A defined serum-free culture system for human long-term haematopoietic stem cells.

Long-term repopulating haematopoietic stem cells (LT-HSCs) have the ability to reconstitute the entire haematopoietic system following transplantation permanently. Despite great achievements in HSC transplantation, the limited transplantable HSC number, especially LT-HSCs, remains critical for successful transplantation and broader applications. In this study, we established a defined serum-free culture system for in vitro expansion of LT-HSCs. This culture system (E1) expanded LT-HSCs from umbilical cord blood, human mobilization peripheral blood and bone marrow. These E1-expanded HSCs reconstituted the haematopoietic and immune systems in primary and secondary transplanted mice in a short time. Better haematopoietic reconstitution was observed in secondary xenografted mice. Moreover, we obtained the comprehensive expression profile and cellular components of LT-HSCs from umbilical cord blood. Our study provides a valuable tool for LT-HSC research and may improve clinical applications of HSCs.

De novo genome assembly of a Han Chinese male and genome-wide detection of structural variants using Oxford Nanopore sequencing.

Advances in third-generation sequencing technologies provide an opportunity to investigate the complex organizational structure of the genome and unravel the genetic mechanisms of disease and physiological traits. Here we report the sequencing and de novo assembly of a healthy male northern Han Chinese genome and detection of structural variants using only nanopore sequencing data. We performed de novo assembly after filtering the raw data. Then, we aligned the assembled contigs to the human reference genome, and visualized chromosomes plot, which illustrated the contiguity of the nanopore assembly. Additionally, genomic structural variants were detected using a structure variation detection tool with long-read sequencing data. Median coverage depth was 30-fold and the read N50 was 27,136 bp. 96.51% of reads had at least one alignment to the human reference genome. The final assembled genome was 2.85 GB in size, with an N50 contig size of 5.4 MB. We identified 20,085 structural variants. Third-generation sequencing technologies have many advantages in de novo whole-genome assembly and detection of structural variants. Our results provide reference data for disease research, and can be used as a novel population-specific dataset of structural variants to support the efficient development of personalized precision medicine.

miR-98 is involved in missed abortion by targeting GDF6 and FAPP2.

Missed abortion (MA) is a common disease in obstetrics and gynecology. More and more studies have focused on the relationship between miRNAs and pregnancy maintenance and its related diseases. The aim of this article is to explore the relationship between miRNA and MA. The expression of miR-98 were detected by in situ hybridization and real-time PCR. Cell proliferation, activity and migration were measured via Edu, MTT, and transwell assays. The target genes of miR-98 are identified by dual-luciferase activity assay. And the expression levels of target genes were determined by Western blot, real-time PCR and immunohistochemistry. miR-98 was significantly up-regulated in placental villi from over 35 years old MA patients compared with the age-matched normal pregnant women. Up-regulation of miR-98 suppressed the proliferation, activity and migration of the human trophoblast HTR-8/SVneo cell in vitro. miR-98 could bind to GDF6 and FAPP2 mRNA 3'-UTR and negatively regulate their expression. The downregulation of miR-98 promoted cell proliferation, then knockdown of GDF6 or FAPP2 inhibited miR-98-mediated cell proliferation. GDF6 and FAPP2 expression in the placental villi from MA patients were decreased compared to normal placental tissues. The expression of miR-98 in MA had an opposite relationship with the expression of GDF6 and FAPP2. Overexpression of miR-98 is associated with the occurrence of MA. miR-98 prevents proliferation, viability and migration of trophoblast cells partially through targeting GDF6 and FAPP2.

DACE: a scalable DP-means algorithm for clustering extremely large sequence data.

Motivation: Advancements in next-generation sequencing technology have produced large amounts of reads at low cost in a short time. In metagenomics, 16S and 18S rRNA gene have been widely used as marker genes to profile diversity of microorganisms in environmental samples. Through clustering of sequencing reads we can determine both number of OTUs and their relative abundance. In many applications, clustering of very large sequencing data with high efficiency and accuracy is essential for downstream analysis. Results: Here, we report a scalable D irichlet Process Means (DP-means) a lgorithm for c lustering e xtremely large sequencing data, termed . With an efficient random projection partition strategy for parallel clustering, DACE can cluster billions of sequences within a couple of hours. Experimental results show that DACE runs between 6 and 80 times faster than state-of-the-art programs, while maintaining overall better clustering accuracy. Using 80 cores, DACE clustered the Lake Taihu 16S rRNA gene sequencing data (∼316M reads, 30 GB) in 25 min, and the Ocean TARA Eukaryotic 18S rRNA gene sequencing data (∼500M reads, 88 GB) into ∼100 000 clusters within an hour. When applied to the IGC gene catalogs in human gut microbiome (∼10M genes), DACE produced 9.8M clusters with 52K redundant genes in 1.5 hours of running time. Availability and Implementation: DACE is available at https://github.com/tinglab/DACE . Contacts: tingchen@mail.tsinghua.edu.cn or ningchen@mail.tsinghua.edu.cn. Supplementary information: Supplementary data are available at Bioinformatics online.

Dimethyloxallyl Glycine Preconditioning Promotes the Anti-inflammatory and Anti-fibrotic Effects of Human Umbilical Cord Mesenchymal Stem Cells on Kidney Damage in Systemic Lupus Erythematosus Related to TGF-ß/Smad Signaling Pathway.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease lacking effective treatments without adverse effects. Dimethyloxallyl glycine (DMOG) enhanced mesenchymal stem cells (MSC) capabilities, but it remains unclear how DMOG-pretreatment of MSCs augments their SLE treatment. Here, we explore the therapeutic potential of DMOG-pretreated human umbilical cord MSCs (hUC-MSCs) in a mouse lupus nephritis (LN) model. In vitro experiments showed that DMOG could alleviate the mRNA levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6 and increase the mRNA level of IL-13 in lipopolysaccharide (LPS)-induced inflammation in hUC-MSCs. DMOG enhanced the migratory and invasive abilities of the hUC-MSCs. In vivo animal studies revealed that DMOG-pretreated hUC-MSCs exhibited more pronounced inhibition of lymphadenectasis and reduced kidney weight and urinary protein content than MSCs alone. DMOG-pretreated hUC-MSCs improved renal morphological structure and alleviated inflammatory cell infiltration and renal fibrosis, evidenced by the reduced mRNA levels of fibrosis markers, including fibronectin (Fn), collagen alpha-1 chain (Colα1), collagen alpha-3 chain (Colα3), and TNF-α, IFN-γ, and IL-6 cytokines. Further investigation revealed that DMOG-pretreated hUC-MSCs down-regulated the expressions of transforming growth factor (Tgf)-ß1 and its downstream effectors Smad2 and Smad3, recognized as central mediators in renal fibrosis (P < 0.05). The findings suggest that DMOG-pretreated hUC-MSCs can augment the therapeutic efficacy of hUC-MSCs in LN by enhancing their anti-inflammatory and antifibrotic effects, and the TGF-ß/Smad signaling pathway may be involved in this process.

Transplantation of umbilical cord-derived mesenchymal stem cells promotes the recovery of thin endometrium in rats.

The endometrium plays a critical role in embryo implantation and pregnancy, and a thin uterus is recognized as a key factor in embryo implantation failure. Umbilical cord mesenchymal stem cells (UC-MSCs) have attracted interest for the repair of intrauterine adhesions. The current study investigated the repair of thin endometrium in rats using the UC-MSCs and the mechanisms involved. Rats were injected with 95% ethanol to establish a model of thin endometrium. The rats were randomly divided into normal, sham, model, and UC-MSCs groups. Endometrial morphological alterations were observed by hematoxylin-eosin staining and Masson staining, and functional restoration was assessed by testing embryo implantation. The interaction between UC-MSCs and rat endometrial stromal cells (ESCs) was evaluated using a transwell 3D model and immunocytochemistry. Microarray mRNA and miRNA platforms were used for miRNA-mRNA expression profiling. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed to identify the biological processes, molecular functions, cellular components, and pathways of endometrial injury and UC-MSCs transplantation repair and real-time quantitative reverse transcription PCR (qRT-PCR) was performed to further identify the expression changes of key molecules in the pathways. Endometrium thickness, number of glands, and the embryo implantation numbers were improved, and the degree of fibrosis was significantly alleviated by UC-MSCs treatment in the rat model of thin endometrium. In vitro cell experiments showed that UC-MSCs migrated to injured ESCs and enhanced their proliferation. miRNA microarray chip results showed that expression of 45 miRNAs was downregulated in the injured endometrium and upregulated after UC-MSCs transplantation. Likewise, expression of 39 miRNAs was upregulated in the injured endometrium and downregulated after UC-MSCs transplantation. The miRNA-mRNA interactions showed the changes in the miRNA and mRNA network during the processes of endometrial injury and repair. GO and KEGG analyses showed that the process of endometrial injury was mainly attributed to the decomposition of the extracellular matrix (ECM), protein degradation and absorption, and accompanying inflammation. The process of UC-MSCs transplantation and repair were accompanied by the reconstruction of the ECM, regulation of chemokines and inflammation, and cell proliferation and apoptosis. The key molecules involved in ECM-receptor interaction pathways were further verified by qRT-PCR. Itga1 and Thbs expression decreased in the model group and increased by UC-MSCs transplantation, while Laminin and Collagen expression increased in both the model group and MSCs group, with greater expression observed in the latter. This study showed that UC-MSCs transplantation could promote recovery of thin endometrial morphology and function. Furthermore, it revealed the expression changes of miRNA and mRNA after endometrial injury and UC-MSCs transplantation repair processed, and signaling pathways that may be involved in endometrial injury and repair.

Down-regulation of microRNA-30d-5p is associated with gestational diabetes mellitus by targeting RAB8A.

Gestational Diabetes Mellitus (GDM) is a complicated clinical process, and metabolic disorders during pregnancy are closely related to the structure and function of the placenta. The aberrant expression of miRNAs in the placenta may play a role in the occurrence and development of GDM. Analysis of microRNA (miRNA) expression signature in placenta showed that the level of miR-30d-5p was significantly down-regulated in GDM patients. This study aims to explore the possible mechanism of GDM under the regulation of miR-30d-5p. In situ hybridization and qRT-PCR assay showed that miR-30d expression down-regulated in the placentas from GDM patients compared with normal control group. The trophoblast cells proliferation and glucose uptake capacity were increased, the ability of migration and invasion were also improved after inhibiting the function of endogenous mature miR-30d-5p. Bioinformatics analysis and luciferase reporter assays showed that miR-30d-5p binds to the 3'UTR of RAB8A mRNA, resulting in RAB8A suppression. Moreover, the down-regulation of RAB8A could attenuate the increase in trophoblast cell proliferation, migration, invasion and glucose uptake induced by miR-30d-5p functional inhibitor. These data imply that miR-30d-5p expression is down-regulated in placental tissue from GDM patients and affects trophoblast cell functions by targeting RAB8A, which may provide new insight into the pathogenesis of GDM.

Genome-Wide Association Identifies Risk Pathways for SAPHO Syndrome.

SAPHO syndrome is a rare chronic inflammatory disease which is characterized by the comprehensive manifestations of bone, joint, and skin. However, little is known about the pathogenesis of SAPHO syndrome. A genome-wide association study (GWAS) of 49 patients and 121 control subjects have primarily focused on identification of common genetic variants associated with SAPHO, the data were analyzed by classical multiple logistic regression. Later, GWAS findings were further validated using whole exome sequencing (WES) in 16 patients and 15 controls to identify potentially functional pathways involved in SAPHO pathogenesis. In general, 40588 SNPs in genomic regions were associated with P < 0.05 after filter process, only 9 SNPs meet the expected cut-off P-value, however, none of them had association with SAPHO syndrome based on published literatures. And then, 15 pathways were found involved in SAPHO pathogenesis, of them, 6 pathways including osteoclast differentiation, bacterial invasion of epithelial cells, et al., had strong association with skin, osteoarticular manifestations of SAPHO or inflammatory reaction based published research. This study identified aberrant osteoclast differentiation and other pathways were involved in SAPHO syndrome. This finding may give insight into the understanding of pathogenic genes of SAPHO and provide the basis for SAPHO research and treatment.

The polymorphism of rs11614913 T/T in pri-miR-196a-2 alters the miRNA expression and associates with recurrent spontaneous abortion in a Han-Chinese population.

Rs11614913 in pri-miR-196a-2 is involved in the occurrence of many diseases, especially in cancers. However, it remains unknown whether miR-196a-2 is associated with human recurrent spontaneous abortion (RSA) in Chinese Han population. Our study found that rs11614913 T/T in pri-miR-196a-2 was associated with the increase risk of human unexplained RSA (URSA) in recessive mode in Chinese Han population. The T allele of rs11614913 increased the production of mature miR-196a-3p. Rs11614913 T/T inhibited HTR-8/SVneo cells proliferation and migration and promoted cells apoptosis. Further investigation discovered that dihydrofolate reductase (DHFR) was the target of miR-196a-3p and inversely regulated by miR-196a-3p. Dual-luciferase assay indicated that T allele in miR-196a-2 rs11614913 could more effectively suppress DHFR expression than C allele. In addition, C to T substitution in miR-196a-2 rs11614913 attenuated the sensibility of cells to mifepristone. Collectively, our data suggest that miR-196a-2 rs11614913 T/T in pri-miR-196a-2 may be conductive to the genetic predisposition to RSA by disrupting the production of mature miR-196a-3p and reinforcing the expression of DHFR.

Analysis of clinical features and early warning signs in patients with severe COVID-19: A retrospective cohort study.

Coronavirus disease 2019 (COVID-19) was first identified in Wuhan, China, in December 2019. Although previous studies have described the clinical aspects of COVID-19, few studies have focused on the early detection of severe COVID-19. Therefore, this study aimed to identify the predictors of severe COVID-19 and to compare clinical features between patients with severe COVID-19 and those with less severe COVID-19. Patients admitted to designated hospital in the Henan Province of China who were either discharged or died prior to February 15, 2020 were enrolled retrospectively. Additionally, patients who underwent at least one of the following treatments were assigned to the severe group: continuous renal replacement therapy, high-flow oxygen absorption, noninvasive and invasive mechanical ventilation, or extracorporeal membrane oxygenation. The remaining patients were assigned to the non-severe group. Demographic information, initial symptoms, and first visit examination results were collected from the electronic medical records and compared between the groups. Multivariate logistic regression analysis was performed to determine the predictors of severe COVID-19. A receiver operating characteristic curve was used to identify a threshold for each predictor. Altogether,104 patients were enrolled in our study with 30 and 74 patients in the severe and non-severe groups, respectively. Multivariate logistic analysis indicated that patients aged ≥63 years (odds ratio = 41.0; 95% CI: 2.8, 592.4), with an absolute lymphocyte value of ≤1.02×109/L (odds ratio = 6.1; 95% CI = 1.5, 25.2) and a C-reactive protein level of ≥65.08mg/L (odds ratio = 8.9; 95% CI = 1.0, 74.2) were at a higher risk of severe illness. Thus, our results could be helpful in the early detection of patients at risk for severe illness, enabling the implementation of effective interventions and likely lowering the morbidity of COVID-19 patients.

Profiling of the pattern of the human TRB/IGH-CDR3 repertoire in primary biliary cholangitis patients.

INTRODUCTION: Primary biliary cholangitis (PBC) is characterized by lymphocyte cell-induced immune destruction of cholangiole. However, the immunological characteristics of peripheral blood cells in PBC patients remain unknown. This study was designed to reveal the differences in the immunological characteristics between PBC patients and healthy adults. METHODS: We performed high-throughput sequencing to determine the TRB-CDR3 and IGH-CDR3 repertoires of T and B cells in 19 healthy controls and 29 PBC patients. Different immunological characteristics, such as distinctive complementarity determining region 3 (TRB-CDR3) lengths, usage bias of V and J segments, and random nucleotide addition were identified in PBC and healthy control (HC) groups. RESULTS: The diversity of TRB-CDR3 was significantly lower in the PBC group compared with the HC group. CDR3 and the N addition length distribution were significantly changed compared with the HC group. It appeared that the PBC group had more short N additions and the HC group had more long N additions in the TRB-CDR3 repertoire. The results also revealed a set of PBC-associated clonotypes compared with the HC group. CONCLUSION: This study suggested that PBC is a complex autoimmune disease process with evidence of different TRB-CDR3 rearrangements compared with healthy adults that share IGH-CDR3 peptides with some autoimmune diseases. This new insight may contribute to a better understanding of the immune functions of PBC patients and benefit efficient applications of PBC diagnosis and treatments.