Mutation characteristics of cancer susceptibility genes in Chinese ovarian cancer patients.

Introduction: The association between mutations in susceptibility genes and the occurrence of ovarian cancer has been extensively studied. Previous research has primarily concentrated on genes involved in the homologous recombination repair pathway, particularly BRCA1 and BRCA2. However, a wider range of genes related to the DNA damage response pathways has not been fully explored. Methods: To investigate the mutation characteristics of cancer susceptibility genes in the Chinese ovarian cancer population and the associations between gene mutations and clinical data, this study initially gathered a total of 1171 Chinese ovarian cancer samples and compiled a dataset of germline mutations in 171 genes. Results: In this study, it was determined that MC1R and PRKDC were high-frequency ovarian cancer susceptibility genes in the Chinese population, exhibiting notable distinctions from those in European and American populations; moreover high-frequency mutation genes, such as MC1R: c.359T>C and PRKDC: c.10681T>A, typically had high-frequency mutation sites. Furthermore, we identified c.8187G>T as a characteristic mutation of BRCA2 in the Chinese population, and the CHEK2 mutation was significantly associated with the early onset of ovarian cancer, while the CDH1 and FAM175A mutations were more prevalent in Northeast China. Additionally, Fanconi anemia pathway-related genes were significantly associated with ovarian carcinogenesis. Conclusion: In summary, this research provided fundamental data support for the optimization of ovarian cancer gene screening policies and the determination of treatment, and contributed to the precise intervention and management of patients.

Multiome in the Same Cell Reveals the Impact of Osmotic Stress on Arabidopsis Root Tip Development at Single-Cell Level.

Cell-specific transcriptional regulatory networks (TRNs) play vital roles in plant development and response to environmental stresses. However, traditional single-cell mono-omics techniques are unable to directly capture the relationships and dynamics between different layers of molecular information within the same cells. While advanced algorithm facilitates merging scRNA-seq and scATAC-seq datasets, accurate data integration remains a challenge, particularly when investigating cell-type-specific TRNs. By examining gene expression and chromatin accessibility simultaneously in 16,670 Arabidopsis root tip nuclei, the TRNs are reconstructed that govern root tip development under osmotic stress. In contrast to commonly used computational integration at cell-type level, 12,968 peak-to-gene linkage is captured at the bona fide single-cell level and construct TRNs at an unprecedented resolution. Furthermore, the unprecedented datasets allow to more accurately reconstruct the coordinated changes of gene expression and chromatin states during cellular state transition. During root tip development, chromatin accessibility of initial cells precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for subsequent differentiation steps. Pseudo-time trajectory analysis reveal that osmotic stress can shift the functional differentiation of trichoblast. Candidate stress-related gene-linked cis-regulatory elements (gl-cCREs) as well as potential target genes are also identified, and uncovered large cellular heterogeneity under osmotic stress.

Single-cell transcriptomic analysis reveals transcriptional and cell subpopulation differences between human and pig immune cells.

BACKGROUND: The pig is a promising donor candidate for xenotransplantation. Understanding the differences between human and swine immune systems is critical for addressing xenotransplant rejection and hematopoietic reconstitution. The gene transcriptional profile differences between human and pig immune cell subpopulations have not been studied. To assess the similarities and differences between pigs and humans at the levels of gene transcriptional profiles or cell subpopulations are important for better understanding the cross-species similarity of humans and pigs, and it would help establish the fundamental principles necessary to genetically engineer donor pigs and improve xenotransplantation. OBJECTIVE: To assess the gene transcriptional similarities and differences between pigs and humans. METHODS: Two pigs and two healthy humans' PBMCs were sorted for 10 × genomics single-cell sequence. We generated integrated human-pig scRNA-seq data from human and pig PBMCs and defined the overall gene expression landscape of pig peripheral blood immune cell subpopulations by updating the set of human-porcine homologous genes. The subsets of immune cells were detected by flow cytometry. RESULTS: There were significantly less T cells, NK cells and monocytes but more B cells in pig peripheral blood than those in human peripheral blood. High oxidative phosphorylation, HIF-1, glycolysis, and lysosome-related gene expressions in pig CD14+ monocytes were observed, whereas pig CD14+ monocytes exhibited lower levels of cytokine receptors and JAK-STAT-related genes. Pig activated CD4+T cells decreased cell adhesion and inflammation, while enriched for migration and activation processes. Porcine GNLY+CD8+T cells reduced cytotoxicity and increased proliferation compared with human GNLY+CD8+T cells. Pig CD2+CD8+γδT cells were functionally homologous to human CD2+CD4+ γδT cells. Pig CD2-CD8-γδT cells expressed genes with quiescent and precursor characteristics, while CD2-CD8+γδT cells expressed migration and memory-related molecules. Pig CD24+ and CD5+B cells are associated with inflammatory responses. CONCLUSION: Our research with integrated scRNA-seq assays identified the different distribution of pig immune cell subpopulations and the different transcriptional profiles of human and pig immune cells. This study enables a deeper understanding of the development and function of porcine immune cells.

Dissecting embryonic and extraembryonic lineage crosstalk with stem cell co-culture.

Embryogenesis necessitates harmonious coordination between embryonic and extraembryonic tissues. Although stem cells of both embryonic and extraembryonic origins have been generated, they are grown in different culture conditions. In this study, utilizing a unified culture condition that activates the FGF, TGF-ß, and WNT pathways, we have successfully derived embryonic stem cells (FTW-ESCs), extraembryonic endoderm stem cells (FTW-XENs), and trophoblast stem cells (FTW-TSCs) from the three foundational tissues of mouse and cynomolgus monkey (Macaca fascicularis) blastocysts. This approach facilitates the co-culture of embryonic and extraembryonic stem cells, revealing a growth inhibition effect exerted by extraembryonic endoderm cells on pluripotent cells, partially through extracellular matrix signaling. Additionally, our cross-species analysis identified both shared and unique transcription factors and pathways regulating FTW-XENs. The embryonic and extraembryonic stem cell co-culture strategy offers promising avenues for developing more faithful embryo models and devising more developmentally pertinent differentiation protocols.

Single-cell analysis reveals specific neuronal transition during mouse corticogenesis.

Background: Currently, the mechanism(s) underlying corticogenesis is still under characterization. Methods: We curated the most comprehensive single-cell RNA-seq (scRNA-seq) datasets from mouse and human fetal cortexes for data analysis and confirmed the findings with co-immunostaining experiments. Results: By analyzing the developmental trajectories with scRNA-seq datasets in mice, we identified a specific developmental sub-path contributed by a cell-population expressing both deep- and upper-layer neurons (DLNs and ULNs) specific markers, which occurred on E13.5 but was absent in adults. In this cell-population, the percentages of cells expressing DLN and ULN markers decreased and increased, respectively, during the development suggesting direct neuronal transition (namely D-T-U). Whilst genes significantly highly/uniquely expressed in D-T-U cell population were significantly enriched in PTN/MDK signaling pathways related to cell migration. Both findings were further confirmed by co-immunostaining with DLNs, ULNs and D-T-U specific markers across different timepoints. Furthermore, six genes (co-expressed with D-T-U specific markers in mice) showing a potential opposite temporal expression between human and mouse during fetal cortical development were associated with neuronal migration and cognitive functions. In adult prefrontal cortexes (PFC), D-T-U specific genes were expressed in neurons from different layers between humans and mice. Conclusion: Our study characterizes a specific cell population D-T-U showing direct DLNs to ULNs neuronal transition and migration during fetal cortical development in mice. It is potentially associated with the difference of cortical development in humans and mice.

A novel computational method enables RNA editome profiling during human hematopoiesis from scRNA-seq data.

RNA editing is a post-transcriptional modification with a cell-specific manner and important biological implications. Although single-cell RNA-seq (scRNA-seq) is an effective method for studying cellular heterogeneity, it is difficult to detect and study RNA editing events from scRNA-seq data because of the low sequencing coverage. To overcome this, we develop a computational method to systematically identify RNA editing sites of cell types from scRNA-seq data. To demonstrate its effectiveness, we apply it to scRNA-seq data of human hematopoietic stem/progenitor cells (HSPCs) with an annotated lineage differentiation relationship according to previous research and study the impacts of RNA editing on hematopoiesis. The dynamic editing patterns reveal the relevance of RNA editing on different HSPCs. For example, four microRNA (miRNA) target sites on 3' UTR of EIF2AK2 are edited across all HSPC populations, which may abolish the miRNA-mediated inhibition of EIF2AK2. Elevated EIF2AK2 may thus activate the integrated stress response (ISR) pathway to initiate global translational attenuation as a protective mechanism to maintain cellular homeostasis during HSPCs' differentiation. Besides, our findings also indicate that RNA editing plays an essential role in the coordination of lineage commitment and self-renewal of hematopoietic stem cells (HSCs). Taken together, we demonstrate the capacity of scRNA-seq data to exploit RNA editing events of cell types, and find that RNA editing may exert multiple modules of regulation in hematopoietic processes.

Noninvasive preeclampsia prediction using plasma cell-free RNA signatures.

BACKGROUND: Preeclampsia, especially preterm preeclampsia and early-onset preeclampsia, is a life-threating pregnancy disorder, and the heterogeneity and complexity of preeclampsia make it difficult to predict risk and to develop treatments. Plasma cell-free RNA carries unique information from human tissue and may be useful for noninvasive monitoring of maternal, placental, and fetal dynamics during pregnancy. OBJECTIVE: This study aimed to investigate various RNA biotypes associated with preeclampsia in plasma and to develop classifiers to predict preterm preeclampsia and early-onset preeclampsia before diagnosis. STUDY DESIGN: We performed a novel, cell-free RNA sequencing method termed polyadenylation ligation-mediated sequencing to investigate the cell-free RNA characteristics of 715 healthy pregnancies and 202 pregnancies affected by preeclampsia before symptom onset. We explored differences in the abundance of different RNA biotypes in plasma between healthy and preeclampsia samples and built preterm preeclampsia and early-onset preeclampsia prediction classifiers using machine learning methods. Furthermore, we validated the performance of the classifiers using the external and internal validation cohorts and assessed the area under the curve and positive predictive value. RESULTS: We detected 77 genes, including messenger RNA (44%) and microRNA (26%), that were differentially expressed in healthy mothers and mothers with preterm preeclampsia before symptom onset, which could separate participants with preterm preeclampsia from healthy samples and that played critical functional roles in preeclampsia physiology. We developed 2 classifiers for predicting preterm preeclampsia and early-onset preeclampsia before diagnosis based on 13 cell-free RNA signatures and 2 clinical features (in vitro fertilization and mean arterial pressure), respectively. Notably, both classifiers showed enhanced performance when compared with the existing methods. The preterm preeclampsia prediction model achieved 81% area under the curve and 68% positive predictive value in an independent validation cohort (preterm, n=46; control, n=151); the early-onset preeclampsia prediction model had an area under the curve of 88% and a positive predictive value of 73% in an external validation cohort (early-onset preeclampsia, n=28; control, n=234). Furthermore, we demonstrated that downregulation of microRNAs may play vital roles in preeclampsia through the upregulation of preeclampsia-relevant target genes. CONCLUSION: In this cohort study, a comprehensive transcriptomic landscape of different RNA biotypes in preeclampsia was presented and 2 advanced classifiers with substantial clinical importance for preterm preeclampsia and early-onset preeclampsia prediction before symptom onset were developed. We demonstrated that messenger RNA, microRNA, and long noncoding RNA can simultaneously serve as potential biomarkers of preeclampsia, holding the promise of prevention of preeclampsia in the future. Abnormal cell-free messenger RNA, microRNA, and long noncoding RNA molecular changes may help to elucidate the pathogenic determinants of preeclampsia and open new therapeutic windows to effectively reduce pregnancy complications and fetal morbidity.

Establishment of bovine trophoblast stem cells.

Here, we report that a chemical cocktail (LCDM: leukemia inhibitory factor [LIF], CHIR99021, dimethinedene maleate [DiM], minocycline hydrochloride), previously developed for extended pluripotent stem cells (EPSCs) in mice and humans, enables de novo derivation and long-term culture of bovine trophoblast stem cells (TSCs). Bovine TSCs retain developmental potency to differentiate into mature trophoblast cells and exhibit transcriptomic and epigenetic (chromatin accessibility and DNA methylome) features characteristic of trophectoderm cells from early bovine embryos. The bovine TSCs established in this study will provide a model to study bovine placentation and early pregnancy failure.

Dissecting embryonic and extra-embryonic lineage crosstalk with stem cell co-culture.

Faithful embryogenesis requires precise coordination between embryonic and extraembryonic tissues. Although stem cells from embryonic and extraembryonic origins have been generated for several mammalian species(Bogliotti et al., 2018; Choi et al., 2019; Cui et al., 2019; Evans and Kaufman, 1981; Kunath et al., 2005; Li et al., 2008; Martin, 1981; Okae et al., 2018; Tanaka et al., 1998; Thomson et al., 1998; Vandevoort et al., 2007; Vilarino et al., 2020; Yu et al., 2021b; Zhong et al., 2018), they are grown in different culture conditions with diverse media composition, which makes it difficult to study cross-lineage communication. Here, by using the same culture condition that activates FGF, TGF-ß and WNT signaling pathways, we derived stable embryonic stem cells (ESCs), extraembryonic endoderm stem cells (XENs) and trophoblast stem cells (TSCs) from all three founding tissues of mouse and cynomolgus monkey blastocysts. This allowed us to establish embryonic and extraembryonic stem cell co-cultures to dissect lineage crosstalk during early mammalian development. Co-cultures of ESCs and XENs uncovered a conserved and previously unrecognized growth inhibition of pluripotent cells by extraembryonic endoderm cells, which is in part mediated through extracellular matrix signaling. Our study unveils a more universal state of stem cell self-renewal stabilized by activation, as opposed to inhibition, of developmental signaling pathways. The embryonic and extraembryonic stem cell co-culture strategy developed here will open new avenues for creating more faithful embryo models and developing more developmentally relevant differentiation protocols.

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism.

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.

Stemness-related genes revealed by single-cell profiling of naïve and stimulated human CD34+ cells from CB and mPB.

BACKGROUND: Hematopoietic stem cells (HSCs) from different sources show varied repopulating capacity, and HSCs lose their stemness after long-time ex vivo culture. A deep understanding of these phenomena may provide helpful insights for HSCs. METHODS: Here, we applied single-cell RNA-seq (scRNA-seq) to analyse the naïve and stimulated human CD34+ cells from cord blood (CB) and mobilised peripheral blood (mPB). RESULTS: We collected over 16 000 high-quality single-cell data to construct a comprehensive inference map and characterised the HSCs under a quiescent state on the hierarchy top. Then, we compared HSCs in CB with those in mPB and HSCs of naïve samples to those of cultured samples, and identified stemness-related genes (SRGs) associated with cell source (CS-SRGs) and culture time (CT-SRGs), respectively. Interestingly, CS-SRGs and CT-SRGs share genes enriched in the signalling pathways such as mRNA catabolic process, translational initiation, ribonucleoprotein complex biogenesis and cotranslational protein targeting to membrane, suggesting dynamic protein translation and processing may be a common requirement for stemness maintenance. Meanwhile, CT-SRGs are enriched in pathways involved in glucocorticoid and corticosteroid response that affect HSCs homing and engraftment. In contrast, CS-SRGs specifically contain genes related to purine and ATP metabolic process, which is crucial for HSC homeostasis in the stress settings. Particularly, when CT-SRGs are used as reference genes for the construction of the development trajectory of CD34+ cells, lymphoid and myeloid lineages are clearly separated after HSCs/MPPs. Finally, we presented an application through a small-scale drug screening using Connectivity Map (CMap) against CT-SRGs. A small molecule, cucurbitacin I, was found to efficiently expand HSCs ex vivo while maintaining its stemness. CONCLUSIONS: Our findings provide new perspectives for understanding HSCs, and the strategy to identify candidate molecules through SRGs may be applicable to study other stem cells.

Single-cell transcriptome reveals core cell populations and androgen-RXFP2 axis involved in deer antler full regeneration.

Deer antlers constitute a unique mammalian model for the study of both organ formation in postnatal life and annual full regeneration. Previous studies revealed that these events are achieved through the proliferation and differentiation of antlerogenic periosteum (AP) cells and pedicle periosteum (PP) cells, respectively. As the cells resident in the AP and the PP possess stem cell attributes, both antler generation and regeneration are stem cell-based processes. However, the cell composition of each tissue type and molecular events underlying antler development remain poorly characterized. Here, we took the approach of single-cell RNA sequencing (scRNA-Seq) and identified eight cell types (mainly THY1+ cells, progenitor cells, and osteochondroblasts) and three core subclusters of the THY1+ cells (SC2, SC3, and SC4). Endothelial and mural cells each are heterogeneous at transcriptional level. It was the proliferation of progenitor, mural, and endothelial cells in the activated antler-lineage-specific tissues that drove the rapid formation of the antler. We detected the differences in the initial differentiation process between antler generation and regeneration using pseudotime trajectory analysis. These may be due to the difference in the degree of stemness of the AP-THY1+ and PP-THY1+ cells. We further found that androgen-RXFP2 axis may be involved in triggering initial antler full regeneration. Fully deciphering the cell composition for these antler tissue types will open up new avenues for elucidating the mechanism underlying antler full renewal in specific and regenerative medicine in general.

Case Report: De novo DDX3X mutation caused intellectual disability in a female with skewed X-chromosome inactivation on the mutant allele.

Skewed XCI plays an important role in the phenotypic heterogeneities of many X-linked disorders, even involving in diseases caused by XCI-escaping genes. DDX3X-related intellectual disability is more common in females and less common in males, who usually inherit from unaffected heterozygous mothers. As an X inactivation (XCI) escaping gene, the role of skewed XCI in the phenotype of DDX3X mutant female is unknown. Here we reported a DDX3X: c.694_711dup18 de novo heterozygous mutation in a female with intellectual disability on the maternal X chromosome on the basis of SNPs detected by PCR-sanger sequencing. AR assay revealed that the maternal mutant X chromosome was extremely inactivated in the proband. Using RNA sequencing and whole-exome sequencing, we quantified allelic read counts and allele-specific expression, and confirmed that the mutant X chromosome was inactive. Further, we verified that the mutant DDX3X allele had a lower expression level by RNA sequencing and RT-PCR, and the normal and mutated DDX3X expression accounted for respectively 70% and 30% of total. In conclusion, we found a symptomatic female with extreme skewing XCI in the DDX3X mutant allele. It was discovered that XCI in the mutant allele was insufficient to reverse the phenotype of DDX3X-related neurodevelopmental disorder. It contributed to a better understanding of the role of skewed XCI in phenotypic differences, which can aid in the genetic counseling and prenatal diagnosis of disorders in females with DDX3X defects.

Single-Cell Transcriptomics Reveals Killing Mechanisms of Antitumor Cytotoxic CD4+ TCR-T Cells.

T cell receptor-engineered T cells (TCR-Ts) have emerged as potent cancer immunotherapies. While most research focused on classical cytotoxic CD8+ T cells, the application of CD4+ T cells in adoptive T cell therapy has gained much interest recently. However, the cytotoxic mechanisms of CD4+ TCR-Ts have not been fully revealed. In this study, we obtained an MHC class I-restricted MART-127-35-specific TCR sequence based on the single-cell V(D)J sequencing technology, and constructed MART-127-35-specific CD4+ TCR-Ts and CD8+ TCR-Ts. The antitumor effects of CD4+ TCR-Ts were comparable to those of CD8+ TCR-Ts in vitro and in vivo. To delineate the killing mechanisms of cytotoxic CD4+ TCR-Ts, we performed single-cell RNA sequencing and found that classical granule-dependent and independent cytolytic pathways were commonly used in CD4+ and CD8+ TCR-Ts, while high expression of LTA and various costimulatory receptors were unique features for cytotoxic CD4+ TCR-Ts. Further signaling pathway analysis revealed that transcription factors Runx3 and Blimp1/Tbx21 were crucial for the development and killing function of cytotoxic CD4+ T cells. Taken together, we report the antitumor effects and multifaceted killing mechanisms of CD4+ TCR-Ts, and also indicate that MHC class I-restricted CD4+ TCR-Ts could serve as potential adoptive T cell therapies.

Protoplast technology enables the identification of efficient multiplex genome editing tools in Phalaenopsis.

Phalaenopsis orchids are popular ornamental plants worldwide. The application and optimization of efficient CRISPR-Cas genome editing toolkits in Phalaenopsis greatly accelerate the development of orchid gene function and breeding research. However, these methods are greatly hindered by the deficiency of a rapid screening system. In this study, we established a fast and convenient Phalaenopsis protoplast technology for the identification of functional genome editing tools. Two multiplex genome editing tools, PTG-Cas9-HPG (PTG, polycistronic tRNA-gRNA) system and RMC-Cpf1-HPG (RMC, ribozyme-based multi-crRNA) system, were developed for Phalaenopsis genome editing and further evaluated by established protoplast technology. We successfully detected various editing events comprising substitution and indel at designed target sites of the PDS gene and MADS gene, showing that both PTG-Cas9-HPG and RMC-Cpf1-HPG multiplex genome editing systems are functional in Phalaenopsis. Additionally, by optimizing the promoter that drives Cpf1 expression, we found that Super promoter can significantly improve the editing efficiency of the RMC-Cpf1-HPG system. Altogether, we successfully developed two efficient multiplex genome editing systems, PTG-Cas9-HPG and RMC-Cpf1-HPG, for Phalaenopsis, and the established protoplast-based screening technology provides a valuable foundation for developing more diverse and efficient genome editing toolkits and facilitating the development of orchid precision breeding.

The single-cell stereo-seq reveals region-specific cell subtypes and transcriptome profiling in Arabidopsis leaves.

Understanding the complex functions of plant leaves requires a thorough characterization of discrete cell features. Although single-cell gene expression profiling technologies have been developed, their application in characterizing cell subtypes has not been achieved yet. Here, we present scStereo-seq (single-cell spatial enhanced resolution omics sequencing) that enabled us to show the bona fide single-cell spatial transcriptome profiles of Arabidopsis leaves. Subtle but significant transcriptomic differences between upper and lower epidermal cells have been successfully distinguished. Furthermore, we discovered cell-type-specific gene expression gradients from the main vein to the leaf edge, which led to the finding of distinct spatial developmental trajectories of vascular cells and guard cells. Our study showcases the importance of physical locations of individual cells for exerting complex biological functions in plants and demonstrates that scStereo-seq is a powerful tool to integrate single-cell location and transcriptome information for plant biology study.

A Comprehensive Survey of Genomic Mutations in Breast Cancer Reveals Recurrent Neoantigens as Potential Therapeutic Targets.

Neoantigens are mutated antigens specifically generated by cancer cells but absent in normal cells. With high specificity and immunogenicity, neoantigens are considered as an ideal target for immunotherapy. This study was aimed to investigate the signature of neoantigens in breast cancer. Somatic mutations, including SNVs and indels, were obtained from cBioPortal of 5991 breast cancer patients. 738 non-silent somatic variants present in at least 3 patients for neoantigen prediction were selected. PIK3CA (38%), the highly mutated gene in breast cancer, could produce the highest number of neoantigens per gene. Some pan-cancer hotspot mutations, such as PIK3CA E545K (6.93%), could be recognized by at least one HLA molecule. Since there are more SNVs than indels in breast cancer, SNVs are the major source of neoantigens. Patients with hormone receptor-positive or HER2 negative are more competent to produce neoantigens. Age, but not the clinical stage, is a significant contributory factor of neoantigen production. We believe a detailed description of breast cancer neoantigen signatures could contribute to neoantigen-based immunotherapy development.

Plasma cell-free RNA characteristics in COVID-19 patients.

The pathogenesis of COVID-19 is still elusive, which impedes disease progression prediction, differential diagnosis, and targeted therapy. Plasma cell-free RNAs (cfRNAs) carry unique information from human tissue and thus could point to resourceful solutions for pathogenesis and host-pathogen interactions. Here, we performed a comparative analysis of cfRNA profiles between COVID-19 patients and healthy donors using serial plasma. Analyses of the cfRNA landscape, potential gene regulatory mechanisms, dynamic changes in tRNA pools upon infection, and microbial communities were performed. A total of 380 cfRNA molecules were up-regulated in all COVID-19 patients, of which seven could serve as potential biomarkers (AUC > 0.85) with great sensitivity and specificity. Antiviral (NFKB1A, IFITM3, and IFI27) and neutrophil activation (S100A8, CD68, and CD63)-related genes exhibited decreased expression levels during treatment in COVID-19 patients, which is in accordance with the dynamically enhanced inflammatory response in COVID-19 patients. Noncoding RNAs, including some microRNAs (let 7 family) and long noncoding RNAs (GJA9-MYCBP) targeting interleukin (IL6/IL6R), were differentially expressed between COVID-19 patients and healthy donors, which accounts for the potential core mechanism of cytokine storm syndromes; the tRNA pools change significantly between the COVID-19 and healthy group, leading to the accumulation of SARS-CoV-2 biased codons, which facilitate SARS-CoV-2 replication. Finally, several pneumonia-related microorganisms were detected in the plasma of COVID-19 patients, raising the possibility of simultaneously monitoring immune response regulation and microbial communities using cfRNA analysis. This study fills the knowledge gap in the plasma cfRNA landscape of COVID-19 patients and offers insight into the potential mechanisms of cfRNAs to explain COVID-19 pathogenesis.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion, and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones 2-3 weeks after the onset of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34, and IGHV4-39 in COVID-19 patients were more abundant than those of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.