Clinical characteristics of the host DNA-removed metagenomic next-generation sequencing technology for detecting SARS-CoV-2, revealing host local immune signaling and assisting genomic epidemiology.

Background: Metagenomic next-generation sequencing (mNGS) technology has been central in detecting infectious diseases and helping to simultaneously reveal the complex interplay between invaders and their hosts immune response characteristics. However, it needs to be rigorously assessed for clinical utility. The present study is the first to evaluate the clinical characteristics of the host DNA-removed mNGS technology for detecting SARS-CoV-2, revealing host local immune signaling and assisting genomic epidemiology. Methods: 46 swab specimens collected from COVID-19 patients were assayed by two approved commercial RT-qPCR kits and mNGS. The evolutionary tree of SARS-CoV-2 was plotted using FigTree directly from one sample. The workflow of removing the host and retaining the host was compared to investigate the influence of host DNA removal on the performances of mNGS. Functional enrichment analysis of DEGs and xCell score were used to explore the characteristics of host local immune signaling. Results: The detection rate of mNGS achieved 92.9% (26/28) for 28 samples with a Ct value ≤ 35 and 81.1% (30/37) for all 46 samples. The genome coverage of SARS-CoV-2 could reach up to 98.9% when the Ct value is about 20 in swab samples. Removing the host could enhance the sensitivity of mNGS for detecting SARS-CoV-2 from the swab sample but does not affect the species abundance of microbes RNA. Improving the sequencing depth did not show a positive effect on improving the detection sensitivity of SARS-CoV-2. Cell type enrichment scores found multiple immune cell types were differentially expressed between patients with high and low viral load. Conclusions: The host DNA-removed mNGS has great potential utility and superior performance on comprehensive identification of SARS-CoV-2 and rapid traceability, revealing the microbiome's transcriptional profiles and host immune responses.

MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells.

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene's (mmp1's) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

Comparative genomics reveals cellobiose hydrolysis mechanism of Ruminiclostridium thermocellum M3, a cellulosic saccharification bacterium.

The cellulosome of Ruminiclostridium thermocellum was one of the most efficient cellulase systems in nature. However, the product of cellulose degradation by R. thermocellum is cellobiose, which leads to the feedback inhibition of cellulosome, and it limits the R. thermocellum application in the field of cellulosic biomass consolidated bioprocessing (CBP) industry. In a previous study, R. thermocellum M3, which can hydrolyze cellulosic feedstocks into monosaccharides, was isolated from horse manure. In this study, the complete genome of R. thermocellum M3 was sequenced and assembled. The genome of R. thermocellum M3 was compared with the other R. thermocellum to reveal the mechanism of cellulosic saccharification by R. thermocellum M3. In addition, we predicted the key genes for the elimination of feedback inhibition of cellobiose in R. thermocellum. The results indicated that the whole genome sequence of R. thermocellum M3 consisted of 3.6 Mb of chromosomes with a 38.9% of GC%. To be specific, eight gene islands and 271 carbohydrate-active enzyme-encoded proteins were detected. Moreover, the results of gene function annotation showed that 2,071, 2,120, and 1,246 genes were annotated into the Clusters of Orthologous Groups (COG), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively, and most of the genes were involved in carbohydrate metabolism and enzymatic catalysis. Different from other R. thermocellum, strain M3 has three proteins related to ß-glucosidase, and the cellobiose hydrolysis was enhanced by the synergy of gene BglA and BglX. Meanwhile, the GH42 family, CBM36 family, and AA8 family might participate in cellobiose degradation.

Effect of Hypoxia on the Muscle Fiber Switching Signal Pathways CnA/NFATc1 and Myostatin in Mouse Myocytes.

This study investigated the effects of a CoCl2-simulated hypoxic environment on the muscle fiber switching signaling pathways calcineurin A/nuclear factor of activated T cells cytoplasmic 1 (CnA/NFATc1) and myostatin. In this study, C2C12 muscle cells were cultured in vitro under CoCl2-simulated chemical hypoxic conditions, the expression levels of CnA and myostatin were detected through qRT-PCR and Western blot analyses, and a positioning study of NFATc1 was carried out by immunofluorescence labeling. Results showed that CoCl2 treatment significantly increased the expression levels of CnA and myostatin. Moreover, the position of NFATc1 expression changed; actually, its expression in the nucleus considerably increased. Furthermore, CoCl2-induced hypoxia inhibited the differentiation of C2C12 cells and reduced the expression levels of many slow- and fast-twitch muscles marker genes, but immunofluorescence staining results showed that the proportion of MyHC I type muscle fiber increased after CoCl2 treatment. The hypoxic environment simulated by CoCl2 can activate the signaling pathways CnA/NFATc1 and myostatin and increases the proportion of MyHC I type muscle fibers.