DNA Demethylation and USF Regulate the Meiosis-Specific Expression of the Mouse Miwi

Miwi, a member of the Argonaute family, is required for initiating spermiogenesis; however, the mechanisms that regulate the expression of the Miwi gene remain unknown. By mutation analysis and transgenic models, we identified a 303 bp proximal promoter region of the mouse Miwi gene, which controls specific expression from midpachytene spermatocytes to round spermatids during meiosis. We characterized the binding sites of transcription factors NF-Y (Nuclear Factor Y) and USF (Upstream Stimulatory Factor) within the core promoter and found that both factors specifically bind to and activate the Miwi promoter. Methylation profiling of three CpG islands within the proximal promoter reveals a markedly inverse correlation between the methylation status of the CpG islands and germ cell type–specific expression of Miwi. CpG methylation at the USF–binding site within the E2 box in the promoter inhibits the binding of USF. Transgenic Miwi-EGFP and endogenous Miwi reveal a subcellular co-localization pattern in the germ cells of the Miwi-EGFP transgenic mouse. Furthermore, the DNA methylation profile of the Miwi promoter–driven transgene is consistent with that of the endogenous Miwi promoter, indicating that Miwi transgene is epigenetically modified through methylation in vivo to ensure its spatio-temporal expression. Our findings suggest that USF controls Miwi expression from midpachytene spermatocytes to round spermatids through methylation-mediated regulation. This work identifies an epigenetic regulation mechanism for the spatio-temporal expression of mouse Miwi during spermatogenesis

Genomic data for 78 chickens from 14 populations

Background: Since the domestication of the red jungle fowls (Gallus gallus; dating back to~10 000 B.P.) in Asia, domestic chickens (Gallus gallus domesticus) have been subjected to the combined effects of natural selection and human-driven artificial selection; this has resulted in marked phenotypic diversity in a number of traits, including behavior, body composition, egg production, and skin color. Population genomic variations through diversifying selection have not been fully investigated. Findings: The whole genomes of 78 domestic chickens were sequenced to an average of 18-fold coverage for each bird. By combining this data with publicly available genomes of five wild red jungle fowls and eight Xishuangbanna game fowls, we conducted a comprehensive comparative genomics analysis of 91 chickens from 17 populations. After aligning ~21.30 gigabases (Gb) of high-quality data from each individual to the reference chicken genome, we identified ~6.44 million (M) single nucleotide polymorphisms (SNPs) for each population. These SNPs included 1.10 M novel SNPs in 17 populations that were absent in the current chicken dbSNP (Build 145) entries. Conclusions: The current data is important for population genetics and further studies in chickens and will serve as a valuable resource for investigating diversifying selection and candidate genes for selective breeding in chickens.Peer reviewedAnimal Scienc

Interpretation of the essence of mesenchymal stem cells with the “kidney-triple energizers” system

Kidney and triple energizers are inseparable in structure and function. Therefore, it is proposed that “kidney-triple energizers” might be the essence of mesenchymal stem cells (MSCs) system. As a cardinal, this article aims to interpret the theoretical connotation of the mesenchymal stem cell system from the perspective of “the kidney essence is the body and the triple energizers is the function”. It is reflected in the kidney-three energizers system, and guided by the theory of traditional Chinese medicine, the modern technology of MSCs has achieved good results in the application of refractory diseases such as COVID-19, and a preliminary exploration of the integration of Chinese and Western medicine research has been carried out

Low level laser (LLL) attenuate LPS-induced inflammatory responses in mesenchymal stem cells via the suppression of NF-κB signaling pathway in vitro.

BACKGROUND:Considering promising results in animal models and patients, therapeutic use of MSCs for immune disease is likely to undergo continued evaluation. Low-lever laser (LLL) has been widely applied to retard the inflammatory reaction. LLL treatment can potentially be applied in anti-inflammatory therapy followed by stem cell therapy. AIM OF THE STUDY:The purpose of this study was to investigate the effect of LLL (660 nm) on the inflammatory reaction induced by LPS in human adipose derived mesenchymal stem cells (hADSCs) and pertinent mechanism. MATERIALS AND METHODS:Anti-inflammatory activity of LLL was investigated by LPS-induced mesenchymal stem cells. The production and expression of pro-inflammatory cytokines were evaluated by ELISA kits and RT-qPCR. Nuclear translocation of NF-κB was indicated by immunofluorescent staining. Phosphorylation status of NF-κB p65 and IκBα were illustrated by western blot assay. ROS generation was measured with CM-H2DCFDA, and NO secretion was determined by DAF-FM. We studied surface expression of lymphocyte activation markers when Purified peripheral blood mononuclear cell (PBMC) were activated by phytohaemagglutinin (PHA) in the presence of 3 types of treated MSCs. RESULTS:LLL reduced the secretion of IL-1β, IL-6, IL8, ROS and NO in LPS treated MSCs. Immunofluorescent assay demonstrated the nuclear translocation decrease of NF-κB in LLL treated LPS induced MSCs. Western blot analysis also suggested that LLL suppressed NF-κB activation via regulating the phosphorylation of p65 and IκBα. MSC significantly reduced the expression of activation markers CD25 and CD69 on PHA-stimulated lymphocytes. CONCLUSION:The results indicate that LLL suppressed the activation of NF-κB signaling pathway in LPS treated MSCs through inhibiting phosphorylation of p65 and IκBα, which results in good anti-inflammatory effect. In addition, LLL attenuated activation-associated markers CD25 and CD69 in co-cultures of PBMC and 3 types of treated MSCs

Effect of LLL on mRNA expression and production and of IL-1β, IL4, IL-6, IL-8 and IL-10 by LPS (or not) induced MSCs.

<p>MSCs were incubated with or without LPS (10ng/mL) and simultaneous treated LLL for 1h. Values of *P<0.05, **P<0.01and vs. LPS or control were considered statistically significant.</p

LLL decrease nuclear translocation of NF-κB.

<p>The images show the cytoplasmic localization of NF-κB in the control cells (upper panel), the nuclear translocation of NF-κB in cells treated with LPS (middle panel) and LLL treatment blocked the nuclear translocation of NF-κB caused by LPS stimulation (lower panel).</p

Primers used in study for PCR.

<p>Primers used in study for PCR.</p

Percentage of positive PBMC after stimulation with PHA in the absence or presence of 3 types of treated MSCs.

<p>After reduction of LLL treated MSCs, CD25+ cells decreased (**P < 0.01 vs. PHA) and CD69+ cells were also reduced compared to control groups (**P < 0.01 vs. PHA).</p

LLL suppress ROS and NO promotion induced by LPS in MSCs.

<p>MSCs were incubated with LPS (10ng/mL) and simultaneous treated with LLL for 1 h. Error bars represent the mean±SD (n = 3 per group). Values of **P<0.01 vs. LPS and **P<0.01 vs. control were considered statistically significant.</p