ABSTRACT
Gut microbiome sequencing studies have great potential to translate microbial analysis outcomes into human health research. Sequencing strategies of 16S amplicon and whole-metagenome shotgun (WMS) are two main methods in microbiome research with respective advantages. However, how sample heterogeneity, sequencers and library preparation protocols affect the sequencing reproducibility of gut microbiome needs further investigation. This study aims to provide a reference for the selection of sequencing technologies by comparing differences in microbial composition from different sampling sites. The results of three widely adopted sequencers showed that the technical repetition correlation (r= 0. 94) was high in WMS method, while the biological repetition correlation (r = 0. 69) was low. Bray-Curtis distance identified that dissimilarity from biological replicates was larger than that of technical replicates (P<0. 001). In addition, dissimilarity and specific taxonomic profiles were observed between 16S and WMS datasets. Our results imply that homogenization is a necessary step before sample DNA extraction. The sequencers contributed less to taxonomic variation than the library preparation protocols. We developed an empirical Bayes approach that " borrowed information" in calculations and analyzed batch effect parameters using standardized data and prior distributions of (non-) parameters, which may improve population comparability between 16S and WMS and provide a basis for further application to fusion analysis of published 16S and microbial datasets.
ABSTRACT
Excessive exercise makes the body consume more oxygen and produce excessive free radicals. The increased free radicals lead to oxidative stress injury and dysfunctions in liver tissue. Our previous study showed that Anwulignan, an active monomer in Schisandra sphenanthera Rehd. et Wils. (Schisandra), had anti-fatigue effects in mice. However, whether Anwulignan has a protective effect on liver damage in exhausted mice and the mechanism underlying remain elusive. An exhaustive swimming mice model was used to study the protective effects of Anwulignan on liver damage. The involvement of the nuclear factor (erythroid-derived 2)-like 2 (NRF2)/antioxidant responsive element (ARE) antioxidative pathway in Anwulignan-mediated anti-fatigue was analyzed using NRF2 inhibitor ML385 in HepG2 cells treated with H2O2. Animal welfare and experimental process follow the regulations of the Animal Ethics Committee of Beihua University. Anwulignan significantly lowered serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, reduced liver tissue damages, increased superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and decreased malondialdehyde (MDA) and 8-hydroxy-2 deoxyguanosine (8-OHdG) contents in the livers of exhausted mice, demonstrating a strong antioxidant effect. Furthermore, Anwulignan up-regulated the NRF2/ARE antioxidant pathway in liver tissue, increased B-cell lymphoma 2 (Bcl-2) expression, and decreased Bcl-2-like protein 4 (Bax) and caspase3 expression. In HepG2 cells, Anwulignan improved the cell viability and SOD activity, reduced reactive oxygen species (ROS) and MDA contents, up-regulated the expression of the NRF2/ARE signaling pathway and Bcl-2, and decreased Bax and caspase3 expression in the cells. Furthermore, pretreated ML385 partly abolished all these effects of Anwulignan. Anwulignan protects the liver from damage in the exhausted mice by its antioxidant effects and related to its activation of the NRF2 pathway.
ABSTRACT
Objective To investigate the correlation betw een serum 25-hydroxyvitamin D (25(OH) D) level and enlarged perivascular spaces (EPVS) in patients w ith lacunar infarction. Methods Consecutive inpatients w ith acute lacunar infarction w ere enrolled. Chemiluminescence immunoassay w as used to measure the serum 25(OH)D. Head MRI w as used to assess the severity of EPVS in the centrum ovale and the basal ganglia region (grade 1, 0-10; grade 2, 11-25; grade 3, >25). Univariate analysis w as used to analyze the differences in baseline data among the three groups. Multivariate ordinal logistic regression analysis w as used to investigate the independent correlation betw een the 25(OH)D levels and the severity of EPVS. Results A total of 194 patients w ere enrolled,96 w ere males.Their mean age w as 63.4 ±9.3 years. The mean baseline National Institutes of Health Stroke Scale (NIHSS) score w as 2.0 ±1.7. The mean 25(OH)D level w as(50.25 ±15.50)nmol/L. There w ere 112 patients (57.7%), 45 (23.2%) and 37 (19.1%) w ith EPVS grade 1,grade 2 and grade 3 in the centrum ovale,and 109 (56.2%),53(27.3%) and 32 (16.5%) w ith EPVS grade 1, grade 2 and grade 3 in basal ganglia region, respectively. Multivariate ordinal logistic regression analysis show ed that serum 25(OH)D levels w ere independently associated w ith the severity of EPVS in the centrum ovale (odds ratio 2.898,95% confidence interval 1.345-9.612; P=0.028) and basal ganglia region (odds ratio 2.688, 95% confidence interval 1.182-10.281; P=0.039). Conclusion The low serum 25 (OH) D levels are independently associated w ith the severity of EPVS in patients w ith lacunar infarction.
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<p><b>OBJECTIVE</b>To investigate the effect of transcriptional regulation of aberrant transcription factor AML1-ETO on p14.</p><p><b>METHODS</b>P14expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. Methylation-specific polymerase chain reaction (MSP) was used to analyze the methylation status of p14promoter. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and p14promoter in AML1-ETO positive leukemia cell line. And the p14mRNA expression level was detected by qRT-PCR after treatment with 5-Aza.</p><p><b>RESULTS</b>AML1-ETO-expressing cell subclone displayed low level of p14mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, level of p14mRNA was markedly lower when compared with other acute myeloid leukemias lacking this translocation. P14gene promoter was non-methylated in control group and primary leukemia cells of AML patients without t(8;21) and was hyper-methylated in U937-A/E1-4 and primary leukemia cells of AML patients with t(8;21). The enriched regions in transfected cells were located within p14promoter. 5-Aza could increase the expression of p14.</p><p><b>CONCLUSION</b>P14is a possible target gene of AML1-ETO. The p14silencing induced by hyper-methlylation may be an important factor for occurrence and development of the Msubtype of acute myeloid leukemia.</p>
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Objective To investigate the effect of EGB on SOD, MDA of ventilator-induced lung injury in rats and its possible mechanisms. Methods Thirty male SD rats were randomly divided into 3 groups: control group (C group), high tidal ventilation group (H group) and EGB group (E group). The setting mechanical ventilation was VT=30 mL/kg, RR=40/min, I/E=1/3, PEEP=0 cmH2 O and FiO2=21%. The broncho-alveolar lavage fluid (BLAF) and serum were obtained for determination of the levels of SOD and MDA at the end of 4 h mechanical ventilation. The Lungs were removed, and the wet-to-dry weight ratio (W/D) and pulmonary pathologic changes were measured. Results As compared with C group, W/D and the levels of MDA were significantly increased in H group, but the levels of SOD were reduced in H group. As compared with H group, W/D and the levels of MDA were significantly decreased in E group, but the levels of SOD were increased in E group. Pulmonary pathologic changes were alleviated in E group comparing with H group. Conclusion EGB injection may have a protective role against hyperoxia and induced pulmonary damage in rats.
ABSTRACT
This study was aimed to investigate the effect of AML1-ETO fusion protein on the anti-apoptotic gene BCL-2 in leukemic cells and to explore its role in leukemogenesis. The apoptotic levels of U937-WT, U937-Mock and U937-A/E1-4 cells were examined by flow cytometry. And cleaved caspase-3 protein expression was detected by Western blot. BCL-2 gene expression both in AML1-ETO-expressing cells or U937 nonexpressing cells and in leukemia cells of AML patients with or without t(8;21) was assessed by quantitative PCR. The chromatin immunoprecipitation (ChIP)-based PCR was used to investigate the direct interaction between the AML1-ETO and BCL-2 promoter in AML1-ETO positive leukemia cell line. The results indicated that in U937-A/E cells but not in U937-WT or U937-Mock cells, apoptotic cells statistically significantly increased, and AML1-ETO expression also significantly enhanced activation of caspase-3. AML1-ETO-expressing cell subclones displayed significantly low levels of BCL-2 mRNA in comparison with the non-transfected U937. In primary bone marrow cells of acute myeloid leukemia containing AML1-ETO, levels of BCL-2 mRNA were markedly lower as compared with other acute myeloid leukemias lacking this translocation. The enriched regions in transfected cells were located within BCL-2 promoter. It is concluded that BCL-2 is the direct target gene of AML1-ETO. AML1-ETO can down-regulate the expression of BCL-2.
Subject(s)
Humans , Core Binding Factor Alpha 2 Subunit , Genetics , Metabolism , Gene Expression Regulation, Leukemic , Leukemia, Myeloid, Acute , Genetics , Metabolism , Oncogene Proteins, Fusion , Genetics , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , RUNX1 Translocation Partner 1 Protein , U937 CellsABSTRACT
<p><b>OBJECTIVE</b>To construct a pcDNA3.1-AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells.</p><p><b>METHODS</b>AML1-ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry.</p><p><b>RESULTS</b>The recombinant pcDNA3.1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1-AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0.05). The expression of CD11b in transfected group \[(4.17 ± 0.31)%\] was lower than that in empty plasmid transfected group and non-transfected group \[(11.40 ± 0.17)% and (11.03 ± 0.15)%\] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CDl1b was unchanged in transfected cells treated with TPA (P > 0.05).</p><p><b>CONCLUSION</b>The eukaryotic expression vector for AML1-ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1-ETO in leukemogenesis.</p>