Single-Cell Mapping of Brain Myeloid Cell Subsets Reveals Key Transcriptomic Changes Favoring Neuroplasticity after Ischemic Stroke / 神经科学通报·英文版

Interactions between brain-resident and peripheral infiltrated immune cells are thought to contribute to neuroplasticity after cerebral ischemia. However, conventional bulk sequencing makes it challenging to depict this complex immune network. Using single-cell RNA sequencing, we mapped compositional and transcriptional features of peri-infarct immune cells. Microglia were the predominant cell type in the peri-infarct region, displaying a more diverse activation pattern than the typical pro- and anti-inflammatory state, with axon tract-associated microglia (ATMs) being associated with neuronal regeneration. Trajectory inference suggested that infiltrated monocyte-derived macrophages (MDMs) exhibited a gradual fate trajectory transition to activated MDMs. Inter-cellular crosstalk between MDMs and microglia orchestrated anti-inflammatory and repair-promoting microglia phenotypes and promoted post-stroke neurogenesis, with SOX2 and related Akt/CREB signaling as the underlying mechanisms. This description of the brain's immune landscape and its relationship with neurogenesis provides new insight into promoting neural repair by regulating neuroinflammatory responses.

Visual Analysis of Research on Lung Cancer Immunotherapy by Using CiteSpace / 肿瘤防治研究

Objective To understand the current status of research on lung cancer immunotherapy to provide a reference for further investigation and future topic selection in this field. Methods CiteSpace visualization analysis software was used to analyze 400 Chinese studies in CNKI and 5 001 English studies in the Web of Science database from 2005 to 2021, with "lung cancer" and "immunotherapy" as keywords. Keyword co-occurrence analysis was performed on 17 English studies of "Lung Cancer" "Immunotherapy" and "Single cell sequencing" in the Web of Science database. Results "Non-small cell lung cancer" "immunosuppressants" "PD-L1" "dendritic cells" and "cytokine-induced killer cells" are current research hotspots in lung cancer immunotherapy. Monoclonal antibody drugs including nivolumab, pembrolizumab, atezolizumab, and durvalumab are hotspot drugs. Immunotherapy combined with chemotherapy as well as PD-L1 expression have become the focus of continuous research. The majority of studies on lung cancer immunotherapy are conducted in the United States, followed by China. Conclusion Lung cancer immunotherapy has gradually become a research hot spot in China. In the future, in-depth research is needed to provide cutting-edge directions for lung cancer immunotherapy.

Evaluation of peripheral blood T-lymphocyte subpopulations features in patients with hepatitis B virus-related acute-on-chronic liver failure based on single-cell sequencing technology / 中华肝脏病杂志

Objective: T lymphocyte exhaustion is an important component of immune dysfunction. Therefore, exploring peripheral blood-exhausted T lymphocyte features in patients with hepatitis B virus-related acute-on-chronic liver failure may provide potential therapeutic target molecules for ACLF immune dysfunction. Methods: Six cases with HBV-ACLF and three healthy controls were selected for T-cell heterogeneity detection using the single-cell RNA sequencing method. In addition, exhausted T lymphocyte subpopulations were screened to analyze their gene expression features, and their developmental trajectories quasi-timing. An independent sample t-test was used to compare the samples between the two groups. Results: Peripheral blood T lymphocytes in HBV-ACLF patients had different differentiation trajectories with different features distinct into eight subpopulations. Among them, the CD4(+)TIGIT(+) subsets (P = 0.007) and CD8(+)LAG3(+) (P = 0.010) subsets with highly exhausted genes were significantly higher than those in healthy controls. Quasi-time analysis showed that CD4(+)TIGIT(+) and CD8(+)LAG3(+) subsets appeared in the late stage of T lymphocyte differentiation, suggesting the transition of T lymphocyte from naïve-effector-exhausted during ACLF pathogenesis. Conclusion: There is heterogeneity in peripheral blood T lymphocyte differentiation in patients with HBV-ACLF, and the number of exhausted T cells featured by CD4(+)TIGIT(+)T cell and CD8(+)LAG3(+) T cell subsets increases significantly, suggesting that T lymphocyte immune exhaustion is involved in the immune dysfunction of HBV-ACLF, thereby identifying potential effective target molecules for improving ACLF patients' immune function.

Identification of SULF1 as a Shared Gene in Idiopathic Pulmonary Fibrosis and Lung Adenocarcinoma / 中国肺癌杂志

BACKGROUND@#Idiopathic pulmonary fibrosis (IPF) is an idiopathic chronic, progressive interstitial lung disease with a diagnosed median survival of 3-5 years. IPF is associated with an increased risk of lung cancer. Therefore, exploring the shared pathogenic genes and molecular pathways between IPF and lung adenocarcinoma (LUAD) holds significant importance for the development of novel therapeutic approaches and personalized precision treatment strategies for IPF combined with lung cancer.@*METHODS@#Bioinformatics analysis was conducted using publicly available gene expression datasets of IPF and LUAD from the Gene Expression Omnibus (GEO) database. Weighted gene co-expression network analysis was employed to identify common genes involved in the progression of both diseases, followed by functional enrichment analysis. Subsequently, additional datasets were used to pinpoint the core shared genes between the two diseases. The relationship between core shared genes and prognosis, as well as their expression patterns, clinical relevance, genetic characteristics, and immune-related functions in LUAD, were analyzed using The Cancer Genome Atlas (TCGA) database and single-cell RNA sequencing datasets. Finally, potential therapeutic drugs related to the identified genes were screened through drug databases.@*RESULTS@#A total of 529 shared genes between IPF and LUAD were identified. Among them, SULF1 emerged as a core shared gene associated with poor prognosis. It exhibited significantly elevated expression levels in LUAD tissues, concomitant with high mutation rates, genomic heterogeneity, and an immunosuppressive microenvironment. Subsequent single-cell RNA-seq analysis revealed that the high expression of SULF1 primarily originated from tumor-associated fibroblasts. This study further demonstrated an association between SULF1 expression and tumor drug sensitivity, and it identified potential small-molecule drugs targeting SULF1 highly expressed fibroblasts.@*CONCLUSIONS@#This study identified a set of shared molecular pathways and core genes between IPF and LUAD. Notably, SULF1 may serve as a potential immune-related biomarker and therapeutic target for both diseases.

Characterization of Cell Subsets Associated With Prognosis of Osteosarcoma Based on Single-Cell Sequencing Data / 中国医学科学院学报

Objective To explore the cell subsets and characteristics related to the prognosis of osteosarcoma by analyzing the cellular composition of tumor tissue samples from different osteosarcoma patients.Methods The single-cell sequencing data and bulk sequencing data of different osteosarcoma patients were downloaded.We extracted the information of cell samples for dimensionality reduction,annotation,and cell function analysis,so as to identify the cell subsets and clarify the cell characteristics related to the prognosis of osteosarcoma.The development trajectory of macrophages with prognostic significance was analyzed,and the prognostic model of osteosarcoma was established based on the differentially expressed genes of macrophage differentiation.Results The cellular composition presented heterogeneity in the patients with osteosarcoma.The infiltration of mononuclear phagocytes in osteosarcoma had prognostic significance(P=0.003).Four macrophage subsets were associated with prognosis,and their signature transcription factors included RUNX3(+),ETS1(+),HOXD11(+),ZNF281(+),and PRRX1(+).Prog_Macro2 and Prog_Macro4 were located at the end of the developmental trajectory,and the prognostic ability of macrophage subsets increased with the progression of osteosarcoma.The prognostic model established based on the differentially expressed genes involved in macrophage differentiation can distinguish the survival rate of osteosarcoma patients with different risks(P<0.001).Conclusion Macrophage subsets are closely related to the prognosis of osteosarcoma and can be used as the key target cells for the immunotherapy of osteosarcoma.

Advances in single-cell RNA sequencing in the retina / 中华眼底病杂志

Retina is composed of a heterogeneous population of cell types, each with a unique biological function. Even if the same type of cells, due to genetic heterogeneity will lead to cell function differences. In the past, traditional molecular biological methods cannot resolve variations in their functional roles that arise from these differences, and some cells are difficult to define due to the lack of specific molecular markers or the scarcity of numbers, which hindered the understanding and research of these cells. With the development of biotechnology, single-cell RNA sequencing can analyze and resolve differences in single-cell transcriptome expression profiles, characterize intracellular population heterogeneity, identify new and rare cell subtypes, and more definitely define the characteristics of each cell type. It clarifies the origin, function, and variations in cell phenotypes. Other attributes include pinpointing both disease-related characteristics of cell subtypes and specific differential gene expression patterns, to deepen our understanding of the causes and progression of diseases, as well as to aid clinical diagnosis and targeted therapy.

Comparison of pretreatment techniques for single-cell RNA sequencing of airway aspirate specimens / 中华微生物学和免疫学杂志

Objective:To investigate the appropriate pretreatment methods for single cell RNA sequencing of airway aspirate cells.Methods:Four fresh airway aspirate specimens were collected from four patients with acute respiratory tract infections. These specimens were digested with airway aspirate digester and prepared into single cell suspension. The cells were used for library construction directly (DE), or fixed with 10×Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit and then mixed to construct the library (DF), or cryopreserved, thawed, fixed (FF) before mixed to construct the library. All three methods were treated with oil emulsion using 10 4 cells and subjected to single-cell sequencing using the 10×Genomics platform. The number of obtained cells, data quality, annotated cell types and expression of marker genes were analyzed. Differences in the expression of highly variable genes (HVGs) of the same cell subsets obtained by the three pretreatment methods were compared using Pearson correlation. Expression of the differentially expressed genes in the same cell subpopulation obtained by different pretreatment methods was also compared. The correlation of the expression of differentially expressed genes between the same cell subsets obtained by the three pretreatment methods was analyzed by Pearson correlation. Results:The median numbers of single cells obtained using DE, FF and DF methods were 2 733, 1 140 and 5 897 ( P>0.05). The unique molecular identifiers were higher than 500. The median numbers of genes obtained using the three methods were 801, 887 and 1 259 ( P>0.05). The cells with novelty score over 0.8 accounted for 99%, 87% and 93%, respectively. There were nine cell subsets obtained by the three methods, including squamous cells, secretory cells, ciliated cells, T cells, B cells, macrophages, plasma cells and neutrophils. DF and FF methods could obtain more basal cells with specific high expression of keratin 5 than DE method. The differentially expressed and highly variable genes in the same cell subsets obtained by the three pretreatment methods showed high consistency in their expression with a significant correlation ( P<0.001). Conclusions:Under the same sequencing data volume, the quality of data obtained from fixed airway aspirate single-cell suspensions using the method of probe hybridization and transcriptome sequencing was comparable to that obtained directly from fresh cells. This method was more suitable for the pretreatment of clinical samples used for single-cell RNA sequencing.

Application of single cell RNA sequencing in the treatment of diabetes / 中华内分泌代谢杂志

Single-cell RNA sequencing (scRNA-seq) is used for transcriptome profiling at the individual cell level, which is capable of screening in differentially gene expression that results from genetic mutation. Islet-based developmental atlas and heterogeneity characterization are currently the main applications of scRNA-seq in diabetes. scRNA-seq also can be used to mark and purify the functional β cells from resident adult stem cells in the pancreatic islets, which is expected to improve the outcome of islet β cells transplantation in type 1 diabetic patients. In addition, the technique can aid in learning diabetic β cell dedifferentiation and immunomodulatory functions. Although the study of scRNA-seq in diabetic retinopathy, nephropathy, atherosclerosis, and peripheral neuropathy is still at a nascent stage, scRNA-seq has great potential in a wide range of biomedical and clinical applications.

Investigation of endothelial cell pathological characteristics in murine choroidal neovascularization model based on single-cell RNA sequencing / 中华实验眼科杂志

Objective:To investigate the molecular expression and pathological features of endothelial cell (EC) in a murine model of choroidal neovascularization (CNV) based on single-cell RNA sequencing (scRNA-seq).Methods:Six C57BL/6 mice aged 6-8 weeks were randomly divided into two groups, with 3 mice in each group.Bilateral eyeballs were enucleated.The choroidal tissues from the two groups were isolated by shearing the complex and scraping the choroid, respectively.Single-cell suspension was prepared by continuous digestion with trypsin/type Ⅰ collagenase at 37 ℃, and the cell viability and EC ratio were detected by flow cytometry to determine the preparation method of single-cell suspension.Another 6 mice were randomly assigned into the control group and the CNV group, with 3 mice in each group.The CNV model was induced by laser photocoagulation and single-cell suspensions were prepared 7 days after modeling.Gene expression library construction was performed using the Chromi-um (10x Genomics) instrument.High throughput sequencing was performed using the Illumina Novaseq6000 to obtain the expression matrix.The EC subpopulations were classified according to previous researches and the Cellmarker database.Pseudo-time analysis was performed in EC, revealing the gene expression matrix of different states.CNV-EC were further selected with preliminary analysis of the expression characteristics.Another 6 mice were selected to establish the CNV model and eyeball frozen sections were prepared 7 days after modeling.Expression and distribution as well as the area percentage of EC marker Pecam1, mitochondrial outer membrane proteins Tomm20 and mt-Co1, and capillary markers Kdr and Plvap were observed by immunofluorescence staining, and the vascular diameter was calculated.The use and care of animals followed the ARVO statement.This study protocol was approved by the Experimental Animal Welfare and Ethics Committee of Air Force Military Medical University (No.20200181).Results:The cell viability of the single-cell suspension prepared from choroidal-scleral fragments and choroidal scrapings was 99.4% and 99.1%, respectively, both of which met the sequencing requirements.The percentage of EC detected by flow cytometry was approximately 1.58%.The scRNA-seq result revealed that both the normal control and CNV groups contained 13 choroidal cell clusters.Compared with the normal control group, the proportions of rod/cone photoreceptor cells, EC and hematopoietic cells all increased, while the retinal pigment epithelium (RPE) and Schwan cells reduced in the CNV group.Among all clusters, EC constituted 18.4%.The pseudo-time analysis demonstrated that EC could be further divided into 4 states.The percentage of state 2 EC was 29.1% in the CNV group, which was significantly higher than 9.5% in the normal control group.Differentially expressed gene analysis showed that the expression of mitochondrion-related genes, including mt-Nd4 and mt-Atp6, were upregulated in state 2 EC, while capillary-related genes, including Kdr and Esm1, were downregulated.Immunofluorescent staining revealed that the area of Tomm20 and mt-Co1 in Pecam1-positive EC in the CNV area was (19.50±4.68)% and (4.64±2.82)%, respectively, which were both higher than (3.00±2.09)% and (0.18±0.34)% in normal area ( t=7.88, 3.84; both at P<0.01). The area of Kdr and Plvap in Pecam1-positive EC in the CNV area was (1.50±0.29)% and (0.79±0.97)%, respectively, which were both lower than (31.30±5.44)% and (10.43±2.28)% in the normal area ( t=13.40, 9.48; both at P<0.01). The vascular diameter in the CNV area was (5.52±1.85)μm, which was larger than (4.21±1.84)μm in the normal area ( t=9.57, P<0.001). Conclusions:When CNV occurs, the proportion of EC in choroid increases, and CNV-EC shows pathologic features of mitochondrial metabolic activation and loss of capillary properties, suggesting the mitochondrial activation of EC may play a role in the formation of CNV.

Application of single cell sequencing technology in liver regeneration / 中华消化外科杂志

The liver is a highly proliferative organ. As the liver injured, the hepatocytes can quickly enter the cell cycle to restore the volume and function of liver. Liver regeneration involves complex processes that depend on the interaction of many different cell types. As limited by the average cell change level in tissues, traditional sequencing methods can only acquire the average genetic information reflecting dominant cell subpopulations, but ignore the secondary cell subpopu-lations, which leads to the loss of cellular heterogeneity information. Single-cell sequencing tech-nology can analyze the biological behavior of single cell, which helps to better understand the distri-bution, interaction and cell heterogeneity of different cells during liver regeneration. The authors review the application of single cell sequencing technology in liver regeneration.

Analysis of intercellular communication patterns in facioscapulohumeral muscular dystrophy based on single-cell nuclear transcriptome sequencing / 国际生物医学工程杂志

Objective:To analyze the patterns of intercellular communication in facioscapulohumeral muscular dystrophy (FSHD) by single-cell nuclear transcriptome sequencing.Methods:Bilateral asymmetrical lesions mouth orbicular muscle of two patients with FSHD and mouth orbicular muscle of two healthy patients were selected. Six samples were obtained, and were divided into control group, mild group and severe group. The normal orbicularis muscle sample was collected from 2 healthy individuals (the control group). The muscle samples in the mild group were from two patients with relatively normal muscle sides, and the samples in the severe group were from two patients with more severe muscle damage sides. Single-cell nuclear transcriptome sequencing was performed on all cells of the three groups. Reduced dimension clustering and cell definition were performed to identify differentially expressed genes and enrichment pathways. Intercellular communication patterns among major cell types and key signaling pathways were explored by cellular communication analysis.Results:Differential gene expression analysis of FSHD bilateral muscle samples identified 46 functionally differentially expressed genes associated with the disease in different cell types, related to apoptosis, oxidative stress, immune inflammation, and muscle function. Intercellular communication was generally increased in the severe group. Fibro-adipogenic progenitors (FAPs) and macrophages are important signaling sources in the abnormal muscle microenvironment of FSHD and are closely associated with disease progression. There are six unique signaling pathways in the mild group, including bone morphogenetic proteins (BMP), transforming growth factor-β (TGF-β), CXC motif chemokine ligand (CXCL), adhesion G protein-coupled receptor E5 (ADGRE5), interleukin-16 (IL-16), and wingless-type MMTV integration site family (WNT) signaling pathways. These signaling pathways are mainly involved in the interaction between macrophages, FAPs, and adipocytes and may be involved in the regulation of fat deposition and fibrosis changes in the diseased muscle.Conclusions:Single-cell nuclear transcriptome sequencing provides a relatively comprehensive pattern of intercellular communication between key cell types in FSHD, providing an appropriate reference for understanding the intercellular regulatory mechanisms of the FSHD muscle microenvironment.

Progress of single-cell sequencing in lymphoma / 白血病·淋巴瘤

Single-cell sequencing (SCS) sequences the genetic information of a single cell to better understand the differences amongst cells and reveal the unique changes of each cell type. The specific analysis of cell subsets at the single-cell level can accurately evaluate tumor cells and microenvironment cells to reveal the complexity of molecular components and the difference from the corresponding components in non-malignant tissues. Lymphoma is highly heterogeneous, some have unknown pathological types, etiology and poor prognosis. SCS is helpful to clarify the molecular mechanisms of lymphomagenesis and pathological staging, and guide clinical practice. This article reviews SCS and its application in lymphoma.

Transcriptomic profiles of paraquat-induced Parkinson-like changes in mouse brains based on single-cell RNA sequencing / 环境与职业医学

Background Paraquat (PQ) is one of the most widely used herbicides in the world and a risk factor for Parkinson's disease (PD), but the mechanisms underlying PD are poorly understood. Single-cell RNA sequencing (scRNA-seq) technology can study cellular heterogeneity at genetic level, providing insights into the pathogenesis of PQ-induced PD. Objective To analyze the brain cell grouping of PQ-infected mice and the biological processes involved in the subpopulation of PD-like changes cells by scRNA-seq, and to provide clues for revealing potential mechanisms of PQ-induced PD-like changes in mouse brains. Methods Six male 6-week-old C57BL/6 mice were randomly divided into a control group and an experimental group, three mice in each group, and were intraperitoneally injected with 0 (saline) and 10.0 mg·kg−1 PD respectively, once every two days, for 10 consecutive injections for modeling. After infection, mouse brains were taken and scRNA-seq was performed. Cell segmentation was performed according to gene expression characteristics of different cell types, PD-related cell subsets were screened by bioinformatics tools, and gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set enrichment analysis (GSEA), protein interaction network analysis, and transcription factor prediction were performed on their characteristic genes. Finally, GO and KEGG analyses were performed on the differential genes of PD-associated cell subsets between the PQ-treated group and the control group, and the biological processes in which these genes may participate were analyzed. Results The sequencing data met quality control standards, a total of 55779 cells were obtained, and all cell dimensionality reduction analysis results showed that they could be further divided into 37 clusters, including 5 major cell types. Based on the KEGG analysis of the top 20 characteristic genes of each subpopulation, the specifically expressed Cluster 33 subpopulation (dopaminergic neurons) was screened and found to be significantly associated with PD. The results of GO analysis showed that the biological function of this subpopulation mainly enriched neurotransmitter transport and regulation. The results of GSEA analysis showed that the tyrosine metabolic pathway and the ligand-receptor interaction pathway of neural activity in brain tissues were significantly enriched. The analysis of transcriptional regulatory networks showed that 39 transcription factors were expressed differently. The metabolic pathway of the dopamine neuronal subset, endocytosis, Ras-associated protein 1 (Rap1) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway were all affected by PQ exposure, according to further analysis of its effects on this subpopulation. The GO analysis showed that differential genes were involved in biological processes such as ion transport and synaptic assembly regulation, and were involved in the cellular component formation of cytoplasm and synapses. Conclusion This study has initially mapped the transcriptome of single cells in the mouse brain after PQ exposure, and screened out the specific expression of Cluster 33 subgroup (dopaminergic neurons), which is significantly correlated with PD, and its biological function changes may be one of the mechanisms of PD-like changes in the mouse brain induced by PQ.

Deciphering the dynamic characteristics of non-neuronal cells in dorsal root ganglion of rat at different developmental stage based on single cell transcriptome data / 生物工程学报

Dorsal root ganglia (DRG) is an essential part of the peripheral nervous system and the hub of the peripheral sensory afferent. The dynamic changes of neuronal cells and their gene expression during the development of dorsal root ganglion have been studied through single-cell RNAseq analysis, while the dynamic changes of non-neuronal cells have not been systematically studied. Using single cell RNA sequencing technology, we conducted a research on the non-neuronal cells in the dorsal root ganglia of rats at different developmental stage. In this study, primary cell suspension was obtained from using the dorsal root ganglions (DRGs, L4-L5) of ten 7-day-old rats and three 3-month-old rats. The 10×Genomics platform was used for single cell dissociation and RNA sequencing. Twenty cell subsets were acquired through cluster dimension reduction analysis, and the marker genes of different types of cells in DRG were identified according to previous researches about DRG single cell transcriptome sequencing. In order to find out the non-neuronal cell subsets with significant differences at different development stage, the cells were classified into different cell types according to markers collected from previous researches. We performed pseudotime analysis of 4 types Schwann cells. It was found that subtype Ⅱ Schwann cells emerged firstly, and then were subtype Ⅲ Schwann cells and subtype Ⅳ Schwann cells, while subtype Ⅰ Schwann cells existed during the whole development procedure. Pseudotime analysis indicated the essential genes influencing cell fate of different subtypes of Schwann cell in DRG, such as Ntrk2 and Pmp2, which affected cell fate of Schwann cells during the development period. GO analysis of differential expressed genes showed that the up-regulated genes, such as Cst3 and Spp1, were closely related to biological process of tissue homeostasis and multi-multicellular organism process. The down regulated key genes, such as Col3a1 and Col4a1, had close relationship with the progress of extracellular structure organization and negative regulation of cell adhesion. This suggested that the expression of genes enhancing cell homestasis increased, while the expression of related genes regulating ECM-receptor interaction pathway decreased during the development. The discovery provided valuable information and brand-new perspectives for the study on the physical and developmental mechanism of Schwann cell as well as the non-neuronal cell changes in DRG at different developmental stage. The differential gene expression results provided crucial references for the mechanism of somatosensory maturation during development.

Liver macrophages show an immunotolerance phenotype in nonalcoholic fatty liver combined with Porphyromonas gingivalis-lipopolysaccharide infection / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the functions and potential regulatory targets of local macrophages in nonalcoholic fatty liver combined with Porphyromonas gingivalis (P. gingivalis)infection.@*METHODS@#Single-cell RNA sequencing was used to analyze the phenotypes and functional changes in various cells in the liver tissue of nonalcoholic steatohepatitis (NASH) mice fed with P. gingivalis. Real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and immunofluorescence staining were applied to observe the inflammation and expression levels of macrophage antigen presenting functional markers in the NASH liver. Oil red staining was performed to observe the accumulation of local adipose tissue in the NASH liver. Results were verified through RT-PCRand RNA sequencing using P. gingivalis-lipopolysaccharide treated mouse peritoneal macrophages.@*RESULTS@#In comparison with healthy livers with Kupffer cells, the NASH liver combined with P. gingivalis infection-related macrophages showed significant heterogeneity. C1qb, C1qc, Mafb, Apoe, and Cd14 were highly expressed, but Cd209a, H2-Aa, H2-Ab1, and H2-DMb1, which are related to the antigen presentation function, were weakly expressed. Further in vivo and in vitro investigations indicated that the activation and infiltration of these macrophages may be due to local P. gingivalis-lipopolysaccharide accumulation.@*CONCLUSIONS@#P. gingivalis-lipopolysaccharide induces a local macrophage immunotolerance phenotype in nonalcoholic fatty liver, which may be the key mechanism of periodontitis pathogen infection that promotes NASH inflammation and pathogenesis. This study further clarifies the dysfunction and regulatory mechanisms of macrophages in the pathogenesis of P. gingivalis-infected NASH, thereby providing potential therapeutic targets for its clinical treatment.

Single cell RNA sequencing technology applicated for drug discovery / 药学学报

Single cell RNA sequencing (scRNA-seq) is an advanced technology to study the transcriptome information at the single cell level. The application of this technology can attribute to analyze the heterogeneous map of cells in the process of disease development, and precisely identify the specific cell subsets that are responsive to pharmacological therapy. Currently, scRNA-seq technology has been widely applied in the field of drug research, including studies on therapeutic targets, drug-induced adverse reactions, drug resistance and vaccine. This work reviews the application of scRNA-seq technology in drug discovery, which offers a scientific basis for personalized and accurate medication therapy.

Protective Effects of Zhi Ciweipi Water Extracts on Dextran Sulfate Sodium Salt⁃Induced Ulcerative Colitis in Mice / 中国生物化学与分子生物学报

Ulcerative colitis (UC) is a disease characterized by chronic inflammation of the intestinal mucosa. However, its exact pathogenesis is not fully understood. Zhi Ciweipi (ZCWP), as a traditional Chinese medicine (TCM), has demonstrated suitable anti-inflammatory effects in the treatment of patients with bloody stool and hemorrhoids. However, its therapeutic effects on ulcerative colitis have yet to be investigated in depth. The aim of this study was to investigate the protective effect of the aqueous extract of ZCWP against dextran sulfate sodium salt (DSS)-induced ulcerative colitis in mice and its possible mechanism of action. Successful simulation of ulcerative colitis was achieved by providing mice with drinking water containing 3% DSS. The HE staining results revealed that the aqueous extract of ZCWP could significantly reduce DSS-induced colonic injuries. Transcriptome sequencing analysis identified 10 key inflammation-related genes (IL⁃6, IL⁃1β, CSF2, TNF, IL10, IFN⁃γ, CXCL1, CXCL2, CXCL9, CXCL10), all of which were significantly downregulated (P<0. 05) in response to treatment with the aqueous extract of ZCWP according to qRT-PCR and Western blotting analyses. The immunofluorescence results further indicated that the aqueous extract of ZCWP was able to reduce the DSS-induced increase in the proportion of M1-type macrophages in the colon. Using single-cell sequencing, we thoroughly investigated the signaling relationships between different cell types, which revealed particularly strong communication between M1-type macrophages and fibroblasts. Subsequent qRT-PCR and Western blot analyses verified that the aqueous extract of ZCWP significantly downregulated the expression of DSS-induced fibrosis-related genes in colonic tissues (P<0. 05). In conclusion, the results of this study suggest that the aqueous extract of prepared ZCWP exerts a protective effect against DSS-induced ulcerative colitis by inhibiting M1-type macrophage polarization, downregulating the expression of inflammatory factors, and preventing strong communication between M1-type macrophages and fibroblasts. These findings not only reveal the therapeutic mechanism by which ZCWP aqueous extract treats colitis, but also provide a new theoretical basis for its application in clinical practice.

Improving the prognosis of pancreatic cancer: insights from epidemiology, genomic alterations, and therapeutic challenges / 医学前沿

Pancreatic cancer, notorious for its late diagnosis and aggressive progression, poses a substantial challenge owing to scarce treatment alternatives. This review endeavors to furnish a holistic insight into pancreatic cancer, encompassing its epidemiology, genomic characterization, risk factors, diagnosis, therapeutic strategies, and treatment resistance mechanisms. We delve into identifying risk factors, including genetic predisposition and environmental exposures, and explore recent research advancements in precursor lesions and molecular subtypes of pancreatic cancer. Additionally, we highlight the development and application of multi-omics approaches in pancreatic cancer research and discuss the latest combinations of pancreatic cancer biomarkers and their efficacy. We also dissect the primary mechanisms underlying treatment resistance in this malignancy, illustrating the latest therapeutic options and advancements in the field. Conclusively, we accentuate the urgent demand for more extensive research to enhance the prognosis for pancreatic cancer patients.

Distinct mononuclear diploid cardiac subpopulation with minimal cell-cell communications persists in embryonic and adult mammalian heart / 医学前沿

A small proportion of mononuclear diploid cardiomyocytes (MNDCMs), with regeneration potential, could persist in adult mammalian heart. However, the heterogeneity of MNDCMs and changes during development remains to be illuminated. To this end, 12 645 cardiac cells were generated from embryonic day 17.5 and postnatal days 2 and 8 mice by single-cell RNA sequencing. Three cardiac developmental paths were identified: two switching to cardiomyocytes (CM) maturation with close CM-fibroblast (FB) communications and one maintaining MNDCM status with least CM-FB communications. Proliferative MNDCMs having interactions with macrophages and non-proliferative MNDCMs (non-pMNDCMs) with minimal cell-cell communications were identified in the third path. The non-pMNDCMs possessed distinct properties: the lowest mitochondrial metabolisms, the highest glycolysis, and high expression of Myl4 and Tnni1. Single-nucleus RNA sequencing and immunohistochemical staining further proved that the Myl4+Tnni1+ MNDCMs persisted in embryonic and adult hearts. These MNDCMs were mapped to the heart by integrating the spatial and single-cell transcriptomic data. In conclusion, a novel non-pMNDCM subpopulation with minimal cell-cell communications was unveiled, highlighting the importance of microenvironment contribution to CM fate during maturation. These findings could improve the understanding of MNDCM heterogeneity and cardiac development, thus providing new clues for approaches to effective cardiac regeneration.

Single-cell profiling reveals Müller glia coordinate retinal intercellular communication during light/dark adaptation via thyroid hormone signaling

Light adaptation enables the vertebrate visual system to operate over a wide range of ambient illumination. Regulation of phototransduction in photoreceptors is considered a major mechanism underlying light adaptation. However, various types of neurons and glial cells exist in the retina, and whether and how all retinal cells interact to adapt to light/dark conditions at the cellular and molecular levels requires systematic investigation. Therefore, we utilized single-cell RNA sequencing to dissect retinal cell-type-specific transcriptomes during light/dark adaptation in mice. The results demonstrated that, in addition to photoreceptors, other retinal cell types also showed dynamic molecular changes and specifically enriched signaling pathways under light/dark adaptation. Importantly, Müller glial cells (MGs) were identified as hub cells for intercellular interactions, displaying complex cell‒cell communication with other retinal cells. Furthermore, light increased the transcription of the deiodinase Dio2 in MGs, which converted thyroxine (T4) to active triiodothyronine (T3). Subsequently, light increased T3 levels and regulated mitochondrial respiration in retinal cells in response to light conditions. As cones specifically express the thyroid hormone receptor Thrb, they responded to the increase in T3 by adjusting light responsiveness. Loss of the expression of Dio2 specifically in MGs decreased the light responsive ability of cones. These results suggest that retinal cells display global transcriptional changes under light/dark adaptation and that MGs coordinate intercellular communication during light/dark adaptation via thyroid hormone signaling.