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.

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).

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. (AU)

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.

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.

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/.

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.

Deep learning of cross-species single-cell landscapes identifies conserved regulatory programs underlying cell types.

Despite extensive efforts to generate and analyze reference genomes, genetic models to predict gene regulation and cell fate decisions are lacking for most species. Here, we generated whole-body single-cell transcriptomic landscapes of zebrafish, Drosophila and earthworm. We then integrated cell landscapes from eight representative metazoan species to study gene regulation across evolution. Using these uniformly constructed cross-species landscapes, we developed a deep-learning-based strategy, Nvwa, to predict gene expression and identify regulatory sequences at the single-cell level. We systematically compared cell-type-specific transcription factors to reveal conserved genetic regulation in vertebrates and invertebrates. Our work provides a valuable resource and offers a new strategy for studying regulatory grammar in diverse biological systems.

Systematic identification of cell-fate regulatory programs using a single-cell atlas of mouse development.

Waddington's epigenetic landscape is a metaphor frequently used to illustrate cell differentiation. Recent advances in single-cell genomics are altering our understanding of the Waddington landscape, yet the molecular mechanisms of cell-fate decisions remain poorly understood. We constructed a cell landscape of mouse lineage differentiation during development at the single-cell level and described both lineage-common and lineage-specific regulatory programs during cell-type maturation. We also found lineage-common regulatory programs that are broadly active during the development of invertebrates and vertebrates. In particular, we identified Xbp1 as an evolutionarily conserved regulator of cell-fate determinations across different species. We demonstrated that Xbp1 transcriptional regulation is important for the stabilization of the gene-regulatory networks for a wide range of mouse cell types. Our results offer genetic and molecular insights into cellular gene-regulatory programs and will serve as a basis for further advancing the understanding of cell-fate decisions.

Cell landscape of larval and adult Xenopus laevis at single-cell resolution.

The rapid development of high-throughput single-cell RNA sequencing technology offers a good opportunity to dissect cell heterogeneity of animals. A large number of organism-wide single-cell atlases have been constructed for vertebrates such as Homo sapiens, Macaca fascicularis, Mus musculus and Danio rerio. However, an intermediate taxon that links mammals to vertebrates of more ancient origin is still lacking. Here, we construct the first Xenopus cell landscape to date, including larval and adult organs. Common cell lineage-specific transcription factors have been identified in vertebrates, including fish, amphibians and mammals. The comparison of larval and adult erythrocytes identifies stage-specific hemoglobin subtypes, as well as a common type of cluster containing both larval and adult hemoglobin, mainly at NF59. In addition, cell lineages originating from all three layers exhibits both antigen processing and presentation during metamorphosis, indicating a common regulatory mechanism during metamorphosis. Overall, our study provides a large-scale resource for research on Xenopus metamorphosis and adult organs.

Construction of the axolotl cell landscape using combinatorial hybridization sequencing at single-cell resolution.

The Mexican axolotl (Ambystoma mexicanum) is a well-established tetrapod model for regeneration and developmental studies. Remarkably, neotenic axolotls may undergo metamorphosis, a process that triggers many dramatic changes in diverse organs, accompanied by gradually decline of their regeneration capacity and lifespan. However, the molecular regulation and cellular changes in neotenic and metamorphosed axolotls are still poorly investigated. Here, we develop a single-cell sequencing method based on combinatorial hybridization to generate a tissue-based transcriptomic landscape of the neotenic and metamorphosed axolotls. We perform gene expression profiling of over 1 million single cells across 19 tissues to construct the first adult axolotl cell landscape. Comparison of single-cell transcriptomes between the tissues of neotenic and metamorphosed axolotls reveal the heterogeneity of non-immune parenchymal cells in different tissues and established their regulatory network. Furthermore, we describe dynamic gene expression patterns during limb development in neotenic axolotls. This system-level single-cell analysis of molecular characteristics in neotenic and metamorphosed axolotls, serves as a resource to explore the molecular identity of the axolotl and facilitates better understanding of metamorphosis.

Dietary intervention preserves ß cell function in mice through CTCF-mediated transcriptional reprogramming.

Pancreatic ß cell plasticity is the primary determinant of disease progression and remission of type 2 diabetes (T2D). However, the dynamic nature of ß cell adaptation remains elusive. Here, we establish a mouse model exhibiting the compensation-to-decompensation adaptation of ß cell function in response to increasing duration of high-fat diet (HFD) feeding. Comprehensive islet functional and transcriptome analyses reveal a dynamic orchestration of transcriptional networks featuring temporal alteration of chromatin remodeling. Interestingly, prediabetic dietary intervention completely rescues ß cell dysfunction, accompanied by a remarkable reversal of HFD-induced reprogramming of islet chromatin accessibility and transcriptome. Mechanistically, ATAC-based motif analysis identifies CTCF as the top candidate driving dietary intervention-induced preservation of ß cell function. CTCF expression is markedly decreased in ß cells from obese and diabetic mice and humans. Both dietary intervention and AAV-mediated restoration of CTCF expression ameliorate ß cell dysfunction ex vivo and in vivo, through transducing the lipid toxicity and inflammatory signals to transcriptional reprogramming of genes critical for ß cell glucose metabolism and stress response.

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.

Effect of roscovitine pretreatment for increased utilization of small follicle-derived oocytes on developmental competence of somatic cell nuclear transfer embryos in pigs.

The objective of this study was to evaluate the effect of roscovitine pretreatment on the number of matured oocytes per ovary available for somatic cell nuclear transfer (SCNT) and their developmental competence. Irrespective of reproduction status (prepuberty/puberty), the average number of small follicles per ovary (19.3/17.2) was higher than that of medium follicles (1.5/2.7). In the small follicle group, the in vitro maturation rate of COCs pretreated with 50 µM roscovitine (56.1%) was significantly (P < 0.05) higher than that of the control, 25 or 75 µM treatment (15.5%, 23.7% and 35.2%, respectively), while, in the medium follicle group, there was no significant difference between the control, 25, 50 and 75 µM treatment (76.4%, 78.3%, 80.9% and 60.6%, respectively). As a result, a total number of matured oocytes per ovary was greater for 50 µM treatment (11.8) than for the control, 25 or 75 µM treatment (4.4, 5.0 and 6.3, respectively). In the small follicle group, COCs pretreated with 50 µM roscovitine showed dramatically increased blastocyst rate (16.0%) compared to the control (2.9%) (P < 0.05), whereas, in the medium follicle group, there was no significant difference between groups independent of roscovitine treatment (20.8 vs 23.0%). The cloning efficiency in the roscovitine-treated group was not significantly different from that in the control (2.6 vs 1.4%). In conclusion, the present study indicates that roscovitine treatment increased the number of matured oocytes per ovary available for SCNT and did not have any adverse effect on cloning efficiency in pigs.