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A majority of the variants identified in genome-wide association studies fall in non-coding regions of the genome, indicating their mechanism of impact is mediated via gene expression. Leveraging this hypothesis, transcriptome-wide association studies (TWAS) have assisted in both the interpretation and discovery of additional genes associated with complex traits. However, existing methods for conducting TWAS do not take full advantage of the intra-individual correlation inherently present in multi-context expression studies and do not properly adjust for multiple testing across contexts. We introduce CONTENT-a computationally efficient method with proper cross-context false discovery correction that leverages correlation structure across contexts to improve power and generate context-specific and context-shared components of expression. We apply CONTENT to bulk multi-tissue and single-cell RNA-seq data sets and show that CONTENT leads to a 42% (bulk) and 110% (single cell) increase in the number of genetically predicted genes relative to previous approaches. We find the context-specific component of expression comprises 30% of heritability in tissue-level bulk data and 75% in single-cell data, consistent with cell-type heterogeneity in bulk tissue. In the context of TWAS, CONTENT increases the number of locus-phenotype associations discovered by over 51% relative to previous methods across 22 complex traits., (© 2022. The Author(s).)

Hydrofluoric acid is a highly dangerous and toxic inorganic acid, which is widely used in industrial fields and daily life. The risk of hydrofluoric acid burns is related to hydrofluoric acid mass fraction, duration of exposure to hydrofluoric acid, burn area, burn depth, and burn site, etc. Hydrofluoric acid has strong toxicity and tissue penetration ability. A small area of hydrofluoric acid burns can cause death in a short time. Therefore, improving the understanding of the mechanism of hydrofluoric acid burns and learning how to treat hydrofluoric acid burns in different sites can further improve the cure rate of hydrofluoric acid burns.

Suppressive myeloid cells can contribute to immunotherapy resistance, but their role in response to checkpoint inhibition (CPI) in anti-PD-1 refractory cancers, such as biliary tract cancer (BTC), remains elusive. We use multiplexed single-cell transcriptomic and epitope sequencing to profile greater than 200,000 peripheral blood mononuclear cells from advanced BTC patients (n = 9) and matched healthy donors (n = 8). Following anti-PD-1 treatment, CD14 + monocytes expressing high levels of immunosuppressive cytokines and chemotactic molecules (CD14 CTX ) increase in the circulation of patients with BTC tumors that are CPI resistant. CD14 CTX can directly suppress CD4 + T cells and induce SOCS3 expression in CD4 + T cells, rendering them functionally unresponsive. The CD14 CTX gene signature associates with worse survival in patients with BTC as well as in other anti-PD-1 refractory cancers. These results demonstrate that monocytes arising after anti-PD-1 treatment can induce T cell paralysis as a distinct mode of tumor-mediated immunosuppression leading to CPI resistance., Competing Interests: Declaration of interests L.F. has received research support (to institution) from Abbvie, Amgen, Bavarian Nordic, Bristol Myers Squibb, Dendreon, Janssen, Merck, Roche/Genentech, and Partner Therapeutics. L.F. has received compensation for serving on the scientific advisory boards (to self) for Actym, Alector, Astra Zeneca, Atreca, Bioatla, Bolt, Bristol Myers Squibb, Immunogenesis, Merck, Merck KGA, Nutcracker, RAPT, Scribe, Senti, Soteria, TeneoBio, and Roche/Genentech. C.J.Y. is a co-founder of Dropprint Genomics, is a Scientific Advisory Board member for and holds equity in Related Sciences and ImmunAI, is a consultant for and holds equity in Maze Therapeutics, is a consultant for TReX Bio, and has received research funding from Genentech. B.K. has received research support (to institution) from Partner Therapeutics. R.K.K. has received research support (to institution) from Agios, Astra Zeneca, Bayer, BMS, Eli Lilly, EMD Serono, Exelixis, Genentech/Roche, Merck, Novartis, Partner Therapeutics, QED, Relay Therapeutics, and Taiho. R.K.K. has received compensation for consulting or IDMC membership from Exact Sciences, Genentech/Roche, and Gilead (to self) and from Agios, Astra Zeneca, BMS, and Merck (to institution)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

An effective therapeutic strategy against methicillin-resistant Staphylococcus aureus (MRSA) that does not promote further drug resistance is highly desirable. While phototherapies have demonstrated considerable promise, their application toward bacterial infections can be limited by negative off-target effects to healthy cells. Here, a smart targeted nanoformulation consisting of a liquid perfluorocarbon core stabilized by a lipid membrane coating is developed. Using vancomycin as a targeting agent, the platform is capable of specifically delivering an encapsulated photosensitizer along with oxygen to sites of MRSA infection, where high concentrations of pore-forming toxins trigger on-demand payload release. Upon subsequent near-infrared irradiation, local increases in temperature and reactive oxygen species effectively kill the bacteria. Additionally, the secreted toxins that are captured by the nanoformulation can be processed by resident immune cells to promote multiantigenic immunity that protects against secondary MRSA infections. Overall, the reported approach for the on-demand release of phototherapeutic agents into sites of infection could be applied against a wide range of high-priority pathogens., (© 2022 Wiley-VCH GmbH.)

Objective: The objective of this study is to explore the association of problem gambling with demographics, psychological distress, and gaming behavior in young adult gacha gamers in Hong Kong., Materials and Methods: Cross-sectional data was collected in the first and fifth waves of COVID-19 pandemic in Hong Kong online. Participants who aged 18-25 years and had been playing gacha games over the past 12 months were recruited. Stepwise multiple regression was used to explore the association among risk of problem gambling, gaming behavior, participation in gaming activities and psychological distress. A two-sided p-value <0.05 was considered as statistical significance., Results: Three hundred and thirty-seven completed questionnaires were received with no missing data. 34.7% ( n = 117) of the participants had non/low-risk of problem gambling. About 40% ( n = 136) of them had moderate-risk and the remaining 25% ( n = 84) were at high risk of problem gambling. A higher proportion of female participants (78.6%) were found in high-risk group as compared to 39.7% and 55.6% only in the non/low-risk and moderate-risk groups, respectively. The regression model ( R 2 = 0.513, F = 71.895, p < 0.001) showed that 51.3% of the variance of the total problem gambling score could be explained by stress, anxiety, monthly expenses on gacha purchases, number of motives for gacha purchase and number of gambling activities engaged., Conclusion: The present study provides empirical evidence to support the association between problem gambling and microtransaction especially for gacha which is the most popular type of video game microtransaction in Asia. The established regression model suggests that gacha gamers with higher risk of problem gambling tend to have greater stress, higher anxiety level, spend more on gacha purchase, have more motives for gacha purchases and engage in more gambling activities. In contrast to the extant literature, higher proportion of female participants in high-risk group indicates that female gacha gamers are also at very high risk of becoming problem gamblers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tang, Lee, Lam, Siu, Ye and Lee.)

Maintaining water balance is a real challenge for amphibians in terrestrial environments. Our previous studies with toad Bombina maxima discovered a pore-forming protein and trefoil factor complex βγ-CAT, which is assembled under tight regulation depending on environmental cues. Here we report an unexpected role for βγ-CAT in toad water maintaining. Deletion of toad skin secretions, in which βγ-CAT is a major component, increased animal mortality under hypertonic stress. βγ-CAT was constitutively expressed in toad osmoregulatory organs, which was inducible under the variation of osmotic conditions. The protein induced and participated in macropinocytosis in vivo and in vitro. During extracellular hyperosmosis, βγ-CAT stimulated macropinocytosis to facilitate water import and enhanced exosomes release, which simultaneously regulated aquaporins distribution. Collectively, these findings uncovered that besides membrane integrated aquaporin, a secretory pore-forming protein can facilitate toad water maintaining via macropinocytosis induction and exocytosis modulation, especially in responses to osmotic stress., (© 2022. The Author(s).)

Sialoblastoma (SBL) is an infrequent embryonal malignant tumor originating from the salivary gland, resembling primitive salivary gland anlage, whereas hepatoblastoma (HB) is the most common pediatric liver malignancy. The simultaneous occurrence of both tumors is extremely rare. Here we reported a case of a 6-month-old infant diagnosed with synchronous SBL and HB. The patient received neoadjuvant chemotherapy followed by surgical resection. Fresh tissues of both tumors were collected before and after chemotherapy, which were further profiled by whole exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq). WES analysis revealed potential somatic driver mutation PIK3CA p.Glu454Lys for SBL and canonical mutation CTNNB1 p.Ser45Pro for HB. No shared somatic variants or common copy number alterations were found between SBL and HB primary tumor samples. Though scRNA-seq, single-cell atlases were constructed for both tumors. SBL may recapitulate a pre-acinar stage in the development of salivary gland, including basaloid, duct-like, myoepithelial-like, and cycling phenotypes. In the meantime, HB was composed of tumor cells resembling different stages of the liver, including hepatocyte-like, hepatic progenitor-like, and hepatoblast-like cells. After chemotherapy, both tumors were induced into a more mature phenotype. In terms of transcriptional signatures, SBL and HB showed enhanced expression of epithelial markers KRT8, KRT18 , and essential embryo development genes SDC1, MDK , indicating the disruption of normal embryo epithelium development. Finally, heterozygous deleterious germline mutation BLM and FANCI were identified which could predispose the patient to higher cancer risk. It partially explained the reason for the co-occurrence of SBL and HB. Taken together, we provided valuable resources for deciphering cellular heterogeneity and adaptive change of tumor cells after chemotherapy for synchronous SBL and HB, providing insights into the mechanisms leading to synchronous pediatric tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yang, Zhan, Li, Dai, He, Ye, Meng, Chen, Dong, Chen, Chen, Dong, Li, Zheng, Li, Yao and Dong.)

Juvenile dermatomyositis (JDM) is a rare autoimmune condition with insufficient biomarkers and treatments, in part, due to incomplete knowledge of the cell types mediating disease. We investigated immunophenotypes and cell-specific genes associated with disease activity using multiplexed RNA and protein single-cell sequencing applied to PBMCs from 4 treatment-naïve JDM (TN-JDM) subjects at baseline, 2, 4, and 6 months post-treatment and 4 subjects with inactive disease on treatment. Analysis of 55,564 cells revealed separate clustering of TN-JDM cells within monocyte, NK, CD8 + effector T and naïve B populations. The proportion of CD16 + monocytes was reduced in TN-JDM, and naïve B cells and CD4 + Tregs were expanded. Cell-type differential gene expression analysis and hierarchical clustering identified a pan-cell-type IFN gene signature over-expressed in TN-JDM in all cell types and correlated with disease activity most strongly in cytotoxic cell types. TN-JDM CD16 + monocytes expressed the highest IFN gene score and were highly skewed toward an inflammatory and antigen-presenting phenotype at both the transcriptomic and proteomic levels. A transitional B cell population with a distinct transcriptomic signature was expanded in TN-JDM and characterized by higher CD24 and CD5 proteins and less CD39, an immunoregulatory protein. This data provides new insights into JDM immune dysregulation at cellular resolution and serves as a novel resource for myositis investigators., Competing Interests: CJY was employed by Chan Zuckerberg Biohub. CJY is a Scientific Advisory Board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TReX Bio. CJY has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Neely, Hartoularos, Bunis, Sun, Lee, Kim, Ye and Sirota.)

The host's gene expression and gene regulatory response to pathogen exposure can be influenced by a combination of the host's genetic background, the type of and exposure time to pathogens. Here we provide a detailed dissection of this using single-cell RNA-sequencing of 1.3M peripheral blood mononuclear cells from 120 individuals, longitudinally exposed to three different pathogens. These analyses indicate that cell-type-specificity is a more prominent factor than pathogen-specificity regarding contexts that affect how genetics influences gene expression (i.e., eQTL) and co-expression (i.e., co-expression QTL). In monocytes, the strongest responder to pathogen stimulations, 71.4% of the genetic variants whose effect on gene expression is influenced by pathogen exposure (i.e., response QTL) also affect the co-expression between genes. This indicates widespread, context-specific changes in gene expression level and its regulation that are driven by genetics. Pathway analysis on the CLEC12A gene that exemplifies cell-type-, exposure-time- and genetic-background-dependent co-expression interactions, shows enrichment of the interferon (IFN) pathway specifically at 3-h post-exposure in monocytes. Similar genetic background-dependent association between IFN activity and CLEC12A co-expression patterns is confirmed in systemic lupus erythematosus by in silico analysis, which implies that CLEC12A might be an IFN-regulated gene. Altogether, this study highlights the importance of context for gaining a better understanding of the mechanisms of gene regulation in health and disease., (© 2022. The Author(s).)

The human immune system displays substantial variation between individuals, leading to differences in susceptibility to autoimmune disease. We present single-cell RNA sequencing (scRNA-seq) data from 1,267,758 peripheral blood mononuclear cells from 982 healthy human subjects. For 14 cell types, we identified 26,597 independent cis-expression quantitative trait loci (eQTLs) and 990 trans-eQTLs, with most showing cell type-specific effects on gene expression. We subsequently show how eQTLs have dynamic allelic effects in B cells that are transitioning from naïve to memory states and demonstrate how commonly segregating alleles lead to interindividual variation in immune function. Finally, using a Mendelian randomization approach, we identify the causal route by which 305 risk loci contribute to autoimmune disease at the cellular level. This work brings together genetic epidemiology with scRNA-seq to uncover drivers of interindividual variation in the immune system.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease. Knowledge of circulating immune cell types and states associated with SLE remains incomplete. We profiled more than 1.2 million peripheral blood mononuclear cells (162 cases, 99 controls) with multiplexed single-cell RNA sequencing (mux-seq). Cases exhibited elevated expression of type 1 interferon-stimulated genes (ISGs) in monocytes, reduction of naïve CD4 + T cells that correlated with monocyte ISG expression, and expansion of repertoire-restricted cytotoxic GZMH + CD8 + T cells. Cell type-specific expression features predicted case-control status and stratified patients into two molecular subtypes. We integrated dense genotyping data to map cell type-specific cis-expression quantitative trait loci and to link SLE-associated variants to cell type-specific expression. These results demonstrate mux-seq as a systematic approach to characterize cellular composition, identify transcriptional signatures, and annotate genetic variants associated with SLE.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system 1-7 , although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (T H 2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent T H 17 inflammation. We also observed divergent responses to biologic therapies targeting T H 2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in T H 2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo T H response towards a T H 2-predominant state and prevent aberrant non-T H 2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of T H 17 pathology and unlocked therapeutic responsiveness to targeted anti-T H 2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Early diagnosis of psoriatic arthritis (PSA) is important for successful therapeutic intervention but currently remains challenging due, in part, to the scarcity of non-invasive biomarkers. In this study, we performed single cell profiling of transcriptome and cell surface protein expression to compare the peripheral blood immunocyte populations of individuals with PSA, individuals with cutaneous psoriasis (PSO) alone, and healthy individuals. We identified genes and proteins differentially expressed between PSA, PSO, and healthy subjects across 30 immune cell types and observed that some cell types, as well as specific phenotypic subsets of cells, differed in abundance between these cohorts. Cell type-specific gene and protein expression differences between PSA, PSO, and healthy groups, along with 200 previously published genetic risk factors for PSA, were further used to perform machine learning classification, with the best models achieving AUROC ≥ 0.87 when either classifying subjects among the three groups or specifically distinguishing PSA from PSO. Our findings thus expand the repertoire of gene, protein, and cellular biomarkers relevant to PSA and demonstrate the utility of machine learning-based diagnostics for this disease., Competing Interests: Author WL has received funding from Abbvie, Amgen, Janssen, Leo, Novartis, Pfizer, Regeneron, and TRex Bio. Author CY is a Scientific Advisory Board member for and holds equity in Related Sciences and ImmunAI, a consultant for and holds equity in Maze Therapeutics, and a consultant for TReX Bio. Author CY has received funding from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. Author LG has received funding from Novartis, Pfizer, UCB, AbbVie, Eli Lilly, Gilead, Janssen, Novartis, Pfizer and UCB. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Kumar, Hong, Huang, Paez, Castillo, Calvo, Chang, Cummins, Chung, Yeroushalmi, Bartholomew, Hakimi, Ye, Bhutani, Matloubian, Gensler and Liao.)

Neuroanatomists have long speculated that expanded primate brains contain an increased morphological diversity of inhibitory neurons (INs) 1 , and recent studies have identified primate-specific neuronal populations at the molecular level 2 . However, we know little about the developmental mechanisms that specify evolutionarily novel cell types in the brain. Here, we reconstruct gene expression trajectories specifying INs generated throughout the neurogenic period in macaques and mice by analysing the transcriptomes of 250,181 cells. We find that the initial classes of INs generated prenatally are largely conserved among mammals. Nonetheless, we identify two contrasting developmental mechanisms for specifying evolutionarily novel cell types during prenatal development. First, we show that recently identified primate-specific TAC3 striatal INs are specified by a unique transcriptional programme in progenitors followed by induction of a distinct suite of neuropeptides and neurotransmitter receptors in new-born neurons. Second, we find that multiple classes of transcriptionally conserved olfactory bulb (OB)-bound precursors are redirected to expanded primate white matter and striatum. These classes include a novel peristriatal class of striatum laureatum neurons that resemble dopaminergic periglomerular cells of the OB. We propose an evolutionary model in which conserved initial classes of neurons supplying the smaller primate OB are reused in the enlarged striatum and cortex. Together, our results provide a unified developmental taxonomy of initial classes of mammalian INs and reveal multiple developmental mechanisms for neural cell type evolution., (© 2022. The Author(s).)

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.

Single-cell RNA sequencing (scRNA-seq) has the potential to provide powerful, high-resolution signatures to inform disease prognosis and precision medicine. This paper takes an important first step towards this goal by developing an interpretable machine learning algorithm, CloudPred, to predict individuals' disease phenotypes from their scRNA-seq data. Predicting phenotype from scRNA-seq is challenging for standard machine learning methods-the number of cells measured can vary by orders of magnitude across individuals and the cell populations are also highly heterogeneous. Typical analysis creates pseudo-bulk samples which are biased toward prior annotations and also lose the single cell resolution. CloudPred addresses these challenges via a novel end-to-end differentiable learning algorithm which is coupled with a biologically informed mixture of cell types model. CloudPred automatically infers the cell subpopulation that are salient for the phenotype without prior annotations. We developed a systematic simulation platform to evaluate the performance of CloudPred and several alternative methods we propose, and find that CloudPred outperforms the alternative methods across several settings. We further validated CloudPred on a real scRNA-seq dataset of 142 lupus patients and controls. CloudPred achieves AUROC of 0.98 while identifying a specific subpopulation of CD4 T cells whose presence is highly indicative of lupus. CloudPred is a powerful new framework to predict clinical phenotypes from scRNA-seq data and to identify relevant cells.

Astigmatid mites are economically significant pests of stored products and sources of inhalant allergens causing allergic rhinitis and asthma worldwide. The morphological identification of astigmatid mites at the species level is often a difficult task due to their small size, phenotypic similarity and lack of diagnostic characters. We used multiplex polymerase chain reaction (PCR) to identify astigmatid mite species, which could complement the morphological data for the species-specific identification of mites. Internal ribosomal transcribed spacer (ITS) sequences (i.e., partial 18S, the full length of ITS1-5.8S-ITS2 and partial 28S) from eight astigmatid species ( Acarus siro , Tyrophagus putrescentiae , Suidasia nesbitti , Dermatophagoides pteronyssinus , Dermatophagoides farinae , Lepidoglyphus destructor , Chortoglyphus arcuatus and Gohieria fuscus ) were obtained by DNA extraction and then sequenced after PCR amplification. Specific primers were designed in the ITS2 region manually. Results revealed that an identification method for eight common astigmatid species was established based on multiplex PCR, which should be effective for the identification of other species of mites by redesigning species-specific primers in future experiments., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 Urmia University. All rights reserved.)

We describe an unvaccinated child at risk for life-threatening COVID-19 due to an inherited deficiency of IRF9, which governs ISGF-3-dependent responses to type I and III interferons (IFN). She was admitted, with a high nasal SARS-CoV-2 load on day 1 of upper respiratory tract infection. She was viremic on day 2 and received casirivimab and imdevimab. Her clinical manifestations and viremia disappeared on days 3 and 4, respectively. Circulating SARS-CoV-2 virus induced the expression of IFN-stimulated genes in leukocytes on day 1, whereas the secretion of blood type I IFNs, which peaked on day 4, did not. Antibody-mediated SARS-CoV-2 neutralization is, therefore, sufficient to overcome a deficiency of antiviral IFNs., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Objective: To explore the role of first-aid network construction in the early treatment of patients with critically severe hydrofluoric acid burns. Methods: Twenty-seven fluorine chemical enterprises distributed in Zhejiang province, Jiangxi Province, Fujian Province, and Inner Mongolia Autonomous Region and 22 hospitals with burn/plastic department or professional burn treatment group in Zhejiang province, including Zhejiang Quhua Hospital, and 5 hospitals outside Zhejiang province were involved in the first-aid network construction as member units. As the main unit, Zhejiang Quhua Hospital was responsible for the daily maintenance and technical guidance of the first-aid network. Zhejiang Quhua Hospital was assigned as the designated emergency hospital for 20 fluorine chemical enterprises, a near emergency hospital to the other 7 fluorine chemical enterprises was assigned as the designated hospital for them. Medical records of 56 patients (all males) with critically severe hydrofluoric acid burns who admitted to 5 first-aid network hospitals from January 2006 to June 2021, meeting the inclusion criteria, were involved in the retrospective cohort study. Based on whether the enterprise belonging to the first-aid network construction or not, the patients were divided into first-aid network group (27 cases, aged (41±9) years) and non first-aid network group (29 cases, aged (42±10) years). After the patients in the first-aid network group were injured, the enterprises and hospitals linked up immediately. The hospital where the patient was treated mobilize the treatment force, equipment, materials, and drugs in advance by the first-aid network, thereby realizing seamless joint between pre-hospital first-aid and in-hospital treatment. The hospital started the first-aid process and temporarily mobilized the rescue forces, equipment, materials, and drug after patients in non first-aid network group arrived at the department of emergency of the hospital. The time from injury to medical service, the first detection time of serum calcium, the time staying in department of emergency, the duration of hypocalcemia and hypomagnesemia, and the treatment outcome of patients in the two groups were recorded. Data were statistically analyzed with chi-square test, Fisher's exact probability test, independent-sample t test, and Wilcoxon rank-sum test. Results: The time from injury to medical service, the first detection time of serum calcium, and the time staying in department of emergency of patients in first-aid network group were 40.0 (30.0, 55.0), 23.0 (17.5, 37.5), and 42.0 (37.0, 53.0) min, which were significantly shorter than 180.0 (120.0, 240.0), 31.0 (22.5, 47.5), 61.0 (52.0, 65.5) min in non first-aid network group ( Z =-6.17, -1.98, -4.15, P <0.05 or P <0.01). The duration of hypocalcemia and hypo- magnesemia of patients in first-aid network group were 1.2 (1.1, 1.6) and 1.9 (1.7, 2.1) h, which were significantly shorter than 4.6 (3.1, 6.2) and 3.2 (2.5, 4.6) h in non first-aid network group ( Z =-5.80, -4.81, P <0.01). Three patients (11.1%) in first-aid network group died, among whom 2 patients died at 40 min after injury and 1 patient died 9.0 h after injury. Four patients (13.8%) died in non first-aid network group at 3.0, 3.0, 4.5, and 7.0 h after injury, respectively. The mortality rates of patients in the two groups were similar ( P >0.05). Conclusions: Critically severe hydrofluoric acid burn is an extremely urgent situation encountered in clinical practice. The construction of a first-aid network creates condition for on-site treatment of patients and improves the first-aid efficiency, thereby gaining time to save lives.

BACKGROUNDA previous phase I study showed that the infusion of autologous Tregs expanded ex vivo into patients with recent-onset type 1 diabetes (T1D) had an excellent safety profile. However, the majority of the infused Tregs were undetectable in the peripheral blood 3 months postinfusion (Treg-T1D trial). Therefore, we conducted a phase I study (TILT trial) combining polyclonal Tregs and low-dose IL-2, shown to enhance Treg survival and expansion, and assessed the impact over time on Treg populations and other immune cells.METHODSPatients with T1D were treated with a single infusion of autologous polyclonal Tregs followed by one or two 5-day courses of recombinant human low-dose IL-2 (ld-IL-2). Flow cytometry, cytometry by time of flight, and 10x Genomics single-cell RNA-Seq were used to follow the distinct immune cell populations' phenotypes over time.RESULTSMultiparametric analysis revealed that the combination therapy led to an increase in the number of infused and endogenous Tregs but also resulted in a substantial increase from baseline in a subset of activated NK, mucosal associated invariant T, and clonal CD8+ T cell populations.CONCLUSIONThese data support the hypothesis that ld-IL-2 expands exogenously administered Tregs but also can expand cytotoxic cells. These results have important implications for the use of a combination of ld-IL-2 and Tregs for the treatment of autoimmune diseases with preexisting active immunity.TRIAL REGISTRATIONClinicalTrials.gov NCT01210664 (Treg-T1D trial), NCT02772679 (TILT trial).FUNDINGSean N. Parker Autoimmune Research Laboratory Fund, National Center for Research Resources.

Neutralizing autoantibodies against type I interferons (IFNs) have been found in some patients with critical coronavirus disease 2019 (COVID-19), the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the prevalence of these antibodies, their longitudinal dynamics across the disease severity scale, and their functional effects on circulating leukocytes remain unknown. Here, in 284 patients with COVID-19, we found type I IFN–specific autoantibodies in peripheral blood samples from 19% of patients with critical disease and 6% of patients with severe disease. We found no type I IFN autoantibodies in individuals with moderate disease. Longitudinal profiling of over 600,000 peripheral blood mononuclear cells using multiplexed single-cell epitope and transcriptome sequencing from 54 patients with COVID-19 and 26 non–COVID-19 controls revealed a lack of type I IFN–stimulated gene (ISG-I) responses in myeloid cells from patients with critical disease. This was especially evident in dendritic cell populations isolated from patients with critical disease producing type I IFN–specific autoantibodies. Moreover, we found elevated expression of the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) on the surface of monocytes isolated from patients with critical disease early in the disease course. LAIR1 expression is inversely correlated with ISG-I expression response in patients with COVID-19 but is not expressed in healthy controls. The deficient ISG-I response observed in patients with critical COVID-19 with and without type I IFN–specific autoantibodies supports a unifying model for disease pathogenesis involving ISG-I suppression through convergent mechanisms.

In traumatic brain injury (TBI), a diversity of brain resident and peripherally derived myeloid cells have the potential to worsen damage and/or to assist in healing. We define the heterogeneity of microglia and macrophage phenotypes during TBI in wild-type (WT) mice and Ccr2 -/- mice, which lack macrophage influx following TBI and are resistant to brain damage. We use unbiased single-cell RNA sequencing methods to uncover 25 microglia, monocyte/macrophage, and dendritic cell subsets in acute TBI and normal brains. We find alterations in transcriptional profiles of microglia subsets in Ccr2 -/- TBI mice compared to WT TBI mice indicating that infiltrating monocytes/macrophages influence microglia activation to promote a type I IFN response. Preclinical pharmacological blockade of hCCR2 after injury reduces expression of IFN-responsive gene, Irf7, and improves outcomes. These data extend our understanding of myeloid cell diversity and crosstalk in brain trauma and identify therapeutic targets in myeloid subsets., Competing Interests: Declaration of interests I.C., B.A.Z., J.C., and R.S. are presently or were employees of ChemoCentryx during this study. B.T.W., S.G., L.J., M.C., G.B., and E.D.P. are presently or were employees of Biogen during this study. B.T.W. is currently employed by Moderna. C.J.Y. is a Scientific Advisory Board member for and hold equity in Related Sciences and ImmunAI, a consultant for and hold equity in Maze Therapeutics, and a consultant for TReX Bio. C.J.Y. has received research support from Chan Zuckerberg Initiative, Chan Zuckerberg Biohub, and Genentech. A U.S. Provisional Patent Application No. 63/177,727 for the use of CCX872 in TBI, spinal cord injury, or stroke was filed April 21, 2021., (Published by Elsevier Inc.)

Acute myeloid leukemia patients refractory to induction therapy or relapsed within one year have poor outcomes. Autocrine production of hepatocyte growth factor by myeloid blasts drives leukemogenesis in pre-clinical models. A phase Ib trial evaluated ficlatuzumab, a first-in-class anti-HGF antibody, in combination with cytarabine in this high-risk population. Dose-limiting toxicities were not observed, and 20 mg/kg was established as the recommended phase II dose. The most frequent treatment-related adverse event was febrile neutropenia. Among 17 evaluable patients, the overall response rate was 53%, all complete remissions. Phospho-proteomic mass cytometry showed potent on-target suppression of p-MET after ficlatuzumab treatment and that attenuation of p-S6 was associated with clinical response. Multiplexed single cell RNA sequencing using prospectively acquired patient specimens identified interferon response genes as adverse predictive factors. The ficlatuzumab and cytarabine combination is well-tolerated with favorable efficacy. High-dimensional analyses at single-cell resolution represent promising approaches for identifying biomarkers of response and mechanisms of resistance in prospective clinical studies., Competing Interests: Conflict of Interests: The authors declare no potential conflicts of interest.

Abstract: Rhabdomyosarcoma is the most common soft tissue sarcoma in children, and embryonal rhabdomyosarcoma is the most typical type of rhabdomyosarcoma. The heterogeneity, etiology, and origin of embryonal rhabdomyosarcoma remain unknown.After obtaining the gene expression data of every cell in the tumor tissue by single-cell RNA sequencing, we used the Seurat package in R studio for quality control, analysis, and exploration of the data. All cells are divided into tumor cells and non-tumor cells, and we chose tumor cells by marker genes. Then, we repeated the process to cluster the tumor cells and divided the subgroups by their differentially expressed genes and gene ontology/Kyoto Encyclopedia of Genes and Genomes analysis. Additionally, Monocle 2 was used for pseudo-time analysis to obtain the evolution trajectory of cells in tumor tissues.Tumor cells were divided into 5 subgroups according to their functions, which were characterized by high proliferation, sensing and adaptation to oxygen availability, enhanced epigenetic modification, enhanced nucleoside phosphonic acid metabolism, and ossification. Evolution trajectory of cells in tumor tissues is obtained.We used pseudo-time analysis to distinguish between mesenchymal stem cells and fibroblasts, proved that embryonal rhabdomyosarcoma in the pelvic originated from skeletal muscle progenitor cells, showed the evolutionary trajectory of embryonal rhabdomyosarcoma, and improved the method of evaluating the degree of malignancy of embryonal rhabdomyosarcoma., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that βγ-crystallin fused aerolysin-like protein and trefoil factor complex (βγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima , directly blocks enveloped virus invasion by interfering with cytoplasmic entry. βγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, βγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the βγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that βγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the βγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of βγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that βγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs., (Copyright © 2021 by The American Association of Immunologists, Inc.)

The development of DNA-barcoded antibodies to tag cell surface molecules has enabled the use of droplet-based single-cell sequencing (dsc-seq) to profile protein abundances from thousands of cells simultaneously. As compared to flow and mass cytometry, the high per cell cost of current dsc-seq-based workflows precludes their use in clinical applications and large-scale pooled screens. Here, we introduce SCITO-seq, a workflow that uses splint oligonucleotides (oligos) to enable combinatorially indexed dsc-seq of DNA-barcoded antibodies from over 10 5 cells per reaction using commercial microfluidics. By encoding sample barcodes into splint oligos, we demonstrate that multiplexed SCITO-seq produces reproducible estimates of cellular composition and surface protein expression comparable to those from mass cytometry. We further demonstrate two modified splint oligo designs that extend SCITO-seq to achieve compatibility with commercial DNA-barcoded antibodies and simultaneous expression profiling of the transcriptome and surface proteins from the same cell. These results demonstrate SCITO-seq as a flexible and ultra-high-throughput platform for sequencing-based single-cell protein and multimodal profiling., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis., Competing Interests: AH, AR, LL, SA, JS, SG, XP, HJ, DB, MP, LL, JL, EC, MA, GF, JY, CY, GK, VW, MH No competing interests declared, (© 2021, Hendley et al.)

Systemic lupus erythematosus (SLE) is an autoimmune disease in which outcomes vary among different racial groups. We leverage cell-sorted RNA-seq data (CD14+ monocytes, B cells, CD4+ T cells, and NK cells) from 120 SLE patients (63 Asian and 57 White individuals) and apply a four-tier approach including unsupervised clustering, differential expression analyses, gene co-expression analyses, and machine learning to identify SLE subgroups within this multiethnic cohort. K-means clustering on each cell-type resulted in three clusters for CD4 and CD14, and two for B and NK cells. To understand the identified clusters, correlation analysis revealed significant positive associations between the clusters and clinical parameters including disease activity as well as ethnicity. We then explored differentially expressed genes between Asian and White groups for each cell-type. The shared differentially expressed genes across cells were involved in SLE or other autoimmune-related pathways. Co-expression analysis identified similarly regulated genes across samples and grouped these genes into modules. Finally, random forest classification of disease activity in the White and Asian cohorts showed the best classification in CD4+ T cells in White individuals. The results from these analyses will help stratify patients based on their gene expression signatures to enable SLE precision medicine.

Single-cell RNA sequencing (scRNA-seq) of tissues has revealed remarkable heterogeneity of cell types and states but does not provide information on the spatial organization of cells. To better understand how individual cells function within an anatomical space, we developed XYZeq, a workflow that encodes spatial metadata into scRNA-seq libraries. We used XYZeq to profile mouse tumor models to capture spatially barcoded transcriptomes from tens of thousands of cells. Analyses of these data revealed the spatial distribution of distinct cell types and a cell migration-associated transcriptomic program in tumor-associated mesenchymal stem cells (MSCs). Furthermore, we identify localized expression of tumor suppressor genes by MSCs that vary with proximity to the tumor core. We demonstrate that XYZeq can be used to map the transcriptome and spatial localization of individual cells in situ to reveal how cell composition and cell states can be affected by location within complex pathological tissue., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).)

Although infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has pleiotropic and systemic effects in some individuals 1-3 , many others experience milder symptoms. Here, to gain a more comprehensive understanding of the distinction between severe and mild phenotypes in the pathology of coronavirus disease 2019 (COVID-19) and its origins, we performed a whole-blood-preserving single-cell analysis protocol to integrate contributions from all major immune cell types of the blood-including neutrophils, monocytes, platelets, lymphocytes and the contents of the serum. Patients with mild COVID-19 exhibit a coordinated pattern of expression of interferon-stimulated genes (ISGs) 3 across every cell population, whereas these ISG-expressing cells are systemically absent in patients with severe disease. Paradoxically, individuals with severe COVID-19 produce very high titres of anti-SARS-CoV-2 antibodies and have a lower viral load compared to individuals with mild disease. Examination of the serum from patients with severe COVID-19 shows that these patients uniquely produce antibodies that functionally block the production of the ISG-expressing cells associated with mild disease, by activating conserved signalling circuits that dampen cellular responses to interferons. Overzealous antibody responses pit the immune system against itself in many patients with COVID-19, and perhaps also in individuals with other viral infections. Our findings reveal potential targets for immunotherapies in patients with severe COVID-19 to re-engage viral defence.

The thymus' key function in the immune system is to provide the necessary environment for the development of diverse and self-tolerant T lymphocytes. While recent evidence suggests that the thymic stroma is comprised of more functionally distinct subpopulations than previously appreciated, the extent of this cellular heterogeneity in the human thymus is not well understood. Here we use single-cell RNA sequencing to comprehensively profile the human thymic stroma across multiple stages of life. Mesenchyme, pericytes and endothelial cells are identified as potential key regulators of thymic epithelial cell differentiation and thymocyte migration. In-depth analyses of epithelial cells reveal the presence of ionocytes as a medullary population, while the expression of tissue-specific antigens is mapped to different subsets of epithelial cells. This work thus provides important insight on how the diversity of thymic cells is established, and how this heterogeneity contributes to the induction of immune tolerance in humans.

Objectives: This study aimed to investigate the association between periodontal indexes and biomarkers in gingival crevicular fluid (GCF) and preterm birth (PTB) in pregnancy, as well as to assess the clinical value of these indexes as predictors of PTB., Methods: A nested case-control study was conducted. A total of 300 systematically healthy pregnant women were selected within 36 weeks of gestation and grouped according to the enrolled weeks. Periodontal indexes, including probing depth (PD), bleeding index (BI), gingival index (GI), and five biomarkers in GCF, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were measured at the enrolled date. The detailed birth outcome was recorded., Results: Only women at 24-28 weeks of gestation per PTB case (four full-term births) were selected as controls subjects, PTB displayed significantly greater GI, BI, and 8-OHdG ( P <0.05). Logistic regression analysis revealed that BI and 8-OHdG were the dependent risk factors of PTB (OR=5.90, P =0.034; OR=1.18, P =0.045, respectively). The areas under the receiver operating characteristic curve (ROC) of BI and 8-OHdG were 0.80 and 0.69, and that of the combined detection was 0.82, which was larger than the individual detection, although the differences were not significant ( P >0.05)., Conclusions: Increased BI and 8-OHdG at 24-28 weeks of gestation are risk factors for PTB. Their combined detection may have some value in the prediction of PTB, but further studies with a larger sample size are needed to explore it and thus provide experiment evidence for establishing an early warning system for PTB in pregnant women with periodontal disease.

Background: Single-cell RNA sequencing (scRNA-seq) provides high-dimensional measurements of transcript counts in individual cells. However, high assay costs and artifacts associated with analyzing samples across multiple sequencing runs limit the study of large numbers of samples. Sample multiplexing technologies such as MULTI-seq and antibody hashing using single-cell multiplexing kit (SCMK) reagents (BD Biosciences) use sample-specific sequence tags to enable individual samples to be sequenced in a pooled format, markedly lowering per-sample processing and sequencing costs while minimizing technical artifacts. Critically, however, pooling samples could introduce new artifacts, partially negating the benefits of sample multiplexing. In particular, no study to date has evaluated whether pooling peripheral blood mononuclear cells (PBMCs) from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) results in significant changes in gene expression resulting from alloreactivity (i.e., response to non-self). The ability to demonstrate minimal to no alloreactivity is crucial to avoid confounded data analyses, particularly for cross-sectional studies evaluating changes in immunologic gene signatures., Results: Here, we applied the 10x Genomics scRNA-seq platform to MULTI-seq and/or SCMK-labeled PBMCs from a single donor with and without pooling with PBMCs from unrelated donors for 30 min at 4 °C. We did not detect any alloreactivity signal between mixed and unmixed PBMCs across a variety of metrics, including alloreactivity marker gene expression in CD4+ T cells, cell type proportion shifts, and global gene expression profile comparisons using Gene Set Enrichment Analysis and Jensen-Shannon Divergence. These results were additionally mirrored in publicly-available scRNA-seq data generated using a similar experimental design. Moreover, we identified confounding gene expression signatures linked to PBMC preparation method (e.g., Trima apheresis), as well as SCMK sample classification biases against activated CD4+ T cells which were recapitulated in two other SCMK-incorporating scRNA-seq datasets., Conclusions: We demonstrate that (i) mixing PBMCs from unrelated donors under standard scRNA-seq sample preparation conditions (e.g., 30 min co-incubation at 4 °C) does not cause an allogeneic response, and (ii) that Trima apheresis and PBMC sample multiplexing using SCMK reagents can introduce undesirable technical artifacts into scRNA-seq data. Collectively, these observations establish important benchmarks for future cross-sectional immunological scRNA-seq experiments.

Whereas the human fetal immune system is poised to generate immune tolerance and suppress inflammation in utero, an adult-like immune system emerges to orchestrate anti-pathogen immune responses in post-natal life. It has been posited that cells of the adult immune system arise as a discrete ontological "layer" of hematopoietic stem-progenitor cells (HSPCs) and their progeny; evidence supporting this model in humans has, however, been inconclusive. Here, we combine bulk and single-cell transcriptional profiling of lymphoid cells, myeloid cells, and HSPCs from fetal, perinatal, and adult developmental stages to demonstrate that the fetal-to-adult transition occurs progressively along a continuum of maturity-with a substantial degree of inter-individual variation at the time of birth-rather than via a transition between discrete waves. These findings have important implications for the design of strategies for prophylaxis against infection in the newborn and for the use of umbilical cord blood (UCB) in the setting of transplantation., Competing Interests: Declaration of Interests C.J.Y. declares partnership at Related Sciences and TReX Bio and owns stock in DroPrint Genomics and Related Sciences. C.J.Y. is supported by research grants from CZ Biohub, CZI, and PICI., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Human regulatory T (T reg ) cells are essential for immune homeostasis. The transcription factor FOXP3 maintains T reg cell identity, yet the complete set of key transcription factors that control T reg cell gene expression remains unknown. Here, we used pooled and arrayed Cas9 ribonucleoprotein screens to identify transcription factors that regulate critical proteins in primary human T reg cells under basal and proinflammatory conditions. We then generated 54,424 single-cell transcriptomes from T reg cells subjected to genetic perturbations and cytokine stimulation, which revealed distinct gene networks individually regulated by FOXP3 and PRDM1, in addition to a network coregulated by FOXO1 and IRF4. We also discovered that HIVEP2, to our knowledge not previously implicated in T reg cell function, coregulates another gene network with SATB1 and is important for T reg cell-mediated immunosuppression. By integrating CRISPR screens and single-cell RNA-sequencing profiling, we have uncovered transcriptional regulators and downstream gene networks in human T reg cells that could be targeted for immunotherapies.

Single-cell RNA-sequencing (scRNA-Seq) is a compelling approach to directly and simultaneously measure cellular composition and state, which can otherwise only be estimated by applying deconvolution methods to bulk RNA-Seq estimates. However, it has not yet become a widely used tool in population-scale analyses, due to its prohibitively high cost. Here we show that given the same budget, the statistical power of cell-type-specific expression quantitative trait loci (eQTL) mapping can be increased through low-coverage per-cell sequencing of more samples rather than high-coverage sequencing of fewer samples. We use simulations starting from one of the largest available real single-cell RNA-Seq data from 120 individuals to also show that multiple experimental designs with different numbers of samples, cells per sample and reads per cell could have similar statistical power, and choosing an appropriate design can yield large cost savings especially when multiplexed workflows are considered. Finally, we provide a practical approach on selecting cost-effective designs for maximizing cell-type-specific eQTL power which is available in the form of a web tool.

Appropriate use and interpretation of serological tests for assessments of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exposure, infection and potential immunity require accurate data on assay performance. We conducted a head-to-head evaluation of ten point-of-care-style lateral flow assays (LFAs) and two laboratory-based enzyme-linked immunosorbent assays to detect anti-SARS-CoV-2 IgM and IgG antibodies in 5-d time intervals from symptom onset and studied the specificity of each assay in pre-coronavirus disease 2019 specimens. The percent of seropositive individuals increased with time, peaking in the latest time interval tested (>20 d after symptom onset). Test specificity ranged from 84.3% to 100.0% and was predominantly affected by variability in IgM results. LFA specificity could be increased by considering weak bands as negative, but this decreased detection of antibodies (sensitivity) in a subset of SARS-CoV-2 real-time PCR-positive cases. Our results underline the importance of seropositivity threshold determination and reader training for reliable LFA deployment. Although there was no standout serological assay, four tests achieved more than 80% positivity at later time points tested and more than 95% specificity.

When discussing chromosomal instability, most of the literature focuses on the characterization of individual molecular mechanisms. These studies search for genomic and environmental causes and consequences of chromosomal instability in cancer, aiming to identify key triggering factors useful to control chromosomal instability and apply this knowledge in the clinic. Since cancer is a phenomenon of new system emergence from normal tissue driven by somatic evolution, such studies should be done in the context of new genome system emergence during evolution. In this perspective, both the origin and key outcome of chromosomal instability are examined using the genome theory of cancer evolution. Specifically, chromosomal instability was linked to a spectrum of genomic and non-genomic variants, from epigenetic alterations to drastic genome chaos. These highly diverse factors were then unified by the evolutionary mechanism of cancer. Following identification of the hidden link between cellular adaptation (positive and essential) and its trade-off (unavoidable and negative) of chromosomal instability, why chromosomal instability is the main player in the macro-cellular evolution of cancer is briefly discussed. Finally, new research directions are suggested, including searching for a common mechanism of evolutionary phase transition, establishing chromosomal instability as an evolutionary biomarker, validating the new two-phase evolutionary model of cancer, and applying such a model to improve clinical outcomes and to understand the genome-defined mechanism of organismal evolution.

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-to-transfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes ~2 months, which includes sequencing turnaround time and data processing with MPRAflow.

To dissect the disease heterogeneity and identify the underlying cellular and molecular events related to metastasis of immature ovarian teratoma in children, single-cell RNA sequencing was performed for a 2-year-old patient with liver metastases from immature ovarian teratoma. A total of 5976 cells were obtained for further analysis, with a median unique molecular identifier count of 6011 per cell and a median number of 1741 genes detected per cell. Fourteen clusters were recognized, with the main lineages comprising epithelial cells, macrophages, fibroblasts, glial cells, and dendritic cells. Ten subclusters of epithelial cells were further defined, originating from the urinary tract, esophagus, bronchus, lung, skin, and gastrointestinal tract. An undefined UBE2C + population in an active state of proliferation was also identified and its biological processes were related to meiosis and maturation of oocytes. Pseudotime analysis revealed different distributions of epithelial cells in the development trajectory. In conclusion, a cluster of UBE2C + epithelial cells in an active state of proliferation was identified in an immature ovarian teratoma in a child, and may contribute to metastasis by regulating epithelial-mesenchymal transition. These findings help toward understanding the origin of the malignant behaviors, offer a potential biomarker for early determination of the tumor nature, and provide new ideas for the therapy of immature ovarian teratoma in children., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Single-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. We observe that snRNA-seq is commonly subject to contamination by high amounts of ambient RNA, which can lead to biased downstream analyses, such as identification of spurious cell types if overlooked. We present a novel approach to quantify contamination and filter droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: (1) human differentiating preadipocytes in vitro, (2) fresh mouse brain tissue, and (3) human frozen adipose tissue (AT) from six individuals. All three data sets showed evidence of extranuclear RNA contamination, and we observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq, our clustering strategy also successfully filtered single-cell RNA-seq data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoires of CD8 + T cells are not distinct in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4 + T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4 + T cell states that are clonally expanded. These CD4 + T cells can kill autologous tumors in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4 + T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1., Competing Interests: Declaration of Interests D.Y.O. has received research support from Roche/Genentech and Merck and has served as a paid consultant for Maze Therapeutics. L.F. has received research support from Roche/Genentech, Abbvie, Bavarian Nordic, Bristol Myers Squibb, Janssen, and Merck. C.J.Y. is a co-founder of Dropprint Genomics., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: During the outbreak of coronavirus disease 2019 (COVID-19), consistent and considerable differences in disease severity and mortality rate of patients treated in Hubei province compared to those in other parts of China have been observed. We sought to compare the clinical characteristics and outcomes of patients being treated inside and outside Hubei province, and explore the factors underlying these differences., Methods: Collaborating with the National Health Commission, we established a retrospective cohort to study hospitalised COVID-19 cases in China. Clinical characteristics, the rate of severe events and deaths, and the time to critical illness (invasive ventilation or intensive care unit admission or death) were compared between patients within and outside Hubei. The impact of Wuhan-related exposure (a presumed key factor that drove the severe situation in Hubei, as Wuhan is the epicentre as well the administrative centre of Hubei province) and the duration between symptom onset and admission on prognosis were also determined., Results: At the data cut-off (31 January 2020), 1590 cases from 575 hospitals in 31 provincial administrative regions were collected (core cohort). The overall rate of severe cases and mortality was 16.0% and 3.2%, respectively. Patients in Hubei (predominantly with Wuhan-related exposure, 597 (92.3%) out of 647) were older (mean age 49.7 versus 44.9 years), had more cases with comorbidity (32.9% versus 19.7%), higher symptomatic burden, abnormal radiologic manifestations and, especially, a longer waiting time between symptom onset and admission (5.7 versus 4.5 days) compared with patients outside Hubei. Patients in Hubei (severe event rate 23.0% versus 11.1%, death rate 7.3% versus 0.3%, HR (95% CI) for critical illness 1.59 (1.05-2.41)) have a poorer prognosis compared with patients outside Hubei after adjusting for age and comorbidity. However, among patients outside Hubei, the duration from symptom onset to hospitalisation (mean 4.4 versus 4.7 days) and prognosis (HR (95%) 0.84 (0.40-1.80)) were similar between patients with or without Wuhan-related exposure. In the overall population, the waiting time, but neither treated in Hubei nor Wuhan-related exposure, remained an independent prognostic factor (HR (95%) 1.05 (1.01-1.08))., Conclusion: There were more severe cases and poorer outcomes for COVID-19 patients treated in Hubei, which might be attributed to the prolonged duration of symptom onset to hospitalisation in the epicentre. Future studies to determine the reason for delaying hospitalisation are warranted., Competing Interests: Conflict of interest: Wen-hua Liang has nothing to disclose. Conflict of interest: Wei-jie Guan has nothing to disclose. Conflict of interest: Cai-chen Li has nothing to disclose. Conflict of interest: Yi-min Li has nothing to disclose. Conflict of interest: Heng-rui Liang has nothing to disclose. Conflict of interest: Yi Zhao has nothing to disclose. Conflict of interest: Xiao-qing Liu has nothing to disclose. Conflict of interest: Ling Sang has nothing to disclose. Conflict of interest: Ru-chong Chen has nothing to disclose. Conflict of interest: Chun-li Tang has nothing to disclose. Conflict of interest: Tao Wang has nothing to disclose. Conflict of interest: Wei Wang has nothing to disclose. Conflict of interest: Qi-hua He has nothing to disclose. Conflict of interest: Zi-sheng Chen has nothing to disclose. Conflict of interest: Sook-San Wong has nothing to disclose. Conflict of interest: Mark Zanin has nothing to disclose. Conflict of interest: Jun Liu has nothing to disclose. Conflict of interest: Xin Xu has nothing to disclose. Conflict of interest: Jun Huang has nothing to disclose. Conflict of interest: Jian-fu Li has nothing to disclose. Conflict of interest: Li-min Ou has nothing to disclose. Conflict of interest: Bo Cheng has nothing to disclose. Conflict of interest: Shan Xiong has nothing to disclose. Conflict of interest: Zhan-hong Xie has nothing to disclose. Conflict of interest: Zheng-yi Ni has nothing to disclose. Conflict of interest: Yu Hu has nothing to disclose. Conflict of interest: Lei Liu has nothing to disclose. Conflict of interest: Hong Shan has nothing to disclose. Conflict of interest: Chun-liang Lei has nothing to disclose. Conflict of interest: Yi-xiang Peng has nothing to disclose. Conflict of interest: Li Wei has nothing to disclose. Conflict of interest: Yong Liu has nothing to disclose. Conflict of interest: Ya-hua Hu has nothing to disclose. Conflict of interest: Peng has nothing to disclose. Conflict of interest: Jian-ming Wang has nothing to disclose. Conflict of interest: Ji-yang Liu has nothing to disclose. Conflict of interest: Zhong Chen has nothing to disclose. Conflict of interest: Gang Li has nothing to disclose. Conflict of interest: Zhi-jian Zheng has nothing to disclose. Conflict of interest: Shao-qin Qiu has nothing to disclose. Conflict of interest: Jie Luo has nothing to disclose. Conflict of interest: Chang-jiang Ye has nothing to disclose. Conflict of interest: Shao-yong Zhu has nothing to disclose. Conflict of interest: Lin-ling Cheng has nothing to disclose. Conflict of interest: Feng Ye has nothing to disclose. Conflict of interest: Shi-yue Li has nothing to disclose. Conflict of interest: Jin-ping Zheng has nothing to disclose. Conflict of interest: Nuo-fu Zhang has nothing to disclose. Conflict of interest: Nan-shan Zhong has nothing to disclose. Conflict of interest: Jian-xing He has nothing to disclose., (Copyright ©ERS 2020.)