Mapping the Mouse Cell Atlas by Microwell-Seq.

Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

Construction of a human cell landscape at single-cell level.

Single-cell analysis is a valuable tool for dissecting cellular heterogeneity in complex systems1. However, a comprehensive single-cell atlas has not been achieved for humans. Here we use single-cell mRNA sequencing to determine the cell-type composition of all major human organs and construct a scheme for the human cell landscape (HCL). We have uncovered a single-cell hierarchy for many tissues that have not been well characterized. We established a 'single-cell HCL analysis' pipeline that helps to define human cell identity. Finally, we performed a single-cell comparative analysis of landscapes from human and mouse to identify conserved genetic networks. We found that stem and progenitor cells exhibit strong transcriptomic stochasticity, whereas differentiated cells are more distinct. Our results provide a useful resource for the study of human biology.

Construction of a cross-species cell landscape at single-cell level.

Individual cells are basic units of life. Despite extensive efforts to characterize the cellular heterogeneity of different organisms, cross-species comparisons of landscape dynamics have not been achieved. Here, we applied single-cell RNA sequencing (scRNA-seq) to map organism-level cell landscapes at multiple life stages for mice, zebrafish and Drosophila. By integrating the comprehensive dataset of > 2.6 million single cells, we constructed a cross-species cell landscape and identified signatures and common pathways that changed throughout the life span. We identified structural inflammation and mitochondrial dysfunction as the most common hallmarks of organism aging, and found that pharmacological activation of mitochondrial metabolism alleviated aging phenotypes in mice. The cross-species cell landscape with other published datasets were stored in an integrated online portal-Cell Landscape. Our work provides a valuable resource for studying lineage development, maturation and aging.

How many cell types are there in nature? How do they change during the life cycle? These are two fundamental questions that researchers have been trying to understand in the area of biology. In this study, single-cell mRNA sequencing data were used to profile over 2.6 million individual cells from mice, zebrafish and Drosophila at different life stages, 1.3 million of which were newly collected. The comprehensive datasets allow investigators to construct a cross-species cell landscape that helps to reveal the conservation and diversity of cell taxonomies at genetic and regulatory levels. The resources in this study are assembled into a publicly available website at http://bis.zju.edu.cn/cellatlas/.

Nicotine Dependence, Perceived Behavioral Control, Descriptive Quitting Norms, and Intentions to Quit Smoking among Chinese Male Regular Smokers.

BACKGROUND: The association between nicotine dependence and intentions to quit smoking is well established. However, the underlying mechanisms and psychosocial factors that moderate this relationship have not been adequately delineated. Reinforcement theory, social learning theory, the theory of planned behavior, and the focus theory of normative conduct suggest that perceived behavioral control (PBC) over smoking cessation is a central mechanism that underlies the relationship between nicotine dependence and quitting intentions. Purpose/Objectives: The present study tested a moderated mediation model to examine the roles of PBC over smoking cessation and social norms, in the relationship between nicotine dependence and quit intentions among male regular smokers. Methods: Self-report measures of nicotine dependence, PBC over smoking cessation, descriptive and injunctive quitting norms, and intentions to quit smoking were administered to 204 Chinese male regular smokers. Results: Nicotine dependence was negatively related to quit intentions, and this relationship was mediated by PBC over smoking cessation. Further, the relationship between nicotine dependence and PBC was moderated by descriptive rather than injunctive quitting norms. The negative effects of nicotine dependence on PBC over smoking cessation were not significant among male regular smokers who reported strong descriptive quitting norms. Conclusions/Importance: Poor PBC over smoking cessation underlies the negative effects of nicotine dependence, and descriptive quitting norms act as a buffer against the adverse effects of nicotine dependence on PBC over smoking cessation. These findings have implications for smoking cessation interventions for male regular smokers.

Generating hESCs with reduced immunogenicity by disrupting TAP1 or TAPBP.

Human embryonic stem cells (hESCs) are thought to be a promising resource for cell therapy, while it has to face the major problem of graft immunological rejection. Major histocompatibility complex (MHC) class I expressed on the cell surface is the major cause of graft rejection. Transporter associated with antigen presentation 1 (TAP1) and TAP-associated glycoprotein (TAPBP) play important roles in regulating MHC class I expression. In this study, we generated TAP1- and TAPBP-deficient hESC lines, respectively, using transcription activator-like effector nucleases technique. These cells showed deficient expression of MHC class I on the cell surface and reduced immunogenicity compared with wild types, but maintained normal pluripotency, karyotypes, and differentiation ability. Thus, our findings are instrumental in developing a universal cell resource with both pluripotency and hypo-immunogenicity for transplantation therapy in the future.

Functional disruption of human leukocyte antigen II in human embryonic stem cell.

BACKGROUND: Theoretically human embryonic stem cells (hESCs) have the capacity to self-renew and differentiate into all human cell types. Therefore, the greatest promise of hESCs-based therapy is to replace the damaged tissues of patients suffering from traumatic or degenerative diseases by the exact same type of cells derived from hESCs. Allograft immune rejection is one of the obstacles for hESCs-based clinical applications. Human leukocyte antigen (HLA) II leads to CD4(+) T cells-mediated allograft rejection. Hence, we focus on optimizing hESCs for clinic application through gene modification. RESULTS: Transcription activator-like effector nucleases (TALENs) were used to target MHC class II transactivator (CIITA) in hESCs efficiently. CIITA (-/-) hESCs did not show any difference in the differentiation potential and self-renewal capacity. Dendritic cells (DCs) derived from CIITA (-/-) hESCs expressed CD83 and CD86 but without the constitutive HLA II. Fibroblasts derived from CIITA (-/-) hESCs were powerless in IFN-γ inducible expression of HLA II. CONCLUSION: We generated HLA II defected hESCs via deleting CIITA, a master regulator of constitutive and IFN-γ inducible expression of HLA II genes. CIITA (-/-) hESCs can differentiate into tissue cells with non-HLA II expression. It's promising that CIITA (-/-) hESCs-derived cells could be used in cell therapy (e.g., T cells and DCs) and escape the attack of receptors' CD4(+) T cells, which are the main effector cells of cellular immunity in allograft.

Dissecting the immune discrepancies in mouse liver allograft tolerance and heart/kidney allograft rejection.

The liver is the most tolerogenic of transplanted organs. However, the mechanisms underlying liver transplant tolerance are not well understood. The comparison between liver transplantation tolerance and heart/kidney transplantation rejection will deepen our understanding of tolerance and rejection in solid organs. Here, we built a mouse model of liver, heart and kidney allograft and performed single-cell RNA sequencing of 66,393 cells to describe the cell composition and immune cell interactions at the early stage of tolerance or rejection. We also performed bulk RNA-seq of mouse liver allografts from Day 7 to Day 60 post-transplantation to map the dynamic transcriptional variation in spontaneous tolerance. The transcriptome of lymphocytes and myeloid cells were characterized and compared in three types of organ allografts. Cell-cell interaction networks reveal the coordinated function of Kupffer cells, macrophages and their associated metabolic processes, including insulin receptor signalling and oxidative phosphorylation in tolerance induction. Cd11b+ dendritic cells (DCs) in liver allografts were found to inhibit cytotoxic T cells by secreting anti-inflammatory cytokines such as Il10. In summary, we profiled single-cell transcriptome analysis of mouse solid organ allografts. We characterized the immune microenvironment of mouse organ allografts in the acute rejection state (heart, kidney) and tolerance state (liver).

Single Nucleus Total RNA Sequencing of Formalin-Fixed Paraffin-Embedded Gliomas.

Gliomas, the predominant form of brain cancer, comprise diverse malignant subtypes with limited curative therapies available. The insufficient understanding of their molecular diversity and evolutionary processes hinders the advancement of new treatments. Technical complexities associated with formalin-fixed paraffin-embedded (FFPE) clinical samples hinder molecular-level analyses of gliomas. Current single-cell RNA sequencing (scRNA-seq) platforms are inadequate for large-scale clinical applications. In this study, automated snRandom-seq is developed, a high-throughput single-nucleus total RNA sequencing platform optimized for archival FFPE samples. This platform integrates automated single-nucleus isolation and droplet barcoding systems with the random primer-based scRNA-seq chemistry, accommodating a broad spectrum of sample types. The automated snRandom-seq is applied to analyze 116 492 single nuclei from 17 FFPE samples of various glioma subtypes, including rare clinical samples and matched primary-recurrent glioblastomas (GBMs). The study provides comprehensive insights into the molecular characteristics of gliomas at the single-cell level. Abundant non-coding RNAs (ncRNAs) with distinct expression profiles across different glioma clusters and uncovered promising recurrence-related targets and pathways in primary-recurrent GBMs are identified. These findings establish automated snRandom-seq as a robust tool for scRNA-seq of FFPE samples, enabling exploration of molecular diversities and tumor evolution. This platform holds significant implications for large-scale integrative and retrospective clinical research.

Fast and flexible profiling of chromatin accessibility and total RNA expression in single nuclei using Microwell-seq3.

Single cell chromatin accessibility profiling and transcriptome sequencing are the most widely used technologies for single-cell genomics. Here, we present Microwell-seq3, a high-throughput and facile platform for high-sensitivity single-nucleus chromatin accessibility or full-length transcriptome profiling. The method combines a preindexing strategy and a penetrable chip-in-a-tube for single nucleus loading and DNA amplification and therefore does not require specialized equipment. We used Microwell-seq3 to profile chromatin accessibility in more than 200,000 single nuclei and the full-length transcriptome in ~50,000 nuclei from multiple adult mouse tissues. Compared with the existing polyadenylated transcript capture methods, integrative analysis of cell type-specific regulatory elements and total RNA expression uncovered comprehensive cell type heterogeneity in the brain. Gene regulatory networks based on chromatin accessibility profiling provided an improved cell type communication model. Finally, we demonstrated that Microwell-seq3 can identify malignant cells and their specific regulons in spontaneous lung tumors of aged mice. We envision a broad application of Microwell-seq3 in many areas of research.

Single-cell RNA sequencing reveals immune cell dysfunction in the peripheral blood of patients with highly aggressive gastric cancer.

Highly aggressive gastric cancer (HAGC) is a gastric cancer characterized by bone marrow metastasis and disseminated intravascular coagulation (DIC). Information about the disease is limited. Here we employed single-cell RNA sequencing to investigate peripheral blood mononuclear cells (PBMCs), aiming to unravel the immune response of patients toward HAGC. PBMCs from seven HAGC patients, six normal advanced gastric cancer (NAGC) patients, and five healthy individuals were analysed by single-cell RNA sequencing. The expression of genes of interest was validated by bulk RNA-sequencing and ELISA. We found a massive expansion of neutrophils in PBMCs of HAGC. These neutrophils are activated, but immature. Besides, mononuclear phagocytes exhibited an M2-like signature and T cells were suppressed and reduced in number. Analysis of cell-cell crosstalk revealed that several signalling pathways involved in neutrophil to T-cell suppression including APP-CD74, MIF-(CD74+CXCR2), and MIF-(CD74+CD44) pathways were increased in HAGC. NETosis-associated genes S100A8 and S100A9 as well as VEGF, PDGF, FGF, and NOTCH signalling that contribute to DIC development were upregulated in HAGC too. This study reveals significant changes in the distribution and interactions of the PBMC subsets and provides valuable insight into the immune response in patients with HAGC. S100A8 and S100A9 are highly expressed in HAGC neutrophils, suggesting their potential to be used as novel diagnostic and therapeutic targets for HAGC.

Pan-Cancer Single-Nucleus Total RNA Sequencing Using snHH-Seq.

Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high-throughput and high-sensitivity method called snHH-seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full-length RNA-seq data is also established. snHH-seq is applied to more than 730 000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

Effects of Smoking Social Cues on Inhibitory Control in Smokers: An Event-Related Potential Study.

Objective: Reduced inhibitory control is a general characteristic of smokers and becomes increasingly pronounced in smoking-related contexts. However, research has rarely considered differences in the effects of various smoking-related cues. To fill this research gap, this study compared the effects of smoking object-related and smoking social-related cues on inhibitory control in smokers. Methods: We used a visual Go/NoGo paradigm with three types of long-lasting backgrounds (neutral, smoking object, and smoking social background) to record the error rates, reaction times, and amplitudes of the N2 and P3 event-related potentials (ERPs) by 25 smokers and 25 non-smokers. Results: (1) Smokers displayed smaller NoGo-N2 amplitudes than controls under the neutral background; (2) smokers displayed smaller NoGo-N2 amplitudes under the smoking social background and smoking object background than they did under the neutral background; (3) relative to neutral and smoking object backgrounds, smokers displayed higher commission error rates, shorter reaction times, and larger NoGo-P3 amplitudes under smoking social background. Conclusion: Smoking-related stimuli impair inhibitory control in smokers, especially when these stimuli are socially related.

Psychometric validation of the Chinese version of the desire thinking questionnaire in adolescent mobile phone users.

BACKGROUND: Desire thinking is a conscious and voluntary cognitive process that is closely linked to levels of craving and addictive behaviors. The Desire Thinking Questionnaire (DTQ) can be used to measure desire thinking in all age groups as well as in addicts. This measurement has also been translated into several languages. This study aimed to test the psychometric properties of the Chinese version of the DTQ (DTQ-C) among adolescent mobile phone users. METHODS: One thousand and ninety-seven adolescents who own a mobile phone and are younger than 18 years old completed the DTQ-C and a battery of questionnaires assessing the big five personality traits, negative affect, brooding, self-control, craving, and problematic mobile phone use (PMPU). The psychometric analyses of the DTQ-C were conducted, including exploratory factor analysis (EFA), confirmatory factor analysis (CFA), reliability, and validity analysis. RESULTS: The EFA revealed a 10-item two-factor structure (i.e., verbal perseveration and imaginal prefiguration) that was confirmed by the CFA. The results of CFA showed fit indexes of χ2/df = 4.83, CFI = 0.967, TLI = 0.954, RMSEA = 0.059, SRMR = 0.032. The total scale had internal consistency reliabilities of 0.93, which demonstrated that DTQ-C presented good reliability. The two dimensions were correlated with PMPU (rverbal perseveration = 0.54; rimaginal prefiguration = 0.45), neuroticism (rverbal perseveration = 0.18; rimaginal prefiguration = 0.14), conscientiousness (rverbal perseveration = -0.19; rimaginal prefiguration = -0.18), depression (rverbal perseveration = 0.22; rimaginal prefiguration = 0.16), anxiety (rverbal perseveration = 0.26; rimaginal prefiguration = 0.22), stress (rverbal perseveration = 0.15; rimaginal prefiguration = 0.10) and self-control (rverbal perseveration = -0.29; rimaginal prefiguration = -0.26), which demonstrated that DTQ-C presented good concurrent validity. The two factors of DTQ-C correlated weakly with brooding (ranging from 0.08 to 0.10). The principal component factor analysis of the two dimensions of desire thinking and craving showed that craving and desire thinking belonged to different dimensions. Both of which showed good divergent validity of desire thinking. Additionally, an examination of incremental validity revealed that two factors were both positively associated with PMPU beyond demographic characteristics, big five personality traits, negative affect, and self-control (Bverbal perseveration = 0.49 and Bimaginal prefiguration = 0.13). CONCLUSIONS: It has been found that the 10-item DTQ-C is a reliable and valid measure of desire thinking in Chinese adolescent mobile phone users.

Characterization of human pluripotent stem cell differentiation by single-cell dual-omics analyses.

In vivo differentiation of human pluripotent stem cells (hPSCs) has unique advantages, such as multilineage differentiation, angiogenesis, and close cell-cell interactions. To systematically investigate multilineage differentiation mechanisms of hPSCs, we constructed the in vivo hPSC differentiation landscape containing 239,670 cells using teratoma models. We identified 43 cell types, inferred 18 cell differentiation trajectories, and characterized common and specific gene regulation patterns during hPSC differentiation at both transcriptional and epigenetic levels. Additionally, we developed the developmental single-cell Basic Local Alignment Search Tool (dscBLAST), an R-based cell identification tool, to simplify the identification processes of developmental cells. Using dscBLAST, we aligned cells in multiple differentiation models to normally developing cells to further understand their differentiation states. Overall, our study offers new insights into stem cell differentiation and human embryonic development; dscBLAST shows favorable cell identification performance, providing a powerful identification tool for developmental cells.

High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq.

Formalin-fixed paraffin-embedded (FFPE) tissues constitute a vast and valuable patient material bank for clinical history and follow-up data. It is still challenging to achieve single cell/nucleus RNA (sc/snRNA) profile in FFPE tissues. Here, we develop a droplet-based snRNA sequencing technology (snRandom-seq) for FFPE tissues by capturing full-length total RNAs with random primers. snRandom-seq shows a minor doublet rate (0.3%), a much higher RNA coverage, and detects more non-coding RNAs and nascent RNAs, compared with state-of-art high-throughput scRNA-seq technologies. snRandom-seq detects a median of >3000 genes per nucleus and identifies 25 typical cell types. Moreover, we apply snRandom-seq on a clinical FFPE human liver cancer specimen and reveal an interesting subpopulation of nuclei with high proliferative activity. Our method provides a powerful snRNA-seq platform for clinical FFPE specimens and promises enormous applications in biomedical research.

A Network Analysis of the Relationships Between Behavioral Inhibition/Activation Systems and Problematic Mobile Phone Use.

Background: It is of great concern to society that individuals can be vulnerable to problematic mobile phone use (PMPU). However, there are a few studies in the field evaluating associations between behavioral inhibition/activation systems (BIS/BAS) and PMPU, and the results have been inconsistent. This study aimed to explore the relationships between BIS/BAS and PMPU by network analysis. Methods: A total of 891 young adults participated in the study. BIS/BAS and PMPU were assessed by using the behavioral inhibition and activation systems scale and smartphone application-based addiction scale, respectively. The structure of the BIS/BAS-PMPU network was characterized using "strength," "closeness" and "betweenness" as centrality indices. Edge-weight accuracy and centrality stability were tested using a bootstrap procedure. Results: The network analysis showed that "mood modification," "tolerance" and "withdrawal symptoms" had high centrality. In addition, the positive connection between BIS and "mood modification" or "tolerance" and between BAS-fun seeking and "mood modification" or "conflict" were also shown in the BIS/BAS-PMPU network. Conclusion: These findings shed light on the central and bridge components between the BIS/BAS and PMPU communities, providing new evidence relevant to potential mechanisms that account for how high-BIS or high-BAS individuals develop PMPU, and inspiring component-based PMPU prevention or interventions.

The Relationship Between Smoker Identity and Smoking Cessation Among Young Smokers: The Role of Smoking Rationalization Beliefs and Cultural Value of Guanxi.

Although the relationship between smoker identity and smoking cessation behavior has been confirmed, the role of smoking-related beliefs and cultural values in this relationship for young smokers is little known. The present study aimed to examine whether the relationship between smoker identity and smoking cessation behavior would be mediated by smoking rationalization beliefs and/or intention to quit smoking and whether the effect of smoker identity on smoking cessation behavior was moderated by cultural value of guanxi. A total of 708 young smokers participated in the study and completed questionnaires that measured smoker identity, smoking rationalization beliefs, intention to quit smoking, smoking cessation behavior and cultural value of guanxi. The results showed: (1) the relationship between smoker identity and smoking cessation behavior was negative and significant. (2) The mediating effect of intention to quit smoking and the serial mediating effect of "smoking rationalization beliefs → intention to quit smoking" on the relationship between smoker identity and smoking cessation behavior was significant. (3) Both the serial mediating effect of "smoking rationalization beliefs → intention to quit smoking" and the direct effect of smoker identity on smoking cessation behavior were moderated by cultural value of guanxi. The current findings increased understanding of psychosocial mechanisms underlying the hindering effect of smoker identity on smoking cessation and suggested the role of smoking rationalization beliefs and cultural value of guanxi should be considered in smoking cessation interventions for young smokers.

Decisional Balance of Smoking and Planning to Quit Smoking: A Moderated Mediation Model among Chinese Male Smokers.

Decisional balance in quitting smoking involves positive and negative attitudes toward smoking. This study explored the relationship between the decisional balance of smoking and planning to quit smoking, and examined the mediating role of willingness to quit smoking and the moderating role of social support. A questionnaire survey was conducted among 326 daily male smokers from China in 2016, including the decisional balance of smoking, willingness to quit smoking, social support, planning to quit, and nicotine dependence. The results showed: (1) willingness to quit smoking mediated the relationship between the decisional balance of smoking and planning to quit smoking; (2) the relationship between the decisional balance of smoking and planning to quit smoking was moderated by emotional support rather than instrumental support. Smokers with a higher level of emotional support for quitting smoking were more likely to progress to planning to quit smoking, while this relationship was not found among smokers with a lower level of emotional support. These findings suggested that smokers' willingness to quit smoking would be key to promoting their planning to quit. Meanwhile, the dynamic process of planning to quit smoking was moderated by emotional support from others.

Socializing with Smoker and Social Smoking Behavior among Chinese Male Smokers with Low Nicotine Dependence: The Mediating Roles of Belief of Smoking Rationalization and Smoker Identity.

BACKGROUND: Previous studies have shown that socializing with other smokers is an essential trigger for social smoking among smokers with a low nicotine dependence. This study further explored the mediating effects of the belief of smoking rationalization and smoker identity on the relationship between socializing with smokers and social smoking behavior. METHODS: A cross-sectional design was conducted. A total of 696 low-nicotine-dependent smokers in China completed questionnaires that assessed socializing with smokers, social smoking behavior, smoker identity, and the belief of smoking rationalization. The mediating roles of the belief of smoking rationalization and smoker identity on the relationship between socializing with smokers and social smoking behavior were assessed by using SPSS 23 and AMOS 23. RESULTS: The belief of smoking rationalization, smoker identity, socializing with smokers, and social smoking behavior were significantly and positively correlated with each other. In addition, this study found an independently mediated role for smoker identity in the relationship with smoker socialization and social smoking behavior, and a sequentially mediated role for smoking rationalization and smoker identity in this relationship. CONCLUSION: Reducing the belief of smoking rationalization and smoker identity may be conducive to reducing social smoking behavior for low-nicotine-dependent smokers when socializing with other smokers.