LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1

Abstract Background H19 is a well-characterized Long noncoding RNA (lncRNA) that has been proven to promote myoblast differentiation in humans and mice. However, its mechanism of action is still not fully interpreted. Methods Using RT-qPCR, we examined H19 RNA levels in various tissues from 1-week, 1-month, 6-month and 36-month old male cattle (i.e., newborn, infant, young and adult). The protein and mRNA levels of MyoG, MyHC, Sirt1 and FoxO1 in the satellite and C2C12 cells with an H19 silencing or overexpression vector were respectively detected using western blot and real-time qPCR. Results H19 was highly expressed in skeletal muscle at all the studied ages. High expression of H19 was required for the differentiation of bovine satellite cells. Knockdown of H19 caused a remarkable increase in the myoblast-inhibitory genes Sirt1/FoxO1, suggesting that H19 suppresses Sirt1/FoxO1 expression during myogenesis. Western blotting analysis of co-transfection of Sirt1 or FoxO1 expression vectors with pcDNA-H19 indicated that Sirt1/FoxO1 overexpression neutralized the promotion of myoblast differentiation through transfection of pcDNA-H19. Conclusion H19 promoted the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1

Additional file 1: Figure S1. of LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1

The expression profiles of H19 in various tissues of cattle at different postnatal stages. The reference genes is 18S RNA. The relative expression levels of H19 in satellite and C2C12 cells during differentiation were calculated according to the method of 2-ΔΔCt. (TIF 498 kb

Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients

Abstract Objective The aim of this study was to evaluate the effectiveness of metagenomic next-generation sequencing (mNGS) for the diagnosis of Pneumocystis jirovecii Pneumonia (PCP) in critically pediatric patients. Methods Seventeen critically pediatric patients with PCP and sixty patients diagnosed with non-PCP pneumonia who were admitted in pediatric intensive care unit between June 2018 and July 2021 were enrolled. Conventional methods and mNGS for detecting Pneumocystis jirovecii (P. jirovecii) were compared. The patients’ demographics, comorbidities, laboratory test results, antibiotic treatment response and 30 day mortality were analyzed. Result The mNGS showed a satisfying diagnostic performance with a sensitivity of 100% in detecting P. jirovecii compared with Gomori methenamine silver staining (5.9%), serum (1,3)-β-D-glucan (86.7%) and and LDH (55.6%). The diagnostic specificity of mNGS for PCP was higher than that of serum BDG (56.7%) and LDH (71.4%). In PCP group, over one thirds’ cases had mixed infections. Compared with survivors, non-survivors had higher stringently mapped read numbers (SMRNs) in bronchoalveolar lavage fluid (BALF) sample (P < 0.05), suggesting SMRNs were closely associated with the severity of response. The detection for P. jirovecii by mNGS both in BALF and blood samples reached a concordance rate of 100%, and the SMRNs in the BALF were remarkably higher than that in blood samples. Initial antimicrobial treatment was modified in 88.2% of PCP patients based on the mNGS results. Conclusion The mNGS is a potential and efficient technology in diagnosing PCP and shows a satisfying performance in the detection of co-pathogens. Both blood and BALF samples for mNGS are suggested for the presumptive diagnosis of PCP

Rapid reconstitution of ubiquitinated nucleosome using a non-denatured histone octamer ubiquitylation approach

Abstract Background Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. Results In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. Conclusions Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology

Rapid reconstitution of ubiquitinated nucleosome using a non-denatured histone octamer ubiquitylation approach.

BACKGROUND: Histone ubiquitination modification is emerging as a critical epigenetic mechanism involved in a range of biological processes. In vitro reconstitution of ubiquitinated nucleosomes is pivotal for elucidating the influence of histone ubiquitination on chromatin dynamics. RESULTS: In this study, we introduce a Non-Denatured Histone Octamer Ubiquitylation (NDHOU) approach for generating ubiquitin or ubiquitin-like modified histone octamers. The method entails the co-expression and purification of histone octamers, followed by their chemical cross-linking to ubiquitin using 1,3-dibromoacetone. We demonstrate that nucleosomes reconstituted with these octamers display a high degree of homogeneity, rendering them highly compatible with in vitro biochemical assays. These ubiquitinated nucleosomes mimic physiological substrates in function and structure. Additionally, we have extended this method to cross-linking various histone octamers and three types of ubiquitin-like proteins. CONCLUSIONS: Overall, our findings offer an efficient strategy for producing ubiquitinated nucleosomes, advancing biochemical and biophysical studies in the field of chromatin biology