters. Individual knockout of ERRα/γ did not have a significant impact on the skeletal muscle. On the other hand, DKO mice exhibit pale muscles compared to wild-type (WT) littermates. RNA-seq analysis revealed a predominant decrease in expression of genes linked to mitochondrial and oxidative metabolism in DKO versus WT muscles. DKO muscles exhibit marked repression of oxidative enzymatic capacity, as well as mitochondrial number and size compared to WT muscles. Mitochondrial function is also impaired in single myofibers isolated from DKO versus WT muscles. In addition, mutant muscles exhibit reduced angiogenic gene expression and decreased capillarity. Consequently, DKO mice have a significantly reduced exercise capacity, further reflected in poor fatigue resistance of DKO mice in in vivo contraction assays. These results show that ERRα and ERRγ together are a critical link between muscle aerobic capacity and exercise tolerance. The ERRα/γ mutant mice could be valuable for understanding the long-term impact of impaired mitochondria and vascular supply on the pathogenesis of muscle-linked disorders.

USP7 substrates identified by proteomics analysis reveal the specificity of USP7.

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

Delineating COVID-19 immunological features using single-cell RNA sequencing.

Understanding the molecular mechanisms of coronavirus disease 2019 (COVID-19) pathogenesis and immune response is vital for developing therapies. Single-cell RNA sequencing has been applied to delineate the cellular heterogeneity of the host response toward COVID-19 in multiple tissues and organs. Here, we review the applications and findings from over 80 original COVID-19 single-cell RNA sequencing studies as well as many secondary analysis studies. We describe that single-cell RNA sequencing reveals multiple features of COVID-19 patients with different severity, including cell populations with proportional alteration, COVID-19-induced genes and pathways, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in single cells, and adaptation of immune repertoire. We also collect published single-cell RNA sequencing datasets from original studies. Finally, we discuss the limitations in current studies and perspectives for future advance.

Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.

Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.

GDF5+ chondroprogenitors derived from human pluripotent stem cells preferentially form permanent chondrocytes.

It has been established in the mouse model that during embryogenesis joint cartilage is generated from a specialized progenitor cell type, distinct from that responsible for the formation of growth plate cartilage. We recently found that mesodermal progeny of human pluripotent stem cells gave rise to two types of chondrogenic mesenchymal cells in culture: SOX9+ and GDF5+ cells. The fast-growing SOX9+ cells formed in vitro cartilage that expressed chondrocyte hypertrophy markers and readily underwent mineralization after ectopic transplantation. In contrast, the slowly growing GDF5+ cells derived from SOX9+ cells formed cartilage that tended to express low to undetectable levels of chondrocyte hypertrophy markers, but expressed PRG4, a marker of embryonic articular chondrocytes. The GDF5+-derived cartilage remained largely unmineralized in vivo. Interestingly, chondrocytes derived from the GDF5+ cells seemed to elicit these activities via non-cell-autonomous mechanisms. Genome-wide transcriptomic analyses suggested that GDF5+ cells might contain a teno/ligamento-genic potential, whereas SOX9+ cells resembled neural crest-like progeny-derived chondroprogenitors. Thus, human pluripotent stem cell-derived GDF5+ cells specified to generate permanent-like cartilage seem to emerge coincidentally with the commitment of the SOX9+ progeny to the tendon/ligament lineage.

Factors affecting the length of stay and hospital readmission rates after an acute exacerbation of chronic obstructive pulmonary disease: a systematic review and meta-analysis.

Background: Patients with chronic obstructive pulmonary disease (COPD) are often readmitted to hospital for treatment due to an acute exacerbation of the disease. However, there are few up-to-date studies investigating the lengths of stay and risk factors for readmission after an acute exacerbation of COPD. This study evaluated the length of stay in patients with an acute exacerbation of COPD and the factors that influenced their readmission. Methods: A search of the PubMed, Cochrane Central Register of Controlled Trials, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), Wanfang, and Weipu databases, carried out using the following search terms: airflow limitation, airway disease, airway obstruction, chronic obstructive pulmonary disease, COPD, length of stay and influencing factors, long-term oxygen therapy, lung disease, readmission, and respiratory system disease. The Cochrane risk of bias tool was used to evaluate the quality of the retrieved studies, and a network meta-analysis was performed using RevMan 5.20. Results: Collectively, they included information on the length of stay for 630 patients who had been readmitted to hospital after an acute exacerbation of COPD (the readmitted group) and information on 688 patients who had not been readmitted (the non-readmitted group). Meta-analysis results showed that there was no difference in patient anxiety [risk ratio (RR) 1.22, 95% confidence interval (CI): 0.70-2.14] or long-term oxygen therapy (RR 1.91, 95% CI: 0.98-3.73) between the readmitted group and the non-readmitted group. However, there was a significant difference between the forced expiratory volume in one second (FEV1) predicted value [mean difference (MD) -5.85, 95% CI: -11.14 to -0.57] and the global initiative for chronic obstructive lung disease (GOLD) classification (C or D) (RR 1.61, 95% CI: 1.05-2.47). Discussion: In summary, no significant relationship was found between patient state of anxiety, long-term oxygen therapy, length of hospital stay, and readmission rate after an acute exacerbation of COPD. However, FEV1 predicted values and GOLD classifications (C or D) had an impact on the length of hospital stay and readmission rate after acute exacerbation of COPD. Larger samples, multiple centers, and further research are needed to confirm the findings of this research.