DBA/1 mice have a higher susceptibility to generalized audiogenic seizures (AGSz) and seizure-induced respiratory arrest (S-IRA) than C57/BL6 mice. The gene expression profile might be potentially related to this difference. This study aimed to investigate the susceptibility difference in AGSz and S-IRA between DBA/1 and C57BL/6 mice by profiling long noncoding RNAs (lncRNAs) and mRNA expression.We compared lncRNAs and mRNAs from the brainstem of the two strains with Arraystar Mouse lncRNA Microarray V3.0 (Arraystar, Rockville, MD). Gene Ontology (GO) and pathway analyses were performed to determine the potentially related biological functions and pathways based on differentially expressed mRNAs. qRT-PCR was carried out to validate the results.A total of 897 lncRNAs and 438 mRNAs were differentially expressed (fold change ≥2, P < 0.05), of which 192 lncRNAs were upregulated and 705 lncRNAs were downregulated. A total of 138 mRNAs were upregulated, and 300 mRNAs were downregulated. In terms of specific mRNAs, Htr5b, Gabra2, Hspa1b and Gfra1 may be related to AGSz or S-IRA. Additionally, lncRNA Neat1 may participate in the difference in susceptibility. GO and pathway analyses suggested that TGF-β signaling, metabolic process and MHC protein complex could be involved in these differences. Coexpression analysis identified 9 differentially expressed antisense lncRNAs and 115 long intergenic noncoding RNAs (lincRNAs), and 2010012P19Rik and its adjacent RNA Tnfsf12-Tnfsf13 may have participated in S-IRA by regulating sympathetic neuron function. The results of the qRT-PCR of five selected lncRNAs (AK038711, Gm11762, 1500004A13Rik, AA388235 and Neat1) and four selected mRNAs (Hspa1b, Htr5b, Gabra2 and Gfra1) were consistent with those obtained by microarray.We concluded that TGF-β signaling and metabolic process may contribute to the differential sensitivity to AGSz and S-IRA. Among mRNAs, Htr5b, Gabra2, Hspa1b and Gfra1 could potentially influence the susceptibility. LncRNA Neat1 and 2010012P19Rik may also contribute to the different response to sound stimulation. Further studies should be carried out to explore the underlying functions and mechanisms of differentially expressed RNAs.
Abstract Macrophage polarization plays a crucial role in inflammatory processes. The histone deacetylase 3 (HDAC3) has a deacetylase‐independent function that can activate pro‐inflammatory gene expression in lipopolysaccharide‐stimulated M1‐like macrophages and cannot be blocked by traditional small‐molecule HDAC3 inhibitors. Here we employed the proteolysis targeting chimera (PROTAC) technology to target the deacetylase‐independent function of HDAC3. We developed a potent and selective HDAC3‐directed PROTAC, P7 , which induces nearly complete HDAC3 degradation at low micromolar concentrations in both THP‐1 cells and human primary macrophages. P7 increases the anti‐inflammatory cytokine secretion in THP‐1‐derived M1‐like macrophages. Importantly, P7 decreases the secretion of pro‐inflammatory cytokines in M1‐like macrophages derived from human primary macrophages. This can be explained by the observed inhibition of macrophage polarization from M0‐like into M1‐like macrophage. In conclusion, we demonstrate that the HDAC3‐directed PROTAC P7 has anti‐inflammatory activity and blocks macrophage polarization, demonstrating that this molecular mechanism can be targeted with small molecule therapeutics.
Angiotensin-converting enzyme has shown altered activity in patients with neurological diseases. An insertion/deletion (I/D) polymorphism of the <i>DCP1</i> gene encoding angiotensin-converting enzyme has been reported to be associated with the risk for Alzheimer’s disease (AD), but ambiguous results have also been presented. We conducted a case-control study in a sample composed of 192 sporadic AD patients and 195 age- and sex-matched controls from Chinese Han population in Beijing and Xi’an districts to investigate the possible effect of the polymorphism. Our data revealed no association between the <i>DCP1</i> polymorphism and AD risk in the total sample. There was no significant difference in the <i>DCP1 </i>allele or genotype frequencies between cases and controls when stratified by gender and <i>APOE</i> ε4 status. However, the D allele and D/D genotype were more frequent among AD patients between 66 and 70 years compared with controls (D allele: OR = 2.8, 95% CI = 1.5–5.2, p = 0.001; D/D genotype: OR = 5.9, 95% CI = 1.7–19.9, p = 0.002). Our results provided new proof that the <i>DCP1 </i>D allele was a probable risk factor for late-onset AD. Its role was independent and was limited to the population at a certain age.
Objective:To construct the recombined plasmid expressing zinc-finger E-box binding homeobox 1(ZEB1) short hairpin RNA(shRNA) and observe the characteristics of epithelial ovarian cancer SKOV3 cells after shRNA-mediated ZEB1 gene silencing in an epithelial ovarian cancer SKOV3 cells.Methods:The shRNA targeting the ZEB1 gene was synthesized,and cloned into plasmid pSUPER-enhanced green fluorescent protein 1(EGFP1).The formed pSUPER-EGFP1-ZEB1-shRNA was transfected into the ovarian cancer SKOV3 cells by Lipofectamine 2000,and the stably transfected cells were isolated by G418 selection.The expression of ZEB1 was detected by RT-PCR and Western blot.The characteristics of ZEB1-shRNA transfected SKOV3 cells were analyzed from the assays of colony formation,wound healing and over expression of miR-200c as well as tumorigenicity in nude mice,respectively.Results:The ZEB1 expression in ZEB1-shRNA transfected SKOV3 cells was significantly lower than that of the scramble control siRNA.The ZEB1-shRNA transfected SKOV3 cells show the abilities of proliferation,migration and tumorigenicity were significantly decreased compared with the scrambled control siRNA(P0.05).ZEB1 low expression resulted in increased miR-200c expression in the ZEB1-shRNA transfected SKOV3 cells.Conclusion:Targeting ZEB1 expression in SKOV3 cells with RNA interference can significantly reduce and inhibit the cell proliferation and the tumorigenicity of SKOV3 cells.These results suggest that the ZEB1 is an effective therapeutic target for human epithelial ovarian caner treatment.
Abstract The mechanisms underlying tissue repair in response to damage have been one of main subjects of investigation. In this study, we leveraged the wound-induced hair neogenesis (WIHN) models in adult mice to explore the inner correlation. Our investigation revealed that heightened release of mitochondrial DNA (mtDNA) accompanying tissue damage activated the toll-like receptor 9 (TLR9) pathway, influencing the repair process and the ultimate number of regenerated hair follicles. Furthermore, our analysis of single-cell RNA sequencing comparisons demonstrated increased TLR9 activation was associated with the recruitment of gamma delta T cells (γδT). Inhibition of γδT cell recruitment led to a reduction in the population of γδT cells and a more fibrotic healing outcome. Notably, these γδT cells exhibited distinctive high production of AREG, contributing to the rapid increase of local AREG levels around the epidermis and influencing the fate commitment of keratinocytes. These findings provide new insights into the roles of TLRs as critical mediators in the sense of tissue damage, the modulation of immune cell activity, and the ultimate influence on healing outcomes. Teaser Starting with how tissue injury stimulates downstream tissue repair and regeneration through relevant signals, this study explored the phenomenon and correlation between tissue damage and TLR9, and the effect of TLR9 on γδT, keratinocytes and the healing outcomes.
Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1-9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.
Objective
To optimize the method of the directional differentiation of adipose-derived stem cells into keratinocytes.
Methods
Adipose-derived stem cells (ADSCs), separated by collagenase digestion method, were isolated and cultured. Then the expression of surface specific markers CD34, CD44 and CD90 were detected by flow cytometer. The effect of different induced mediums cultured for two weeks on the differentiation of ADSCs into KCs was demonstrated: Group 1, the DMEM supplied with 2% FBS and 49% supertant of KCs; group 2, KSFM medium; group 3, DMEM medium supplied with 10% FBS and 5 μM ATRA; 10% FBS DMEM as the control group. Immunofluorescene staining was applied to detect the expression of keratin CK14 and F-actin.
Results
A flattened fibroblast-like morphology was observed in cells, the positive expression rate of CD34 was 0.08%, while those of CD44 and CD90 were 99%. The cells that could differentiate into osteoblasts and chondrocytes, indicated that the cells were ADSCs. There was no significant change in the cell morphology in the group 1 under the induction medium; about 10% of the cells in group 2 were altered; the morphological changes were obvious in group 3, and approximately 20% of the cells showed irregular polygon. The immunofluorescene staining of the cells in group 3 indicated that the cells showed cobblestone-like phenotype and an organized cytoskeletal network with dense actin fibers at the edges; some cells were positive for CK14.
Conclusions
ADSCs show higher induction rate under ATRA stimulation.
Key words:
Adipose tissue; Stem cells; Cell differentiation; Keratinocytes
Cathepsin D (CTSD) is an intracellular aspartyl protease, which is active in the endosomal/lysosomal system. CTSD may play a role in Alzheimer’s disease (AD) through cleaving the amyloid precursor protein into β-amyloid peptide and degrading tau protein into fragments. A functional polymorphism in exon 2 of the cathepsin D gene (C→T, Ala224Val) has recently been reported to increase the risk for AD in some of the Caucasian populations, with a significant overrepresentation of the T allele, but these reports have not been universally duplicated. We performed an association study between CTSD polymorphism and AD in 156 sporadic AD patients and 183 controls of Chinese Han ethnicity. Our data revealed that the distribution of CTSD genotypes and alleles was similar in patients and controls. No direct association was found between CTSD polymorphism and AD risk. There might be a weak synergistic interaction between CTSD T and APOEΕ4 allele in increasing the risk for developing AD.
The periosteum is known as the thin connective tissue covering most bone surfaces. Its extrusive bone regeneration capacity was confirmed from the very first century-old studies. Recently, pluripotent stem cells in the periosteum with unique physiological properties were unveiled. Existing in dynamic contexts and regulated by complex molecular networks, periosteal stem cells emerge as having strong capabilities of proliferation and multipotential differentiation. Through continuous exploration of studies, we are now starting to acquire more insight into the great potential of the periosteum in bone formation and repair in situ or ectopically. It is undeniable that the periosteum is developing further into a more promising strategy to be harnessed in bone tissue regeneration. Here, we summarized the development and structure of the periosteum, cell markers, and the biological features of periosteal stem cells. Then, we reviewed their pivotal role in bone repair and the underlying molecular regulation. The understanding of periosteum-related cellular and molecular content will help enhance future research efforts and application transformation of the periosteum.
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