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.

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.

Single-cell landscape analysis reveals systematic senescence in mammalian Down syndrome.

BACKGROUND: Down syndrome (DS), which is characterized by various malfunctions, is the most common chromosomal disorder. As the DS population continues to grow and most of those with DS live beyond puberty, early-onset health problems have become apparent. However, the cellular landscape and molecular alterations have not been thoroughly studied. METHODS: This study utilized single-cell resolution techniques to examine DS in humans and mice, spanning seven distinct organs. A total of 71 934 mouse and 98 207 human cells were analyzed to uncover the molecular alterations occurring in different cell types and organs related to DS, specifically starting from the fetal stage. Additionally, SA-ß-Gal staining, western blot, and histological study were employed to verify the alterations. RESULTS: In this study, we firstly established the transcriptomic profile of the mammalian DS, deciphering the cellular map and molecular mechanism. Our analysis indicated that DS cells across various types and organs experienced senescence stresses from as early as the fetal stage. This was marked by elevated SA-ß-Gal activity, overexpression of cell cycle inhibitors, augmented inflammatory responses, and a loss of cellular identity. Furthermore, we found evidence of mitochondrial disturbance, an increase in ribosomal protein transcription, and heightened apoptosis in fetal DS cells. This investigation also unearthed a regulatory network driven by an HSA21 gene, which leads to genome-wide expression changes. CONCLUSION: The findings from this study offer significant insights into the molecular alterations that occur in DS, shedding light on the pathological processes underlying this disorder. These results can potentially guide future research and treatment development for DS.

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/.

Tracking the Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in an Emergency Intensive Care Unit by Whole Genome Sequencing.

Purpose: The spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a great threat to human health, especially in the intensive care unit. The aim of this study was to identify the origin and transmission route of a CRKP outbreak in an emergency intensive care unit (EICU), so as to provide prevention and control strategies for CRKP outbreak. Methods: Between Mar and Jun 2018, 10 CRKP isolates from 5 patients in the EICU ward of Shanghai Ruijin hospital north were collected. Modified carbapenem inactivation method (mCIM) and whole-genome sequencing (WGS) were performed on all 10 CRKP isolates. By integrating the genomic and epidemiological data of our isolates and 9 CRKP isolates from an outbreak in another hospital, a putative transmission map was constructed. Results: All 10 outbreak strains were carbapenemase positive in mCIM and belonged to the sequence type 11 (ST11) clone, harbored a set of resistance genes and virulence genes. The phylogenetic tree of CRKP isolates based on two outbreaks revealed that the initial isolate A1 in our EICU ward belonged to one branch of isolates in another hospital, this introductive isolate evolved and caused a subsequent outbreak in our EICU. Conclusion: Integration of genomic and epidemiological data can yield a clear transmission map of CRKP outbreak. Monitoring the rapid evolution of CRKP at the early stage of outbreak, CRKP monitoring after patients are discharged, active surveillance of newly admitted patients, environmental hygiene and efficient antibiotic treatment may be the key to prevent and control of CRKP outbreak.

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.

Effects of MBSR therapy on negative emotions, fatigue, and sleep quality in "post-ICU patients": A randomized controlled clinical trial protocol.

BACKGROUND: Survivors of intensive care unit (ICU) transfer to the common ward are often accompanied by psychological distress, negative emotions, fatigue, and sleep disturbances that affect recovery. Mindfulness-based stress reduction (MBSR) has achieved reliable results in improving physical and mental health. However, no clinical study has been conducted to evaluate the effects of MBSR on negative emotions, fatigue and sleep quality of patients who survived ICU and were transferred to general wards. METHODS: This is a prospective randomized controlled trial (RCT) examining the effects of MBSR on negative emotions, fatigue, and sleep quality in inpatients transferred from ICU to general ward. Participants were randomly divided into the treatment group and the control group in a ratio of 1:1. On the basis of the same nursing plan and health education, the treatment group received MBSR therapy, while the control group received no other interventions, and all the patients were followed up for 3 months after 2 weeks of continuous treatment. The indicators included negative mood indicators [Self-rating Depression Scale (SDS) and Self-Rating Anxiety Scale (SAS)], fatigue index [Fatigue Severity Scale (FSS) and Brief Fatigue Inventory (BFI)], and sleep quality index [Pittsburgh Sleep Quality Index (PSQI)]. Finally, SPSS 20.0 software was used for statistical analysis of the data. DISCUSSION: This study will evaluate the effects of MBSR on negative emotions, fatigue, and sleep quality in hospitalized patients transferred from ICU to general ward. The results of this study will provide a reference for MBSR to improve psychological distress in ICU survivors transferred to general ward. TRIAL REGISTRATION: This study protocol was registered in the Open Science Framework (OSF) (registration number: DOI 10.17605/OSF.IO/PD7SU).

Inhibition of complement C5a receptor protects lung cells and tissues against lipopolysaccharide-induced injury via blocking pyroptosis.

Acute lung injury (ALI) is the injury of alveolar epithelial cells and capillary endothelial cells caused by various factors. Complement system and pyroptosis have been proved to be involved in ALI, and inhibition of C5a/C5a receptor (C5aR) could alleviate ALI. This study aimed to investigate whether C5a/C5aR inhibition could protect against LPS-induced ALI via mediating pyroptosis. Rats were assigned into four groups: Control, LPS, LPS+W-54011 1mg/kg, and LPS+W-54011 5mg/kg. Beas-2B cells pretreated with or without C5a and W-54011, alone and in combination, were challenged with LPS+ATP. Results unveiled that LPS caused lung tissue injury and inflammatory response, increased pyroptotic and apoptotic factors, along with elevated C5a concentration and C5aR expressions. However, W-54011 pretreatment alleviated lung damage and pulmonary edema, reduced inflammation and prevented cell pyroptosis. In vitro studies confirmed that LPS+ATP reduced cell viability, promoted cell death, generated inflammatory factors and promoted expressions of pyroptosis-related proteins, which could be prevented by W-54011 pretreatment while intensified by C5a pretreatment. The co-treatment of C5a and W-54011 could blunt the effects of C5a on LPS+ATP-induced cytotoxicity. In conclusion, inhibition of C5a/C5aR developed protective effects against LPS-induced ALI and the cytotoxicity of Beas-2B cells, and these effects may depend on blocking pyroptosis.

Diagnosis and Analysis of Transabdominal and Intracavitary Ultrasound in Gynecological Acute Abdomen.

In order to explore the effective diagnosis method of gynecological acute abdomen, this paper takes hospital gynecological acute abdomen patients as samples and selects gynecological acute abdomen patients admitted to the hospital to be included in this study. They are divided into transabdominal ultrasound group, intracavitary ultrasound group, and combined group. Moreover, this paper uses mathematical statistics to carry out sample statistics. The statistical data mainly include ectopic pregnancy, torsion of ovarian tumor pedicle, acute suppurative salpingitis, torsion of fallopian tube, hemorrhagic salpingitis, acute pelvic inflammatory disease, rupture of corpus luteum cyst, and diagnosis accuracy rate. In addition, this paper compares the diagnostic accuracy of the abdominal ultrasound group, the intracavitary ultrasound group, and the combined group. The experimental research shows that the combined ultrasound diagnosis method can effectively improve the accuracy of the diagnosis of gynecological acute abdomen.

Characteristics of Injury Patients in the Emergency Department in Shanghai, China: A Retrospective Observational Study.

BACKGROUND This study analyzed the epidemiological characteristics and trends of trauma injuries in Ruijin Hospital North, Shanghai, China, and the feasibility of methods to prevent trauma. MATERIAL AND METHODS In this retrospective cross-sectional study, the electronic databases of Ruijin Hospital North were searched for patients who experienced severe trauma from 2013 to 2016. Characteristics of severe trauma were analyzed, including trauma mechanism, gender, reasons for injury, and injury-associated causes of death. RESULTS Of the 17,093 patients who experienced trauma during the study period, 11,165 (65.3%) were male and 5,928 (34.7%) were female. Analysis by age showed that the highest incidence of traumatic injuries was in subjects aged 25-34 years, whereas analysis by occupation showed the highest incidence of injury in migrant workers without higher education. Classification by Injury Severity Score (ISS) showed that 12,563 (73.5%) subjects had minor injuries, 4,273 (25.0%) had serious injuries, and 256 (1.5%) had severe injuries. In addition, 256 (1.5%) subjects died, with traffic accidents and falling injuries being the main causes of death. The incidence of injury peaked at 9-11 am and 2-4 pm and was significantly higher in autumn and winter than in spring and summer. CONCLUSIONS Most trauma patients were young adults. Injuries due to traffic accidents and falling were the main causes of death, with disregard of driving regulations and other health and safety regulations being the main cause of trauma. Trauma injuries may be prevented by strengthening education and by obeying traffic laws and construction site safety regulations.

A single-cell survey of cellular hierarchy in acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a fatal hematopoietic malignancy and has a prognosis that varies with its genetic complexity. However, there has been no appropriate integrative analysis on the hierarchy of different AML subtypes. METHODS: Using Microwell-seq, a high-throughput single-cell mRNA sequencing platform, we analyzed the cellular hierarchy of bone marrow samples from 40 patients and 3 healthy donors. We also used single-cell single-molecule real-time (SMRT) sequencing to investigate the clonal heterogeneity of AML cells. RESULTS: From the integrative analysis of 191727 AML cells, we established a single-cell AML landscape and identified an AML progenitor cell cluster with novel AML markers. Patients with ribosomal protein high progenitor cells had a low remission rate. We deduced two types of AML with diverse clinical outcomes. We traced mitochondrial mutations in the AML landscape by combining Microwell-seq with SMRT sequencing. We propose the existence of a phenotypic "cancer attractor" that might help to define a common phenotype for AML progenitor cells. Finally, we explored the potential drug targets by making comparisons between the AML landscape and the Human Cell Landscape. CONCLUSIONS: We identified a key AML progenitor cell cluster. A high ribosomal protein gene level indicates the poor prognosis. We deduced two types of AML and explored the potential drug targets. Our results suggest the existence of a cancer attractor.

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.

Maintenance of human haematopoietic stem and progenitor cells in vitro using a chemical cocktail.

Identification of effective culture conditions to maintain and possibly expand human HSPCs in vitro is an important goal. Recent advances highlight the efficacy of chemicals in maintaining and converting cell fates. We screened 186 chemicals and found that a combination of CHIR-99021, Forskolin and OAC1 (CFO) maintained human CD34-positive cells in vitro. Efficiency of the culture system was characterized using flow cytometry for CD34-positive cells, a colony-forming assay and xeno-transplants. We found that human CD34-positive cells treated with this combination had enhanced expression of human HSPC markers and increased haematopoietic re-populating ability in immune-deficient mice. Single-cell RNA-seq analyses showed that the in vitro cultured human CD34-positive cells were heterogeneous. We found that CFO supports maintenance of human CD34-positive cells by activating HOXA9, GATA2 and AKT-cAMP signaling pathway. These data have implications in therapies requiring maintenance and/or expansion of human HSPCs.

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.

A multicenter study of biological effects assessment of pharmacy workers occupationally exposed to antineoplastic drugs in Pharmacy Intravenous Admixture Services.

This multi-centered study was designed to evaluate the biological effects of exposure to antineoplastic drugs (ADs) at PIVAS (Pharmacy Intravenous Admixture Service) across ten Chinese hospitals. 8-hydroxy-2-deoxyguanosine (8-OHdG) was used as a biomarker of DNA oxidative damage and lymphocyte apoptosis assays using peripheral lymphocyte cells were used to detect primary DNA damage. The mutagenicity activity was estimated with the Ames fluctuation test. 158 exposed and 143 unexposed workers participated in this study. The urinary 8-OHdG/Cr concentrations of the exposed group was 22.05±17.89ng/mg Cr, which was significantly higher than controls of 17.36±13.50ng/mg Cr (P<0.05). The rate of early lymphocyte apoptosis was slightly increased in exposed group than that of the control group (P=0.087). The mutagenic activity was significantly higher in the exposed group relative to the control group (P<0.05). Moreover, while no statistically significant difference was observed, higher concentrations of 8-OHdG/Cr in urine and an early lymphocyte apoptosis rate were found in exposed group II as compared to exposed group I. In addition, a significant correlation between early lymphocyte apoptosis and exposure time to ADs was also observed (P<0.05). In conclusion, our study identified elevated biomarkers in PIVAS workers exposed to ADs. However whether these findings could lead to increased incidence of genotoxic responses remains to be further investigated.

A randomized controlled study on the effects of bisoprolol and atenolol on sympathetic nervous activity and central aortic pressure in patients with essential hypertension.

OBJECTIVE: ß-blockers (BBs) with different pharmacological properties may have heterogeneous effects on sympathetic nervous activity (SNA) and central aortic pressure (CAP), which are independent cardiovascular factors for hypertension. Hence, we analyzed the effects of bisoprolol and atenolol on SNA and CAP in hypertensive patients. METHODS: This was a prospective, randomized, controlled study in 109 never-treated hypertensive subjects randomized to bisoprolol (5 mg) or atenolol (50 mg) for 4-8 weeks. SNA, baroreflex sensitivity (BRS) and heart rate (HR) variability (HRV) were measured using power spectral analysis using a Finometer. CAP and related parameters were determined using the SphygmoCor device (pulse wave analysis). RESULTS: Both drugs were similarly effective in reducing brachial BP. However, central systolic BP (-14±10 mm Hg vs -6±9 mm Hg; P<0.001) and aortic pulse pressure (-3±10 mm Hg vs +3±8 mm Hg; P<0.001) decreased more significantly with bisoprolol than with atenolol. The augmentation index at a HR of 75 bpm (AIxatHR75) was significantly decreased (29%±11% to 25%±12%; P = 0.026) in the bisoprolol group only. Furthermore, the change in BRS in the bisoprolol group (3.99±4.19 ms/mmHg) was higher than in the atenolol group (2.66±3.78 ms/mmHg), although not statistically significant (P>0.05). BRS was stable when RHR was controlled (RHR≤65 bpm), and the two treatments had similar effects on the low frequency/high frequency (HF) ratio and on HF. CONCLUSION: BBs seem to have different effects on arterial distensibility and compliance in hypertensive subjects. Compared with atenolol, bisoprolol may have a better effect on CAP. TRIAL REGISTRATION: ClinicalTrials.gov NCT01762436.