COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Single-cell analysis of isoform switching and transposable element expression during preimplantation embryonic development.

Alternative splicing is an essential regulatory mechanism for development and pathogenesis. Through alternative splicing one gene can encode multiple isoforms and be translated into proteins with different functions. Therefore, this diversity is an important dimension to understand the molecular mechanism governing embryo development. Isoform expression in preimplantation embryos has been extensively investigated, leading to the discovery of new isoforms. However, the dynamics of isoform switching of different types of transcripts throughout the development remains unexplored. Here, using single-cell direct isoform sequencing in over 100 single blastomeres from the mouse oocyte to blastocyst stage, we quantified isoform expression and found that 3-prime partial transcripts lacking stop codons are highly accumulated in oocytes and zygotes. These transcripts are not transcription by-products and might play a role in maternal to zygote transition (MZT) process. Long-read sequencing also enabled us to determine the expression of transposable elements (TEs) at specific loci. In this way, we identified 3,894 TE loci that exhibited dynamic changes along the preimplantation development, likely regulating the expression of adjacent genes. Our work provides novel insights into the transcriptional regulation of early embryo development.

Decoding the development of the human hippocampus.

The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and the consolidation of information from short-term memory to long-term memory1,2. Here we use single-cell RNA sequencing and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analysis to illustrate the cell types, cell linage, molecular features and transcriptional regulation of the developing human hippocampus. Using the transcriptomes of 30,416 cells from the human hippocampus at gestational weeks 16-27, we identify 47 cell subtypes and their developmental trajectories. We also identify the migrating paths and cell lineages of PAX6+ and HOPX+ hippocampal progenitors, and regional markers of CA1, CA3 and dentate gyrus neurons. Multiomic data have uncovered transcriptional regulatory networks of the dentate gyrus marker PROX1. We also illustrate spatially specific gene expression in the developing human prefrontal cortex and hippocampus. The molecular features of the human hippocampus at gestational weeks 16-20 are similar to those of the mouse at postnatal days 0-5 and reveal gene expression differences between the two species. Transient expression of the primate-specific gene NBPF1 leads to a marked increase in PROX1+ cells in the mouse hippocampus. These data provides a blueprint for understanding human hippocampal development and a tool for investigating related diseases.

A single-cell RNA-seq survey of the developmental landscape of the human prefrontal cortex.

The mammalian prefrontal cortex comprises a set of highly specialized brain areas containing billions of cells and serves as the centre of the highest-order cognitive functions, such as memory, cognitive ability, decision-making and social behaviour. Although neural circuits are formed in the late stages of human embryonic development and even after birth, diverse classes of functional cells are generated and migrate to the appropriate locations earlier in development. Dysfunction of the prefrontal cortex contributes to cognitive deficits and the majority of neurodevelopmental disorders; there is therefore a need for detailed knowledge of the development of the prefrontal cortex. However, it is still difficult to identify cell types in the developing human prefrontal cortex and to distinguish their developmental features. Here we analyse more than 2,300 single cells in the developing human prefrontal cortex from gestational weeks 8 to 26 using RNA sequencing. We identify 35 subtypes of cells in six main classes and trace the developmental trajectories of these cells. Detailed analysis of neural progenitor cells highlights new marker genes and unique developmental features of intermediate progenitor cells. We also map the timeline of neurogenesis of excitatory neurons in the prefrontal cortex and detect the presence of interneuron progenitors in early developing prefrontal cortex. Moreover, we reveal the intrinsic development-dependent signals that regulate neuron generation and circuit formation using single-cell transcriptomic data analysis. Our screening and characterization approach provides a blueprint for understanding the development of the human prefrontal cortex in the early and mid-gestational stages in order to systematically dissect the cellular basis and molecular regulation of prefrontal cortex function in humans.

Single-cell RNA-seq analysis of mouse preimplantation embryos by third-generation sequencing.

The development of next generation sequencing (NGS) platform-based single-cell RNA sequencing (scRNA-seq) techniques has tremendously changed biological researches, while there are still many questions that cannot be addressed by them due to their short read lengths. We developed a novel scRNA-seq technology based on third-generation sequencing (TGS) platform (single-cell amplification and sequencing of full-length RNAs by Nanopore platform, SCAN-seq). SCAN-seq exhibited high sensitivity and accuracy comparable to NGS platform-based scRNA-seq methods. Moreover, we captured thousands of unannotated transcripts of diverse types, with high verification rate by reverse transcription PCR (RT-PCR)-coupled Sanger sequencing in mouse embryonic stem cells (mESCs). Then, we used SCAN-seq to analyze the mouse preimplantation embryos. We could clearly distinguish cells at different developmental stages, and a total of 27,250 unannotated transcripts from 9,338 genes were identified, with many of which showed developmental stage-specific expression patterns. Finally, we showed that SCAN-seq exhibited high accuracy on determining allele-specific gene expression patterns within an individual cell. SCAN-seq makes a major breakthrough for single-cell transcriptome analysis field.

Multicenter, randomized controlled, open label evaluation of the efficacy and safety of arbidol hydrochloride tablets in the treatment of influenza-like cases.

OBJECTIVE: To study the efficacy and safety of arbidol hydrochloride tablets as a treatment for influenza-like diseases. METHODS: In this multicenter, randomized, controlled, open label study, a total of 412 influenza-like cases were collected from 14 hospitals in seven regions of Hebei Province from September 2021 to March 2022. Patients were randomly divided into two groups. The control group (n = 207) were administered oseltamivir phosphate capsules for five days and the experimental group (n = 205) were administered arbidol hydrochloride tablets for five days. The primary endpoint was the time to normal body temperature, and the secondary endpoints included the time to remission of influenza symptoms, incidence of influenza-like complications, and incidence of adverse reactions. RESULTS: Before treatment, there was no significant difference between the two groups in general conditions, blood routine, body temperature, or symptom severity. After treatment, there was no significant difference between the groups in the mean time to fever remission (59.24 h ± 25.21 vs. 61.05 h ± 29.47) or the mean time to remission of influenza symptoms (57.31 h ± 30.19 vs. 62.02 h ± 32.08). Survival analyses using Log-rank and Wilcoxon bilateral tests showed that there was no significant difference in fever relief time or influenza symptom relief time between the two groups. Regarding the incidence of complications and adverse events, there was only one case of tracheitis, one case of nausea, one case of vomiting, and one case of dizziness in the control group. In the experimental group, there was one case of nausea, one case of vomiting, and one case of drowsiness. In addition, one patient in the control group was hospitalized for urinary calculi. CONCLUSION: There was no significant difference between the patients with influenza-like cases treated with arbidol hydrochloride tablets and those treated with oseltamivir phosphate capsules. Further, the patients treated with arbidol hydrochloride tablets had fewer adverse reactions, and thus, the tablets were safe to use.

Role of CxxC-finger protein 1 in establishing mouse oocyte epigenetic landscapes.

During oogenesis, oocytes gain competence and subsequently undergo meiotic maturation and prepare for embryonic development; trimethylated histone H3 on lysine-4 (H3K4me3) mediates a wide range of nuclear events during these processes. Oocyte-specific knockout of CxxC-finger protein 1 (CXXC1, also known as CFP1) impairs H3K4me3 accumulation and causes changes in chromatin configurations. This study investigated the changes in genomic H3K4me3 landscapes in oocytes with Cxxc1 knockout and the effects on other epigenetic factors such as the DNA methylation, H3K27me3, H2AK119ub1 and H3K36me3. H3K4me3 is overall decreased after knocking out Cxxc1, including both the promoter region and the gene body. CXXC1 and MLL2, which is another histone H3 methyltransferase, have nonoverlapping roles in mediating H3K4 trimethylation during oogenesis. Cxxc1 deletion caused a decrease in DNA methylation levels and affected H3K27me3 and H2AK119ub1 distributions, particularly at regions with high DNA methylation levels. The changes in epigenetic networks implicated by Cxxc1 deletion were correlated with the transcriptional changes in genes in the corresponding genomic regions. This study elucidates the epigenetic changes underlying the phenotypes and molecular defects in oocytes with deleted Cxxc1 and highlights the role of CXXC1 in orchestrating multiple factors that are involved in establishing the appropriate epigenetic states of maternal genome.

The lncRNA Hand2os1/Uph locus orchestrates heart development through regulation of precise expression of Hand2.

Exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here, using multiple knockout mouse models and single cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2os1/Uph has a key complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function. Short deletion of the Hand2os1 promoter in mouse diminishes Hand2os1 transcription to ∼8-32%, but fails to affect HAND2 expression and yields no discernable heart phenotypes. Interestingly, full-length deletion of Hand2os1 in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, leading to profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose that the Hand2os1 locus dampens HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the regulatory complexity of the lncRNA Hand2os1 on HAND2 expression, emphasizing the need for complementary genetic and single cell approaches to delineate the function and primary molecular effects of an lncRNA in animals.

Relationship between non-culprit lesion plaque characteristics changes and in-stent neoatherosclerosis formation: 1-year follow-up optical coherence tomography study.

The relationship between the in-stent neoatherosclerosis (ISNA) formation and the plaque's characteristic changes in the non-culprit lesion is unclear. We aim to investigate the plaque characteristics changes at non-culprit lesions between patients with ISNA and without ISNA formation at 1-year follow-up. We retrospectively enrolled patients who had DES implantation in de novo lesion and underwent immediately after stenting and 1-year follow-up optical coherence tomography (OCT) examination. OCT-defined ISNA was defined as the presence of lipid-laden neointima or calcification within the culprit stent with a longitudinal extension of ≥1 mm. Non-culprit lesions were divided into two groups: ISNA group (with ISNA) and non-ISNA group (without ISNA). Plaque characteristics of non-culprit lesions were evaluated at baseline and 1-year follow-up. In total, 89 patients with 89 non-culprit lesions (ISNA: n = 37; non-ISNA: n = 52) were included in the analyses. The lesions in the ISNA group show a smaller minimum lumen area compared to the non-ISNA group at 1-year follow-up (2.57 ± 1.08 mm2 versus 3.20 ± 1.62 mm2, p = 0.044). The lesions of the ISNA group show a significant decrease in minimum lumen area changes percent (-7.25% versus 6.46%, p = 0.039). And there are more lesions with minimum lumen area (64.9% versus 38.5%, p = 0.014) and minimum lumen diameter (64.9% versus 40.4%, p = 0.023) decrease in the ISNA group. Furthermore, the lesions in ISNA group have more plaques with lipid core length increase (25.0% versus 10.0%, p = 0.040), more plaques with FCT decrease (50.0% versus 74.0%, p = 0.027) and less TCFA change to non-TCFA (33.3% versus 87.5%, p = 0.010). The plaque characteristic changes in non-culprit lesions are closely related to ISNA formation. The ISNA formation may accompany by a tardier plaque stabilization process in non-culprit lesions.

Xist Intron 1 Repression by Transcriptional-Activator-Like Effectors Designer Transcriptional Factor Improves Somatic Cell Reprogramming in Mice.

Xist is the master regulator of X chromosome inactivation. In order to further understand the Xist locus in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) and in somatic cell nuclear transfer (SCNT), we tested transcription-activator-like effectors-based designer transcriptional factors (dTFs), which were specific to numerous regions at the Xist locus. We report that the selected dTF repressor 6 (R6) binding the intron 1 of Xist, which caused higher H3K9me3 followed by X chromosome opening and repression of X-linked genes in mouse embryonic fibroblasts, rather than affecting Xist expression, substantially improved the iPSC generation and the SCNT preimplantation embryo development. Conversely, the dTF activator targeting the same genomic region of R6 decreased iPSC formation and blocked SCNT-embryo development. These results thus uncover the critical requirement for the Xist locus in epigenetic resetting, which is not directly related to Xist transcription. This may provide a unique route to improving the reprogramming. Stem Cells 2019;37:599-608.

The DNA methylation landscape of human early embryos.

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

Regional GABA Concentrations Modulate Inter-network Resting-state Functional Connectivity.

Coordinated activity within and differential activity between large-scale neuronal networks such as the default mode network (DMN) and the control network (CN) is a critical feature of brain organization. The CN usually exhibits activations in response to cognitive tasks while the DMN shows deactivations; in addition, activity between the two networks is anti-correlated at rest. To address this issue, we used functional MRI to measure whole-brain BOLD signal during resting-state and task-evoked conditions, and MR spectroscopy (MRS) to quantify GABA and glutamate concentrations, in nodes within the DMN and CN (MPFC and DLPFC, respectively) in 19 healthy individuals at 3 Tesla. We found that GABA concentrations in the MPFC were significantly associated with DMN deactivation during a working memory task and with anti-correlation between DMN and CN at rest and during task performance, while GABA concentrations in the DLPFC weakly modulated DMN-CN anti-correlation in the opposite direction. Highlighting specificity, glutamate played a less significant role related to brain activity. These findings indicate that GABA in the MPFC is potentially involved in orchestrating between-network brain activity at rest and during task performance.

Single-cell RNA-seq analysis reveals the progression of human osteoarthritis.

OBJECTIVES: Understanding the molecular mechanisms underlying human cartilage degeneration and regeneration is helpful for improving therapeutic strategies for treating osteoarthritis (OA). Here, we report the molecular programmes and lineage progression patterns controlling human OA pathogenesis using single-cell RNA sequencing (scRNA-seq). METHODS: We performed unbiased transcriptome-wide scRNA-seq analysis, computational analysis and histological assays on 1464 chondrocytes from 10 patients with OA undergoing knee arthroplasty surgery. We investigated the relationship between transcriptional programmes of the OA landscape and clinical outcome using severity index and correspondence analysis. RESULTS: We identified seven molecularly defined populations of chondrocytes in the human OA cartilage, including three novel phenotypes with distinct functions. We presented gene expression profiles at different OA stages at single-cell resolution. We found a potential transition among proliferative chondrocytes, prehypertrophic chondrocytes and hypertrophic chondrocytes (HTCs) and defined a new subdivision within HTCs. We revealed novel markers for cartilage progenitor cells (CPCs) and demonstrated a relationship between CPCs and fibrocartilage chondrocytes using computational analysis. Notably, we derived predictive targets with respect to clinical outcomes and clarified the role of different cell types for the early diagnosis and treatment of OA. CONCLUSIONS: Our results provide new insights into chondrocyte taxonomy and present potential clues for effective and functional manipulation of human OA cartilage regeneration that could lead to improved health.

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development.

Comparison of triple antithrombotic therapy and dual antiplatelet therapy for patients with atrial fibrillation after percutaneous coronary stenting.

The aim of this study was to evaluate the safety and efficacy of triple antithrombotic therapy with warfarin, aspirin and clopidogrel in patients with atrial fibrillation undergoing percutaneous coronary intervention (PCI). We retrospectively reviewed clinical and follow-up data of 156 consecutive patients with atrial fibrillation after percutaneous coronary stenting. Patients were followed up at 2 and 12 months. A total of 156 consecutive patients were identified. There were 70 patients (dual antiplatelet therapy group, DAPT), warfarin was not used and 86 patients (triple antithrombotic therapy group, TT), both dual antiplatelet therapy and warfarin therapy were prescribed. The baseline data and PCI data were similar in the two groups. The outcome events were similar in the two groups except for bleeding events. There was a significant difference in bleeding risk in the two groups. In summary, triple antithrombotic therapy increases the bleeding risk. Dual antiplatelet therapy decreased this bleeding risk but tended to increase the risk of stroke.

Categorizing Extrachromosomal Circular DNA as Biomarkers in Serum of Cancer.

Extrachromosomal circular DNA (eccDNA), a double-stranded circular DNA molecule found in multiple organisms, has garnered an increasing amount of attention in recent years due to its close association with the initiation, malignant progression, and heterogeneous evolution of cancer. The presence of eccDNA in serum assists in non-invasive tumor diagnosis as a biomarker that can be assessed via liquid biopsies. Furthermore, the specific expression patterns of eccDNA provide new insights into personalized cancer therapy. EccDNA plays a pivotal role in tumorigenesis, development, diagnosis, and treatment. In this review, we comprehensively outline the research trajectory of eccDNA, discuss its role as a diagnostic and prognostic biomarker, and elucidate its regulatory mechanisms in cancer. In particular, we emphasize the potential application value of eccDNA in cancer diagnosis and treatment and anticipate the development of novel tumor diagnosis strategies based on serum eccDNA in the future.

Systematic evaluation of single-cell RNA-seq analyses performance based on long-read sequencing platforms.

INTRODUCTION: The rapid development of next-generation sequencing (NGS)-based single-cell RNA sequencing (scRNA-seq) allows for detecting and quantifying gene expression in a high-throughput manner, providing a powerful tool for comprehensively understanding cellular function in various biological processes. However, the NGS-based scRNA-seq only quantifies gene expression and cannot reveal the exact transcript structures (isoforms) of each gene due to the limited read length. On the other hand, the long read length of third-generation sequencing (TGS) technologies, including Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), enable direct reading of intact cDNA molecules. OBJECTIVES: Both ONT and PacBio have been used in conjunction with scRNA-seq, but their performance in single-cell analyses has not been systematically evaluated. METHODS: To address this, we generated ONT and PacBio data from the same single-cell cDNA libraries containing different amount of cells. RESULTS: Using NGS as a control, we assessed the performance of each platform in cell type identification. Additionally, the reliability in identifying novel isoforms and allele-specific gene/isoform expression by both platforms was verified, providing a systematic evaluation to design the sequencing strategies in single-cell transcriptome studies. CONCLUSION: Beyond gene expression analysis, which the NGS-based scRNA-seq only affords, TGS-based scRNA-seq achieved gene splicing analyses, identifying novel isoforms. Attribute to higher sequencing quality of PacBio, it outperforms ONT in accuracy of novel transcripts identification and allele-specific gene/isoform expression.

Inhibition of HDAC activity directly reprograms murine embryonic stem cells to trophoblast stem cells.

Embryonic stem cells (ESCs) can differentiate into all cell types of the embryonic germ layers. ESCs can also generate totipotent 2C-like cells and trophectodermal cells. However, these latter transitions occur at low frequency due to epigenetic barriers, the nature of which is not fully understood. Here, we show that treating mouse ESCs with sodium butyrate (NaB) increases the population of 2C-like cells and enables direct reprogramming of ESCs into trophoblast stem cells (TSCs) without a transition through a 2C-like state. Mechanistically, NaB inhibits histone deacetylase activities in the LSD1-HDAC1/2 corepressor complex. This increases acetylation levels in the regulatory regions of both 2C- and TSC-specific genes, promoting their expression. In addition, NaB-treated cells acquire the capacity to generate blastocyst-like structures that can develop beyond the implantation stage in vitro and form deciduae in vivo. These results identify how epigenetics restrict the totipotent and trophectoderm fate in mouse ESCs.

Protective effect of ulinastatin on myocardial injuries in children with severe pneumonia.

BACKGROUND: This study aimed to investigate the effect of ulinastatin on myocardial protection in children with severe pneumonia. METHODS: In this retrospective study, children with severe pneumonia were divided into two groups based on their treatment methods. The control group (n=39) received anti-infection therapy, while the experimental group (n=43) received anti-infection therapy combined with ulinastatin. The clinical treatment efficacy, levels of peripheral inflammatory factors, T lymphocyte subsets, QT dispersion and adverse reactions of the two groups were observed and compared before and after treatment. RESULTS: The clinical efficacy was improved after intervention (P<0.05), and the total response rate was 88.4% (38/43) in the experimental group and 64.1% in the control group. The post-treatment levels of interleukin-8 (IL-8), interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) in the peripheral blood were lower than those before treatment, with significant differences (P<0.05). After treatment, the serum levels of CD3+, CD4+ and CD4+/CD8+ in the experimental group were significantly higher than those in the control group, whereas the levels of IL-6, IL-8 and hscRP in the peripheral blood were lower in the experimental group than those in the control group, with significant differences (P<0.05). The QT dispersion indexes, such as QTmax, QTmin, QTd, QTcmax, QTcd and QTcmin in the experimental group were shorter than those in the control group (P<0.05). CONCLUSION: Ulinastatin has significant therapeutic efficacy and safety in the clinical treatment of children with severe pneumonia, which may be related to inhibition of the release of inflammatory factors, shortened QT dispersion and the improvement of immune function of peripheral blood T lymphocyte subsets.