Large-scale genomic survey and characterization of mcr genes carried by foodborne Cronobacter isolates.

IMPORTANCE: Cronobacter is an emerging foodborne opportunistic pathogen, which can cause neonatal meningitis, bacteremia, and NEC by contaminating food. However, the entire picture of foodborne Cronobacter carriage of the mcr genes is not known. Here, we investigated the mcr genes of Cronobacter isolates by whole-genome sequencing and found 133 previously undescribed Cronobacter isolates carrying mcr genes. Further genomic analysis revealed that these mcr genes mainly belonged to the mcr-9 and mcr-10. Genomic analysis of the flanking structures of mcr genes revealed that two core flanking structures were prevalent in foodborne Cronobacter isolates, and the flanking structure carrying IS1R was found for the first time in this study.

A food poisoning caused by ST7 Staphylococcal aureus harboring sea gene in Hainan province, China.

ST7 Staphylococcus aureus is highly prevalent in humans, pigs, as well as food in China; however, staphylococcal food poisoning (SFP) caused by this ST type has rarely been reported. On May 13, 2017, an SFP outbreak caused by ST7 S. aureus strains occurred in two campuses of a kindergarten in Hainan Province, China. We investigated the genomic characteristics and phylogenetic analysis of ST7 SFP strains combined with the 91 ST7 food-borne strains from 12 provinces in China by performing whole-genome sequencing (WGS). There was clear phylogenetic clustering of seven SFP isolates. Six antibiotic genes including blaZ, ANT (4')-Ib, tetK, lnuA, norA, and lmrS were present in all SFP strains and also showed a higher prevalence rate in 91 food-borne strains. A multiple resistance plasmid pDC53285 was present in SFP strain DC53285. Among 27 enterotoxin genes, only sea and selx were found in all SFP strains. A ФSa3int prophage containing type A immune evasion cluster (sea, scn, sak, and chp) was identified in SFP strain. In conclusion, we concluded that this SFP event was caused by the contamination of cakes with ST7 S. aureus. This study indicated the potential risk of new emergencing ST7 clone for SFP.

Computational predicting the human infectivity of H7N9 influenza viruses isolated from avian hosts.

The genome composition of a given avian influenza virus is the primary determinant of its potential for cross-species transmission from birds to humans. Here, we introduce a viral genome-based computational tool that can be used to evaluate the human infectivity of avian isolates of influenza A H7N9 viruses, which can enable prediction of the potential risk of these isolates infecting humans. This tool, which is based on a novel class weight-biased logistic regression (CWBLR) algorithm, uses the sequences of the eight genome segments of an H7N9 strain as the input and gives the probability of this strain infecting humans (reflecting its human infectivity). We examined the replication efficiency and the pathogenicity of several H7N9 avian isolates that were predicted to have very low or high human infectivity by the CWBLR model in cell culture and in mice, and found that the strains with high predicted human infectivity replicated more efficiently in mammalian cells and were more infective in mice than those that were predicted to have low human infectivity. These results demonstrate that our CWBLR model can serve as a powerful tool for predicting the human infectivity and cross-species transmission risks of H7N9 avian strains.

Could Environment Affect the Mutation of H1N1 Influenza Virus?

H1N1 subtype influenza A viruses are the most common type of influenza A virus to infect humans. The two major outbreaks of the virus in 1918 and 2009 had a great impact both on human health and social development. Though data on their complete genome sequences have recently been obtained, the evolution and mutation of A/H1N1 viruses remain unknown to this day. Among many drivers, the impact of environmental factors on mutation is a novel hypothesis worth studying. Here, a geographically disaggregated method was used to explore the relationship between environmental factors and mutation of A/H1N1 viruses from 2000-2019. All of the 11,721 geo-located cases were examined and the data was analysed of six environmental elements according to the time and location (latitude and longitude) of those cases. The main mutation value was obtained by comparing the sequence of the influenza virus strain with the earliest reported sequence. It was found that environmental factors systematically affect the mutation of A/H1N1 viruses. Minimum temperature displayed a nonlinear, rising association with mutation, with a maximum ~15 °C. The effects of precipitation and social development index (nighttime light) were more complex, while population density was linearly and positively correlated with mutation of A/H1N1 viruses. Our results provide novel insight into understanding the complex relationships between mutation of A/H1N1 viruses and environmental factors.

gcMeta: a Global Catalogue of Metagenomics platform to support the archiving, standardization and analysis of microbiome data.

Meta-omics approaches have been increasingly used to study the structure and function of the microbial communities. A variety of large-scale collaborative projects are being conducted to encompass samples from diverse environments and habitats. This change has resulted in enormous demands for long-term data maintenance and capacity for data analysis. The Global Catalogue of Metagenomics (gcMeta) is a part of the 'Chinese Academy of Sciences Initiative of Microbiome (CAS-CMI)', which focuses on studying the human and environmental microbiome, establishing depositories of samples, strains and data, as well as promoting international collaboration. To accommodate and rationally organize massive datasets derived from several thousands of human and environmental microbiome samples, gcMeta features a database management system for archiving and publishing data in a standardized way. Another main feature is the integration of more than ninety web-based data analysis tools and workflows through a Docker platform which enables data analysis by using various operating systems. This platform has been rapidly expanding, and now hosts data from the CAS-CMI and a number of other ongoing research projects. In conclusion, this platform presents a powerful and user-friendly service to support worldwide collaborative efforts in the field of meta-omics research. This platform is freely accessible at https://gcmeta.wdcm.org/.

MiR-218 Inhibits Erythroid Differentiation and Alters Iron Metabolism by Targeting ALAS2 in K562 Cells.

microRNAs (miRNAs) are involved in a variety of biological processes. The regulatory function and potential role of miRNAs targeting the mRNA of the 5'-aminolevulinate synthase 2 (ALAS2) in erythropoiesis were investigated in order to identify miRNAs which play a role in erythroid iron metabolism and differentiation. Firstly, the role of ALAS2 in erythroid differentiation and iron metabolism in human erythroid leukemia cells (K562) was confirmed by ALAS2 knockdown. Through a series of screening strategies and experimental validations, it was identified that hsa-miR-218 (miR-218) targets and represses the expression of ALAS2 by binding to the 3'-untranslated region (UTR). Overexpression of miR-218 repressed erythroid differentiation and altered iron metabolism in K562 cells similar to that seen in the ALAS2 knockdown in K562 cells. In addition to iron metabolism and erythroid differentiation, miR-218 was found to be responsible for a reduction in K562 cell growth. Taken together, our results show that miR-218 inhibits erythroid differentiation and alters iron metabolism by targeting ALAS2 in K562 cells.

Functional validation of a constitutive autonomous silencer element.

Sequences of the genome that are capable of silencing gene expression are thought to play a key role in gene regulation. However, very few silencer elements capable of functioning in mammalian cells have been described, and only a fraction of these have been tested for the ability to function in an autonomous fashion. We report here the characterization and functional validation of a constitutive autonomous silencer element from the human genome called T39, and the comparison of T39 to three other putative silencer elements previously described by others. Functional analysis included one assay for enhancer-blocking insulator activity and two independent assays for silencer activity, all based on stable transfection and comparison to a neutral spacer control. In erythroid K562 cells, T39 exhibited potent silencer activity, the previously described element PRE2-S5 exhibited modest silencer activity, and the two other previously described elements exhibited no silencer activity. T39 was further found to be capable of silencing three disparate promoters, of silencing gene expression in three disparate cell lines, and of functioning as a single copy in a topology-independent manner. Of the four elements analyzed, only T39 exhibits a constitutive pattern of DNase hypersensitivity and binding by CTCF. In its native location the T39 element also exhibits a unique interaction profile with a subset of distal putative regulatory elements. Taken together, these studies validate T39 as a constitutive autonomous silencer, identify T39 as a defined control for future studies of other regulatory elements such as insulators, and provide a basic chromatin profile for one highly potent silencer element.

Knockdown of transcription factor forkhead box O3 (FOXO3) suppresses erythroid differentiation in human cells and zebrafish.

Our previous study on the dynamic transcriptomes activated during human erythropoiesis suggested that transcription factor forkhead box O3 (FOXO3) possibly plays a role in erythroid differentiation. Functional studies in human cell line TF-1 indicated that FOXO3 knockdown repressed erythropoietin (EPO)-induced erythroid differentiation by activating promoter region of B-cell translocation gene 1 (BTG1), thereby regulating its expression. In zebrafish, injection of foxo3b-specific morpholinos (foxo3b MO) resulted in reduced globin (hbae1 and hbbe2) and gata1 gene expression. Transcriptome analyses of erythroid lineage cells isolated from the control and foxo3b morphants revealed the dynamic regulation of foxo3b. Further study suggested that BTG1 is partially responsible for FOXO3 regulation in erythroid differentiation of TF-1 cells but is inconsequential in zebrafish. Taken together, we found that FOXO3 plays an important role in erythroid differentiation in both human TF-1 cells and zebrafish, but the mechanism underlying this regulation still remains unclear.

Genomic discovery of potent chromatin insulators for human gene therapy.

Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

Transcriptome dynamics during human erythroid differentiation and development.

To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs-ESERs, ESERs-FLERs, and FLERs-PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell-cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.

Comprehensive characterization of erythroid-specific enhancers in the genomic regions of human Krüppel-like factors.

BACKGROUND: Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify cis-regulatory elements (CREs). Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood. RESULTS: In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses. CONCLUSION: Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells.

[Role of chromatin conformation in eukaryotic gene regulation].

Gene expression in eukaryotes is regulated at multiple levels, which involves various cis-regulatory elements and trans-acting factors at transcriptional level. In addition, DNA methylation and histone modifications also play crucial roles in epigenetic regulation of eukaryotic genes. It is pivotal for evaluating the regulation of gene expression to understand the structural properties and spatial organization of chromatin at 3-D level. The dynamic alternations of chromatin conformation can either activate gene expression by facilitating the interactions between enhancers or other cis-regulatory elements and their target genes or suppress gene expression by blocking the interactions due to steric hindrance. Although the precise molecular mechanisms underlying the gene regulation via conformational changes of chromatin remain obscure, epigenetic studies including histone modification, nucleosome positioning, chromosome territories as well as chromatin interactions, have provided accumulating evidence to demonstrate the significance of chromatin conformation in eukaryotic gene regulation. Here, we reviewed the recent advances on the role of dynamic alterations of chromatin in gene regulation , which occur at different levels from the primary structure to three dimensional conformation.