Abstract Background DNA methylation is an important epigenetic modification that plays a critical role in most eukaryotic organisms. Parental alleles in haploid genomes may exhibit different methylation patterns, which can lead to different phenotypes and even different therapeutic and drug responses to diseases. However, to our knowledge, no software is available for the identification of DNA methylation haplotype regions with combined allele-specific DNA methylation, single nucleotide polymorphisms (SNPs) and high-throughput chromosome conformation capture (Hi-C) data. Results In this paper, we developed a new method, MethHaplo, that identify DNA methylation haplotype regions with allele-specific DNA methylation and SNPs from whole-genome bisulfite sequencing (WGBS) data. Our results showed that methylation haplotype regions were ten times longer than haplotypes with SNPs only. When we integrate WGBS and Hi-C data, MethHaplo could call even longer haplotypes. Conclusions This study illustrates the usefulness of methylation haplotypes. By constructing methylation haplotypes for various cell lines, we provide a clearer picture of the effect of DNA methylation on gene expression, histone modification and three-dimensional chromosome structure at the haplotype level. Our method could benefit the study of parental inheritance-related disease and hybrid vigor in agriculture.
Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or l-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.
To investigate the impact of heteroresistance on the results of genotype drug susceptibility test for Mycobacterium tuberculosis (M. tuberculosis).A total of 80 phenotype ofloxacin-resistant M. tuberculosis isolates obtained from Shanghai Municipal Centers for Disease Control and Prevention were included in the study. The mutations of gyrA and gyrB in each isolate were identified by gene sequencing. Molecular cloning and multiplex real-time PCR melting curve assay were applied to analyze the presence of heteroresistance in those isolates that had showed no mutations by gene sequencing.The sequencing results showed that 15 of these 80 isolates had no mutations in gyrA or gyrB. By analyzing the sequencing chromatogram, we found that 7 of the 15 isolates had gyrA mutations. By molecular cloning and melting curve assay, we identified another 7 isolates with low frequency mutations in gyrA that had been previously identified. In total, 14 isolates with heteroresistance were detected in these 15 isolates. The consistency between the results of phenotype and genotype drug susceptibility tests was increased from 81.3% (65/80) to 98.8% (79/80).Heteroresistance is an important factor for the inconsistency between the results of phenotype and genotype drug susceptibility tests. For improving the rapid detection of drug resistant tuberculosis, novel genotypic drug susceptibility test methods with higher sensitivity should be developed and applied.
Recent studies indicate that the long intergenic non-coding RNA LINC00152 plays crucial roles in various human diseases. Here, we investigated whether levels of LINC00152 or its genetic variants correlate with the clinical features of tuberculosis (TB) in western Chinese patients. We genotyped the single nucleotide polymorphism LINC00152 rs80292941 in 476 TB patients and 475 healthy subjects using a custom-by-design 48-Plex SNPscan Kit, and measured relative levels of LINC00152 using RT-qPCR. We observed that LINC00152 levels were lower in TB patients than controls. Moreover, rs80292941 TT genotype carriers had the lowest LINC00152 levels among TB patients, and rs80292941 AA genotype carriers are more likely to suffer from hepatotoxicity induced by antituberculosis therapy [OR = 3.97, 95% = 1.53-10.13, p = 0.002]. Our findings strongly suggest that LINC00152 may promote TB progression and highlight rs80292941 single nucleotide polymorphism as a novel predisposition marker for antituberculosis drug-induced hepatotoxicity.
Abstract Background Mannheimia haemolytica (M. haemolytica) is the primary pathogen responsible for respiratory diseases in ruminants. As an opportunistic pathogen, it often co-infects with other bacteria and viruses, leading to severe pneumonia. In this study, a suspected M. haemolytica pathogen was isolated from the lungs of sheep on a farm in Luoyang that exhibited respiratory symptoms and died acutely. The species classification, biological characteristics, and genome sequence analysis of the pathogen were determined. Results Morphological observations, biochemical tests, and phylogenetic analysis confirmed that the isolate was closely related to serotype A1 M. haemolytica GCA-900474405.1. Furthermore, sequence comparison of the capsular gene region revealed that the bacterium belonged to serotype A5 M. haemolytica and was named MH-1. Antibiotic sensitivity tests showed that MH-1 was resistant to tetracycline, erythromycin, spectinomycin and penicillin-G, and sensitive to other selected antibiotics. In animal experiments conducted on mice via intraperitoneal inoculation with MH-1, depression symptoms and dishevelled hair were observed in all mice in the highest-dose group, leading to death. The LD50 value for mice was determined to be 1.27×109 CFU. Whole-genome sequence analysis revealed that MH-1 had a total of 20 open reading frames (ORFs) encoded genes related to pathogenicity, including proteins involved in adhesion, invasion, iron uptake, and antiphagocytosis. While 8 ORFs were responsible for drug resistance genes, such as the macrolide resistance gene macB, the tetracycline resistance gene tet(35), the aminoglycoside resistance gene APH(3')-Ia, and the β-lactam resistance gene CRP. These findings were consistent with the results obtained from the antibiotic susceptibility test. Conclusion In conclusion, we successfully isolated and identified a strain of M. haemolytica serotype A5 from sheep. Through whole-genome sequencing and biological characterization analysis, we have enriched the understanding of the pathogenic properties of M. haemolytica in sheep. This information provides valuable insights for prevention and treatment strategies against M. haemolytica infections in sheep.
Abstract Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR–Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR–dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.
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