Data analysis guidelines for single-cell RNA-seq in biomedical studies and clinical applications.

The application of single-cell RNA sequencing (scRNA-seq) in biomedical research has advanced our understanding of the pathogenesis of disease and provided valuable insights into new diagnostic and therapeutic strategies. With the expansion of capacity for high-throughput scRNA-seq, including clinical samples, the analysis of these huge volumes of data has become a daunting prospect for researchers entering this field. Here, we review the workflow for typical scRNA-seq data analysis, covering raw data processing and quality control, basic data analysis applicable for almost all scRNA-seq data sets, and advanced data analysis that should be tailored to specific scientific questions. While summarizing the current methods for each analysis step, we also provide an online repository of software and wrapped-up scripts to support the implementation. Recommendations and caveats are pointed out for some specific analysis tasks and approaches. We hope this resource will be helpful to researchers engaging with scRNA-seq, in particular for emerging clinical applications.

Transitioning EPMA applications in a large multisite teaching hospital.

INTRODUCTION: University Hospitals of Leicester (UHL) has co-developed and deployed a novel Electronic Prescribing and Medicines Administration (EPMA) application as part of the trust electronic patient record (EPR) programme that meets specific clinical demands and interoperability standards of the National Health Service (NHS) despite clinical pressures from the COVID-19 pandemic. METHODS: Following an initial limited pilot deployment, a big-bang whole site-based approach allowed transition of 1844 acute adult inpatients beds from an existing standalone EMPA to the new system. This project used a frontline driven and agile management strategy. Clinical risk was managed using a combination of standard risk logs, robust clinical prototyping and robust disaster recovery plans. Early engagement with clinical teams allowed for advanced product configuration before live deployment and reduced the need for sustained transition support for clinical staff. RESULTS: An iterative, well-governed approach, led by a combination of information technology (IT) and clinical staff with a responsive vendor, enabled a complex new EPMA system in a large acute NHS trust to be deployed with limited resources despite the ongoing COVID-19 pandemic. DISCUSSION: The development and deployment of EMPA and EPR systems across NHS trusts is a key enabler for better healthcare delivery. This case study shows it is possible to deploy a new clinical IT system at scale without interruption of clinical services and with a relatively modest deployment team. Sustainability of the project was also ensured through a clear clinically led governance structure to manage risk quickly and carry lessons learnt onto new developments.

Post COVID-19 pandemic recovery of intracity human mobility in Wuhan: Spatiotemporal characteristic and driving mechanism.

After successfully inhibiting the first wave of COVID-19 transmission through a city lockdown, Wuhan implemented a series of policies to gradually lift restrictions and restore daily activities. Existing studies mainly focus on the intercity recovery under a macroscopic view. How does the intracity mobility return to normal? Is the recovery process consistent among different subareas, and what factor affects the post-pandemic recovery? To answer these questions, we sorted out policies adopted during the Wuhan resumption, and collected the long-time mobility big data in 1105 traffic analysis zones (TAZs) to construct an observation matrix (A). We then used the nonnegative matrix factorization (NMF) method to approximate A as the product of two condensed matrices (WH). The column vectors of W matrix were visualized as five typical recovery curves to reveal the temporal change. The row vectors of H matrix were visualized to identify the spatial distribution of each recovery type, and were analyzed with variables of population, GDP, land use, and key facility to explain the recovery driving mechanisms. We found that the "staggered time" policies implemented in Wuhan effectively staggered the peak mobility of several recovery types ("staggered peak"). Besides, different TAZs had heterogeneous response intensities to these policies ("staggered area") which were closely related to land uses and key facilities. The creative policies taken by Wuhan highlight the wisdom of public health crisis management, and could provide an empirical reference for the adjustment of post-pandemic intervention measures in other cities.

Multiomics approach reveals the ubiquitination-specific processes hijacked by SARS-CoV-2.

The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global pandemic that seriously threatens health and socioeconomic development, but the existed antiviral drugs and vaccines still cannot yet halt the spread of the epidemic. Therefore, a comprehensive and profound understanding of the pathogenesis of SARS-CoV-2 is urgently needed to explore effective therapeutic targets. Here, we conducted a multiomics study of SARS-CoV-2-infected lung epithelial cells, including transcriptomic, proteomic, and ubiquitinomic. Multiomics analysis showed that SARS-CoV-2-infected lung epithelial cells activated strong innate immune response, including interferon and inflammatory responses. Ubiquitinomic further reveals the underlying mechanism of SARS-CoV-2 disrupting the host innate immune response. In addition, SARS-CoV-2 proteins were found to be ubiquitinated during infection despite the fact that SARS-CoV-2 itself didn't code any E3 ligase, and that ubiquitination at three sites on the Spike protein could significantly enhance viral infection. Further screening of the E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) library revealed four E3 ligases influencing SARS-CoV-2 infection, thus providing several new antiviral targets. This multiomics combined with high-throughput screening study reveals that SARS-CoV-2 not only modulates innate immunity, but also promotes viral infection, by hijacking ubiquitination-specific processes, highlighting potential antiviral and anti-inflammation targets.

Spike-mediated ACE2 down-regulation was involved in the pathogenesis of SARS-CoV-2 infection.

The ongoing global pandemic of Coronavirus disease 2019 (COVID-19) poses a serious threat to human health, with patients reportedly suffering from thrombus, vascular injury and coagulation in addition to acute and diffuse lung injury and respiratory diseases. Angiotensin converting enzyme 2 (ACE2) as the receptor for SARS-CoV-2 entry, is also an important regulator of renin-angiotensin system (RAS) homeostasis, which plays an unsettled role in the pathogenesis of COVID-19. Here, we demonstrated that SARS-CoV-2 Spike protein activated intracellular signals to degrade ACE2 mRNA. The decrease of ACE2 and higher level of angiotensin (Ang) II were verified in COVID-19 patients. High dose of Ang II induced pulmonary artery endothelial cell death in vitro, which was also observed in the lung of COVID-19 patients. Our finding indicates that the downregulation of ACE2 potentially links COVID-19 to the imbalance of RAS.

Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19.

Respiratory immune characteristics associated with Coronavirus Disease 2019 (COVID-19) severity are currently unclear. We characterized bronchoalveolar lavage fluid immune cells from patients with varying severity of COVID-19 and from healthy people by using single-cell RNA sequencing. Proinflammatory monocyte-derived macrophages were abundant in the bronchoalveolar lavage fluid from patients with severe COVID-9. Moderate cases were characterized by the presence of highly clonally expanded CD8+ T cells. This atlas of the bronchoalveolar immune microenvironment suggests potential mechanisms underlying pathogenesis and recovery in COVID-19.

Live streaming to sustain clinical learning.

BACKGROUND: The COVID-19 pandemic has necessitated the need to develop teaching innovations that provide safe, authentic clinical encounters which facilitate experiential learning. In tandem with the dissemination of teleconsultation and online teaching, this pilot study describes, evaluates and justifies a multi-camera live-streaming teaching session to medical students from the clinical environment. APPROACH: Multiple audio and video inputs capturing an outpatient clinic setting were routed through Open Broadcast Software (OBS) to create a customised feed streamed to remote learners through a videoconferencing platform. Sessions were conducted between September 2020 and March 2021. Twelve students sequentially interacted with a patient who held an iPad. Higher quality Go-Pro cameras captured the scene, allowing students to view the consultation from the patient and doctor's perspective. A consultant then conducted a 'gold standard' patient consultation observed by students. A faculty member remotely facilitated the session, providing pre-clinic teaching and debriefing. The equipment required with costing for a standard and low-cost version is described, as well as a set-up schematic and overview of ideal conditions and barriers encountered during trials. EVALUATION: All students completed a post-participation questionnaire, rating the overall quality of the sessions as 9.7/10. The quality of online facilitation, utility of observing peers' and consultant interaction with the patient, opportunity for peer-to-peer learning and availability of multiple camera angles were particularly valued by students. IMPLICATIONS: This innovation permits an authentic clinical interaction to be experienced by multiple students remotely, promoting equitable access to high-quality teaching, while maintaining the safety of students and patients.

SARS-CoV-2 helicase NSP13 hijacks the host protein EWSR1 to promote viral replication by enhancing RNA unwinding activity

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in December 2019 and has led to a global coronavirus disease 2019 (COVID-19) pandemic. Currently, incomplete understanding of how SARS-CoV-2 arrogates the host cell to establish its life cycle has led to slow progress in the development of effective drugs. Results In this study, we found that SARS-CoV-2 hijacks the host protein EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) to promote the activity of its helicase NSP13 to facilitate viral propagation. NSP13 is highly conserved among coronaviruses and is crucial for virus replication, providing chemical energy to unwind viral RNA replication intermediates. Treatment with different SARS-CoV-2 NSP13 inhibitors in multiple cell lines infected with SARS-CoV-2 effectively suppressed SARS-CoV-2 infection. Using affinity-purification mass spectrometry, the RNA binding protein EWSR1 was then identified as a potent host factor that physically associated with NSP13. Furthermore, silencing EWSR1 dramatically reduced virus replication at both viral RNA and protein levels. Mechanistically, EWSR1 was found to bind to the NTPase domain of NSP13 and potentially enhance its dsRNA unwinding ability. Conclusion In conclusion, our results pinpoint EWSR1 as a novel host factor for NSP13 that could potentially be used for drug repurposing as a therapeutic target for COVID-19.

Impact of Long-Term Home Quarantine on Mental Health and Physical Activity of People in Shanghai During the COVID-19 Pandemic.

This study aimed to investigate the effects of long-term home quarantine on the mental health of people during the COVID-19 epidemic in Shanghai. We conducted an online questionnaire survey on March 26 2020 and collected data on demographics, level of physical activity (PA), and mental health status of the participants. We assessed the mental health status using the Patient Health Questionnaire (PHQ-9) and Generalized Anxiety Disorder Scale (GAD-7), whereas PA was assessed using International Physical Activity Questionnaire Short Form (IPAQ-SF). Of all 2,409 valid samples, participants reported performing a total of 2015.20 metabolic equivalent of task (MET)-minutes/week of total PA before the outbreak period and 1720.29 MET-minutes/week of total PA during the outbreak period (p < 0.001). Participants who spent a longer time at home reported to have a better performance on the PHQ-9 (p = 0.087) and GAD-7 (p < 0.001). A high level of PA was considered an protective factor against depression (OR = 0.755, 95% CI 0.603-0.944, p < 0.001). Additionally, a high level of PA had a preventative effect on anxiety (OR = 0.741, 95% CI 0.568-0.967, p < 0.001), and a longer working period during the outbreak was shown to be a risk factor for anxiety (11-29 days, OR 1.455, 95% CI 1.110-1.909; 30-60 days OR 1.619, 95% CI 1.227-2.316). Home confinement during the pandemic might not have a negative effect on mental health provided that people engage in more PA indoors. This study encourages interventions for mental health problems through physical exercise.

The Transient IFN Response and the Delay of Adaptive Immunity Feature the Severity of COVID-19.

COVID-19 patients show heterogeneous and dynamic immune features which determine the clinical outcome. Here, we built a single-cell RNA sequencing (scRNA-seq) dataset for dissecting these complicated immune responses through a longitudinal survey of COVID-19 patients with various categories of outcomes. The data reveals a highly fluctuating peripheral immune landscape in severe COVID-19, whereas the one in asymptomatic/mild COVID-19 is relatively steady. Then, the perturbed immune landscape in peripheral blood returned to normal state in those recovered from severe COVID-19. Importantly, the imbalance of the excessively strong innate immune response and delayed adaptive immunity in the early stage of viral infection accelerates the progression of the disease, indicated by a transient strong IFN response and weak T/B-cell specific response. The proportion of abnormal monocytes appeared early and rose further throughout the severe disease. Our data indicate that a dynamic immune landscape is associated with the progression and recovery of severe COVID-19, and have provided multiple immune biomarkers for early warning of severe COVID-19.

SARS-CoV-2 NSP13 Inhibits Type I IFN Production by Degradation of TBK1 via p62-Dependent Selective Autophagy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.

Codeveloping an effective EMPA to maturity in an acute NHS Trust: an implementer report.

INTRODUCTION: University Hospitals Leicester has codeveloped, with Nervecentre, an Electronic Prescribing and Medicines Administration System that meets specific clinical and interoperability demands of the National Health Service (NHS). METHODS: The system was developed through a frontline-led and agile approach with a project team consisting of clinicians, Information Technology (IT) specialists and the vendor's representatives over an 18-month period. RESULTS: The system was deployed successfully with more than a thousand transcriptions during roll-out. Despite the high caseload and novelty of the system, there was no increase in error rates within the first 3 months of roll-out. Healthcare professionals perceived the new system as efficient with improved clinical workflow, and safe through an integrated medication alert system. DISCUSSION: This case study demonstrates how NHS trusts can successfully co-develop, with vendors, new IT systems which meet interoperability standards such as Fast Healthcare Interoperability Resources, while improving front line clinical experience. CONCLUSION: Alternative methods to the 'big bang' deployment of IT projects, such as 'gradual implementation', must be demonstrated and evaluated for their ability to deliver digital transformation projects in the NHS successfully.

Dysregulated hematopoiesis in bone marrow marks severe COVID-19.

Severe coronavirus disease 2019 (COVID-19) is often indicated by lymphopenia and increased myelopoiesis; however, the underlying mechanism is still unclear, especially the alteration of hematopoiesis. It is important to explore to what extent and how hematopoietic stem cells contribute to the impairment of peripheral lymphoid and myeloid compartments in COVID-19 patients. In this study, we used single-cell RNA sequencing to assess bone marrow mononuclear cells from COVID-19 patients with peripheral blood mononuclear cells as control. The results showed that the hematopoietic stem cells in these patients were mainly in the G1 phase and prone to apoptosis, with immune activation and anti-viral responses. Importantly, a significant accumulation of immature myeloid progenitors and a dramatic reduction of lymphoid progenitors in severe cases were identified, along with the up-regulation of transcription factors (such as SPI1, LMO4, ETS2, FLI1, and GATA2) that are important for the hematopoietic stem cell or multipotent progenitor to differentiate into downstream progenitors. Our results indicate a dysregulated hematopoiesis in patients with severe COVID-19.

Spatial disparities of self-reported COVID-19 cases and influencing factors in Wuhan, China.

The lack of detailed COVID-19 cases at a fine spatial resolution restricts the investigation of spatial disparities of its attack rate. Here, we collected nearly one thousand self-reported cases from a social media platform during the early stage of COVID-19 epidemic in Wuhan, China. We used kernel density estimation (KDE) to explore spatial disparities of epidemic intensity and adopted geographically weighted regression (GWR) model to quantify influences of population dynamics, transportation, and social interactions on COVID-19 epidemic. Results show that self-reported COVID-19 cases concentrated in commercial centers and populous residential areas. Blocks with higher population density, higher aging rate, more metro stations, more main roads, and more commercial point-of-interests (POIs) have a higher density of COVID-19 cases. These five explanatory variables explain 76% variance of self-reported cases using an OLS model. Commercial POIs have the strongest influence, which increase COVID-19 cases by 28% with one standard deviation increase. The GWR model performs better than OLS model with the adjusted R 2 of 0.96. Spatial heterogeneities of coefficients in the GWR model show that influencing factors play different roles in diverse communities. We further discussed potential implications for the healthy city and urban planning for the sustainable development of cities.

SARS-CoV-2 promotes RIPK1 activation to facilitate viral propagation.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the ongoing global pandemic that poses substantial challenges to public health worldwide. A subset of COVID-19 patients experience systemic inflammatory response, known as cytokine storm, which may lead to death. Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is an important mediator of inflammation and cell death. Here, we examined the interaction of RIPK1-mediated innate immunity with SARS-CoV-2 infection. We found evidence of RIPK1 activation in human COVID-19 lung pathological samples, and cultured human lung organoids and ACE2 transgenic mice infected by SARS-CoV-2. Inhibition of RIPK1 using multiple small-molecule inhibitors reduced the viral load of SARS-CoV-2 in human lung organoids. Furthermore, therapeutic dosing of the RIPK1 inhibitor Nec-1s reduced mortality and lung viral load, and blocked the CNS manifestation of SARS-CoV-2 in ACE2 transgenic mice. Mechanistically, we found that the RNA-dependent RNA polymerase of SARS-CoV-2, NSP12, a highly conserved central component of coronaviral replication and transcription machinery, promoted the activation of RIPK1. Furthermore, NSP12 323L variant, encoded by the SARS-CoV-2 C14408T variant first detected in Lombardy, Italy, that carries a Pro323Leu amino acid substitution in NSP12, showed increased ability to activate RIPK1. Inhibition of RIPK1 downregulated the transcriptional induction of proinflammatory cytokines and host factors including ACE2 and EGFR that promote viral entry into cells. Our results suggest that SARS-CoV-2 may have an unexpected and unusual ability to hijack the RIPK1-mediated host defense response to promote its own propagation and that inhibition of RIPK1 may provide a therapeutic option for the treatment of COVID-19.

SCIGA: Software for large-scale, single-cell immunoglobulin repertoire analysis.

BACKGROUND: B-cell immunoglobulin repertoires with paired heavy and light chain can be determined by means of 10X single-cell V(D)J sequencing. Precise and quick analysis of 10X single-cell immunoglobulin repertoires remains a challenge owing to the high diversity of immunoglobulin repertoires and a lack of specialized software that can analyze such diverse data. FINDINGS: In this study, specialized software for 10X single-cell immunoglobulin repertoire analysis was developed. SCIGA (Single-Cell Immunoglobulin Repertoire Analysis) is an easy-to-use pipeline that performs read trimming, immunoglobulin sequence assembly and annotation, heavy and light chain pairing, statistical analysis, visualization, and multiple sample integration analysis, which is all achieved by using a 1-line command. Then SCIGA was used to profile the single-cell immunoglobulin repertoires of 9 patients with coronavirus disease 2019 (COVID-19). Four neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were identified from these repertoires. CONCLUSIONS: SCIGA provides a complete and quick analysis for 10X single-cell V(D)J sequencing datasets. It can help researchers to interpret B-cell immunoglobulin repertoires with paired heavy and light chain.

Dynamics of TCR repertoire and T cell function in COVID-19 convalescent individuals.

SARS-CoV-2 outbreak has been declared by World Health Organization as a worldwide pandemic. However, there are many unknowns about the antigen-specific T-cell-mediated immune responses to SARS-CoV-2 infection. Here, we present both single-cell TCR-seq and RNA-seq to analyze the dynamics of TCR repertoire and immune metabolic functions of blood T cells collected from recently discharged COVID-19 patients. We found that while the diversity of TCR repertoire was increased in discharged patients, it returned to basal level ~1 week after becoming virus-free. The dynamics of T cell repertoire correlated with a profound shift of gene signatures from antiviral response to metabolism adaptation. We also demonstrated that the top expanded T cell clones (~10% of total T cells) display the key anti-viral features in CD8+ T cells, confirming a critical role of antigen-specific T cells in fighting against SARS-CoV-2. Our work provides a basis for further analysis of adaptive immunity in COVID-19 patients, and also has implications in developing a T-cell-based vaccine for SARS-CoV-2.