Tuberculosis (TB) has one of the highest mortality rates among infectious diseases worldwide. The immune response in the host after infection is proposed to contribute significantly to the progression of TB, but the specific mechanisms involved remain to be elucidated. Single-cell RNA sequencing (scRNA-seq) provides unbiased transcriptome sequencing of large quantities of individual cells, thereby defining biological comprehension of cellular heterogeneity and dynamic transcriptome state of cell populations in the field of immunology and is therefore increasingly applied to lung disease research. Here, we first briefly introduce the concept of scRNA-seq, followed by a summarization on the application of scRNA-seq to TB. Furthermore, we underscore the potential of scRNA-seq for clinical biomarker exploration, host-directed therapy, and precision therapy research in TB and discuss the bottlenecks that need to be overcome for the broad application of scRNA-seq to TB-related research.
Humans ; Single-Cell Analysis/methods* ; Tuberculosis/genetics* ; Sequence Analysis, RNA/methods* ; Transcriptome/genetics*

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Over the past decade, high-throughput RNA sequencing (RNA-seq) has vastly expanded our understanding of transcriptome dynamics in human physiology and disease. As a powerful tool for investigating systematic changes in RNA biology, RNA-seq has facilitated the discovery of novel functional RNA species. Mature RNA transcripts, which transmit genetic information from DNA to proteins, undergo intricate transcriptional and post-transcriptional regulation. This process allows a single gene to produce multiple RNA transcripts, each performing specific functions depending on the physiological or pathological context. Specific RNA transcripts (SRTs) are uniquely expressed in particular tissues or tumors and are closely associated with tissue-specific functions or disease states, particularly cancer. This review explores the generation of SRTs through key mechanisms, such as alternative splicing (AS), transcriptional regulation, polyadenylation (polyA), and the influence of transposable elements (TEs). We also examine their critical roles in normal tissue development and diseases, with an emphasis on their relevance to cancer. Furthermore, the potential applications of SRTs in diagnosing and treating diseases, especially malignancies, are discussed. By serving as diagnostic markers and therapeutic targets, SRTs hold significant promise in the development of personalized medicine and precision therapies. This review aims to provide new insights into the importance of SRTs in advancing the understanding and treatment of human diseases.
Humans ; Neoplasms/genetics* ; Alternative Splicing/genetics* ; RNA/genetics* ; Animals ; Sequence Analysis, RNA/methods* ; Polyadenylation/genetics*

Humans ; Consensus ; High-Throughput Nucleotide Sequencing/standards* ; Neoplasms/diagnosis* ; Quality Control ; Sequence Analysis, DNA/methods* ; Sequence Analysis, RNA/methods*

OBJECTIVES: Recent advancements in single-cell RNA sequencing (scRNA-seq) have revolutionized the study of cellular heterogeneity, particularly within the hematological system. However, accurately annotating cell types remains challenging due to the complexity of immune cells. To address this challenge, we develop a PAN-blood single-cell Data Annotator (scPANDA), which leverages a comprehensive 10-million-cell atlas to provide precise cell type annotation. METHODS: The atlas, constructed from data collected in 16 studies, incorporated rigorous quality control, preprocessing, and integration steps to ensure a high-quality reference for annotation. scPANDA utilizes a three-layer inference approach, progressively refining cell types from broad compartments to specific clusters. Iterative clustering and harmonization processes were employed to maintain cell type purity throughout the analysis. Furthermore, the performance of scPANDA was evaluated in three external datasets. RESULTS: The atlas was structured hierarchically, consisting of 16 compartments, 54 classes, 4,‍460 low-level clusters (pd_cc_cl_tfs), and 611 high-level clusters (pmid_cts). Robust performance of the tool was demonstrated in annotating diverse immune scRNA-seq datasets, analyzing immune-tumor coexisting clusters in renal cell carcinoma, and identifying conserved cell clusters across species. CONCLUSIONS scPANDA exemplifies effective reference mapping with a large-scale atlas, enhancing the accuracy and reliability of blood cell type identification.
Humans ; Single-Cell Analysis/methods* ; Sequence Analysis, RNA/methods* ; Blood Cells

OBJECTIVE: To distinguish the ambiguous genotyping results of human leukocyte antigen (HLA), identify a novel HLA-B allele and analyze the nucleotide sequence. METHODS: A total of 2 076 umbilical core blood samples from the Zhejiang Cord Blood Bank in 2022 were detected using the next generation sequencing technology (NGS) based on the Ion Torrent S5 platform. Among these a rare HLA-B allele with ambiguous combination result containing a base mutation was identified, and was further confimed by the third-generation sequencing (TGS) based on the nanopore technology. RESULTS: The NGS typing result of HLA-B locus showed HLA-B* 46:18, 54:06 or HLA-B*46:01, 54:XX (including a base mutation), and nanopore sequencing confirmed the typing as HLA-B*46:01, 54:XX (including a base mutation). Compared with HLA-B*54:01:01:01, the HLA-B*54:XX allele showed one single nucleotide substitution at position 1014 T>C in exon 6, with no amino acid change. The nucleotide sequence of the novel HLA-B*54:XX has been submitted to the GenBank nucleotide sequence database and the accession number OP853532 was assigned. CONCLUSION A ambiguous genotyping of the HLA-B Locus detected by NGS was distinguished by nanopore sequencing and a new HLA-B allele was successfully identified, which was officially named as HLA-B*54:01:11 by the World Health Organization Nomenclature Committee for Factors of the HLA System.
Humans ; High-Throughput Nucleotide Sequencing ; Alleles ; HLA-B Antigens/genetics* ; Genotype ; Mutation ; Sequence Analysis, DNA ; Base Sequence

OBJECTIVE: To investigate the molecular alterations of splenocytes associated with anti-factor Ⅷ (FⅧ) immune response and the underlying mechanisms based on hemophilia A (HA) murine model via RNA sequencing (RNA-seq) technology. METHODS: Severe HA mice were immunized with recombinant human factor Ⅷ (rhF8) weekly for 4 weeks to establish an FⅧ inhibitor model. High quality raw data were obtained by using bulk RNA-seq and CASAVA base identification technology, and the differentially expressed genes (DEGs) were identified. The DEGs were statistically classified by gene ontology (GO) annotation to obtain information on the major signaling pathways and biological processes involved in anti-FⅧ immune response in HA mouse splenocytes. The cell clusters, genes, and signaling pathway datasets were comprehensively analyzed by GO, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and single cell RNA-seq (ScRNA-seq) analysis, respectively. Flow cytometry analysis was used to verify the changes in T follicular helper cells (Tfh) and regulatory T cells (Treg). RESULTS: A total of 3731 DEGs was identified, including 2275 genes with up-regulated expression and 1456 genes with down-regulated expression. The DEGs were enriched in helper T cell differentiation, cytokine receptor, T cell receptor signaling pathway, ferroptosis, etc. Uniform Manifold Approximation and Project (UMAP) downscaling and visualization analysis yielded a total number of 11 T/NK cell subsets, visualizing the overall expression distribution of C-X-C chemokine-specific receptor gene cxcr5 among these T/NK cell subsets. Higher expression of cxcr5 was found in activated Tfh from FⅧ inhibitor mice, in comparison to the control group. The visualization using Upset plot R language showed a close interaction between Tfh and Treg. Moreover, the increased frequencies of Tfh and the decreased frequencies of Treg in inhibitor mouse splenocytes were further verified by flow cytometry analysis. CONCLUSION Multiple immune cell subsets, signaling pathways, and characteristic genes may be involved in the process of anti-FⅧ immune response in HA mouse splenocytes. The molecules involved in the regulation of Tfh/Treg may play key roles, which provide potential biological targets and therapeutic strategies for HA patients with inhibitors in the future.
Animals ; Hemophilia A/genetics* ; Mice ; Sequence Analysis, RNA ; Disease Models, Animal ; Spleen/cytology* ; T-Lymphocytes, Regulatory/immunology* ; Humans ; Signal Transduction ; Factor VIII/immunology* ; T-Lymphocytes, Helper-Inducer/immunology*

Objective： To investigate the relationship between the lipid metabolism-related gene sterol carrier protein 2(SCP2) and prostate cancer (PCa) from a multi-omics perspective using single-cell transcriptomes combined with Mendelian randomization. Methods： Single-cell transcriptome data of benign and malignant prostate tissues were obtained from GSE120716, GSE157703 and GSE141445 datasets, respectively. Integration, quality control and annotation were performed on the data to categorize the epithelial cells into high and low SCP2 expression groups, followed by further differential and trajectory analyses. Single nucleotide polymorphism (SNP) data for SCP2 expression quantitative trait loci (eQTL) were subsequently downloaded from Genotype-Tissue Expression (GTEx) and investigated from the PCa Society Cancer-Related Genomic Alteration Panel for the Investigation of Cancer-Related Alterations (PRACTICAL) to obtain PCa outcome data for Mendelian randomization analysis to validate the causal relationship between SCP2 and PCa. Results： High SCP2-expressing epithelial cells had higher energy metabolism and proliferation capacity with low immunotherapy response and metastatic tendency. Trajectory analysis showed that epithelial cells with high SCP2 expression may have a higher degree of malignancy, and SCP2 may be a key marker gene for differentiation of malignant epithelial cells in the prostate. Further Mendelian randomization results showed a significant causal relationship between SCP2 and PCa development (OR=1.045, 95% CI: 1.010 －1.083, P=0.011). Conclusion： By combining single-cell transcriptome and Mendelian randomization, the role of the lipid metabolism-related gene SCP2 in PCa development has been confirmed, and new targets and therapeutic directions for PCa treatment have been provided.
Humans ; Prostatic Neoplasms/genetics* ; Male ; Mendelian Randomization Analysis ; Polymorphism, Single Nucleotide ; Quantitative Trait Loci ; Single-Cell Analysis ; Sequence Analysis, RNA ; Carrier Proteins/genetics* ; Transcriptome ; Lipid Metabolism

Nasopharyngeal carcinoma（NPC） is a distinct type of head and neck cancer closely associated with Epstein-Barr virus（EBV） infection and exhibits significant geographic variations in its incidence. Despite recent advancements in radiotherapy techniques and precision medicine for NPC, the overall survival rate remains unsatisfactory due to tumor metastasis, recurrence, and drug resistance. Single-cell RNA sequencing（scRNA-seq） is an emerging technology that allows for the analysis of gene expression at single-cell resolution, providing a clearer understanding of tumor cell subpopulations, the evolutionary trajectory of tumor cells, and the functional roles and interactions of cells within the tumor microenvironment. This provides new ideas for the development of precision medicine in NPC. Here, we review the applications of scRNA-seq in exploring the mechanisms of NPC pathogenesis, tumor heterogeneity, the tumor microenvironment, drug resistance, and therapeutic response.
Humans ; Nasopharyngeal Neoplasms/genetics* ; Tumor Microenvironment ; Nasopharyngeal Carcinoma ; Single-Cell Analysis ; Sequence Analysis, RNA ; Precision Medicine ; Drug Resistance, Neoplasm ; Epstein-Barr Virus Infections ; Herpesvirus 4, Human ; Single-Cell Gene Expression Analysis

OBJECTIVES: To explore the expression profile of circular RNAs (circRNAs) and their potential roles in prognosis and progression of Wilms' tumor (WT). METHODS: Four pairs of WT and adjacent tissues were collected for high-throughput circRNA sequencing to identify the differentially expressed circular RNAs. RT-qPCR was used to verify the expression levels of the top 6 candidate circRNAs in the clinical samples. hsa_circ_0001900 was selected for analysis of its correlation with clinicopathological features and prognosis in 34 patients with WT. Sanger sequencing and RNase R digestion experiments were used to verify the cycling site and structural stability of hsa_circ_0001900 molecule. RESULTS: A total of 23 978 circular RNA molecules were identified in WT tissues by high-throughput circular RNA sequencing, and among them 614 were differentially expressed in WT. hsa_circ_0001900 showed the highest expression level among the differentially expressed circRNAs, which was consistent with the findings in clinical tumor samples and the sequencing results. Correlation analysis showed that hsa_circ_0001900 expression level was positively correlated with WT volume, and the children with high hsa_circ_0001900 expression had a lowered recurrence-free survival rate. The results of Sanger sequencing verified the circular splice site sequence of the molecule, and Rnase R digestion assay confirmed its stable covalent structure. CONCLUSIONS This study presents a comprehensive expression profile of circular RNAs in WT, and the expression level of hsa_circ_0001900 is related to the size of WT and the patients' prognosis, suggesting its possible role as a key driving gene in WT progression.
Humans ; RNA, Circular ; Wilms Tumor/pathology* ; Prognosis ; High-Throughput Nucleotide Sequencing ; Kidney Neoplasms/genetics* ; Sequence Analysis, RNA ; Male ; Female