SENP1-Sirt3 Signaling Controls Mitochondrial Protein Acetylation and Metabolism.

Sirt3, as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolic adaption to various stresses. However, how to regulate Sirt3 activity responding to metabolic stress remains largely unknown. Here, we report Sirt3 as a SUMOylated protein in mitochondria. SUMOylation suppresses Sirt3 catalytic activity. SUMOylation-deficient Sirt3 shows elevated deacetylation on mitochondrial proteins and increased fatty acid oxidation. During fasting, SUMO-specific protease SENP1 is accumulated in mitochondria and quickly de-SUMOylates and activates Sirt3. SENP1 deficiency results in hyper-SUMOylation of Sirt3 and hyper-acetylation of mitochondrial proteins, which reduces mitochondrial metabolic adaption responding to fasting. Furthermore, we find that fasting induces SENP1 translocation into mitochondria to activate Sirt3. The studies on mice show that Sirt3 SUMOylation mutation reduces fat mass and antagonizes high-fat diet (HFD)-induced obesity via increasing oxidative phosphorylation and energy expenditure. Our results reveal that SENP1-Sirt3 signaling modulates Sirt3 activation and mitochondrial metabolism during metabolic stress.

Monoclonal antibody-based systematic identification of SUMO1-modification sites reveals TFII-I SUMOylation is involved in tumor growth.

SUMOylation plays an essential role in diverse physiological and pathological processes. Identification of wild-type SUMO1-modification sites by mass spectrometry is still challenging. In this study, we produced a monoclonal SUMO1C-K antibody recognizing SUMOylated peptides and proposed an efficient streamline for identification of SUMOylation sites. We identified 471 SUMOylation sites in 325 proteins from five raw data. These identified sites exhibit a high positive rate when evaluated by mutation-verified SUMOylation sites. We identified many SUMOylated proteins involved in mitochondrial metabolism and non-membrane-bounded organelles formation. We proposed a SUMOylation motif, ΨKXD/EP, where proline is required for efficient SUMOylation. We further revealed SUMOylation of TFII-I was stimulated by growth signals and was required for nucleus-localization of p-ERK1/2. Mutation of SUMOylation sites of TFII-I suppressed tumor cell growth in vitro and in vivo. Taken together, we provided a strategy for personalized identification of wild-type SUMO1-modification sites and revealed the physiological significance of TFII-I SUMOylation in this study.

Porphyromonas gingivalis promotes the progression of oral squamous cell carcinoma by stimulating the release of neutrophil extracellular traps in the tumor immune microenvironment.

BACKGROUND: The aim of this study was to investigate the impact of Porphyromonas gingivalis (P. gingivalis) on the progression of oral squamous cell carcinoma (OSCC) through neutrophil extracellular traps (NETs) in the tumor immune microenvironment. METHODS: The expression of NETs-related markers was identified through immunohistochemistry, immunofluorescence, and Western blotting in different clinical stages of OSCC samples. The relationship between NETs-related markers and clinicopathological characteristics in 180 samples was analyzed using immunohistochemistry data. Furthermore, the ability to predict the prognosis of OSCC patients was determined by ROC curve analysis and survival analysis. The effect of P. gingivalis on the release of NETs was identified through immunofluorescence and immunohistochemistry, both in vitro and in vivo. CAL27 and SCC25 cell lines were subjected to NETs stimulation to elucidate the influence of NETs on various cellular processes, including cell proliferation, migration, invasion, and metastasis in vitro. Furthermore, the impact of NETs on the growth and metastatic potential of OSCC was assessed using in vivo models involving tumor-bearing mice and tumor metastasis mouse models. RESULTS: Immunochemistry analysis revealed a significant correlation between the NETs-related markers and clinical stage, living status as well as TN stage. P. gingivalis has demonstrated its ability to effectively induce the release of NETs both in vivo and in vitro. NETs have the potential to facilitate cell migration, invasion, and colony formation. Moreover, in vivo experiments have demonstrated that NETs play a pivotal role in promoting tumor metastasis. CONCLUSION: High expression of NETs-related markers demonstrates a strong correlation with the progression of OSCC. Inhibition of the NETs release process stimulated by P. gingivalis and targeted NETs could potentially open up a novel avenue in the field of immunotherapy for patients afflicted with OSCC.

Long Non-coding RNA LINC00342 Promotes the Proliferation, Invasion, and Migration of Primary Hepatocellular Carcinoma Cells by Regulating the Expression of miRNA-19a-3p, miRNA-545-5p, and miRNA-203a-3p.

This study aimed to investigate the role of the long non-coding RNA (lncRNA) LINC00342-207 (LINC00342) in the development and progression of primary hepatocellular carcinoma (HCC). Forty-two surgically resected HCC tissues and corresponding paracancerous tissues were collected from October 2019 to December 2020 and examined for lncRNA LINC00342, microRNA (miR)-19a-3p, miR-545-5p, miR-203a-3p, cell cycle protein D1 (CyclinD1/CCND1), murine double minute 2 (MDM2), and fibroblast growth factor 2 (FGF2) expression. The disease-free survival and overall survival of patients with HCC were followed up. HCC cell lines and the normal hepatocyte cell line HL-7702 were cultured and the expression level of LINC00342 was measured. HepG2 cells were transfected with LINC00342 siRNA, LINC00342 overexpression plasmid, miR-19a-3p mimics and their corresponding suppressors, miR-545-5p mimics and their corresponding suppressors, and miR-203a-3p mimics and their corresponding suppressors. The proliferation, apoptosis, migration, and invasion of HepG2 cells were detected. Stably transfected HepG2 cells were inoculated into the left axilla of male BALB/c nude mice, and the volume and quality of transplanted tumors as well as the expression levels of LINC00342, miR-19a-3p, miR-545-5p, miR-203a-3p, CCND1, MDM2, and FGF2 were examined. LINC00342 played an oncogenic role in HCC and exhibited inhibitory effects on proliferation, migration, and invasion, and promoted the apoptosis of HepG2 cells. Moreover, it inhibited the growth of transplanted tumors in vivo in mice. Mechanistically, the oncogenic effect of LINC00342 was associated with the targeted regulation of the miR-19a-3p/CCND1, miR-545-5p/MDM2, and miR-203a-3p/FGF2 axes.

Six-Transmembrane Epithelial Antigen of Prostate 3 Promotes Hepatic Insulin Resistance and Steatosis.

Nonalcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by excessive deposition of fatty acids in the liver. Further deterioration leads to nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma, creating a heavy burden on human health and the social economy. Currently, there are no effective and specific drugs for the treatment of NAFLD. Therefore, it is important to further investigate the pathogenesis of NAFLD and explore effective therapeutic targets for the prevention and treatment of the disease. Six-transmembrane epithelial antigen of prostate 3 (STEAP3), a STEAP family protein, is a metalloreductase. Studies have shown that it can participate in the regulation of liver ischemia-reperfusion injury, hepatocellular carcinoma, myocardial hypertrophy, and other diseases. In this study, we found that the expression of STEAP3 is upregulated in NAFLD. Deletion of STEAP3 inhibits the development of NAFLD in vivo and in vitro, whereas its overexpression promotes palmitic acid/oleic acid stimulation-induced lipid deposition in hepatocytes. Mechanistically, it interacts with transforming growth factor beta-activated kinase 1 (TAK1) to regulate the progression of NAFLD by promoting TAK1 phosphorylation and activating the TAK1-c-Jun N-terminal kinase/p38 signaling pathway. Taken together, our results provide further insight into the involvement of STEAP3 in liver pathology.

Exosomal IRF1-loaded rat adipose-derived stem cell sheet contributes to wound healing in the diabetic foot ulcers.

BACKGROUND: Cell-based therapy has been recognized as a novel technique for the management of diabetic foot ulcers, and cell-sheet engineering leads to improved efficacy in cell transplantation. This study aims to explore the possible molecular mechanism of the rat adipose-derived stem cell (ASC) sheet loaded with exosomal interferon regulatory factor 1 (IRF1) in foot wound healing. METHODS: Rats were rendered diabetic with streptozotocin, followed by measurement of miR-16-5p expression in wound tissues. Relationship between IRF1, microRNA (miR)-16-5p, and trans-acting transcription factor 5 (SP5) was analyzed using luciferase activity, RNA pull-down, and chromatin immunoprecipitation assays. IRF1 was overexpressed in rat ASCs (rASCs) or loaded onto the rASC sheet, and then exosomes were extracted from rASCs. Accordingly, we assessed the effects of IRF1-exosome or IRF1-rASC sheet on the proliferation and migration of the fibroblasts along with endothelial cell angiogenesis. RESULTS: miR-16-5p was poorly expressed in the wound tissues of diabetic rats. Overexpression of miR-16-5p promoted fibroblast proliferation and migration as well as endothelial cell angiogenesis, thus expediting wound healing. IRF1 was an upstream transcription factor that could bind to the miR-16-5p promoter and increase its expression. In addition, SP5 was a downstream target gene of miR-16-5p. IRF1-exosome from rASCs or the IRF1-rASC sheet facilitated the foot wound healing in diabetic rats through miR-16-5p-dependent inhibition of SP5. CONCLUSION: The present study demonstrates that exosomal IRF1-loaded rASC sheet regulates miR-16-5p/SP5 axis to facilitate wound healing in diabetic rats, which aids in development of stem cell-based therapeutic strategies for diabetic foot wounds.

Pelargonidin ameliorates inflammatory response and cartilage degeneration in osteoarthritis via suppressing the NF-κB pathway.

Pelargonidin (PG), a derivative of anthocyanins, has anti-oxidant and anti-inflammatory properties. Herein, the protective effect and the mechanism of PG in counteract the osteoarthritis (OA) progression were needed to further evaluate. In the current study, C57BL/6 mice was induced by destabilization of medial meniscus (DMM) surgery to establish the OA model. Primary chondrocytes were acquired from the knee cartilage of newborn mice. Then, PG was administrated to OA mice and IL-1ß-stimulated chondrocytes to evaluate its protective effects, respectively. Results uncovered that no conspicuous cytotoxic effects were observed when chondrocytes were treated with PG at a concentration lower than 40 µM for 24-72 h. Thus, 10 µM, 20 µM, and 40 µM PG were chosen for subsequent experiments in vitro. Then, we observed that 10, 20, and 40 µM PG reduced the levels of IL-6, TNF-α, COX-2 and iNOS in chondrocytes. In line, PG inhibited the IL-1ß-induced ECM catabolism in chondrocytes, as evidenced by deepening toluidine blue staining, increased expression of Collagen II, and decreased expressions of ADAMTS5 and MMP13. Moreover, PG also reduced the IL-1ß-stimulated p-p65 overexpression and nuclear translocation of p65 in chondrocytes. In vivo, Safranin O/Fast green and HE staining showed that articular cartilage surface morphology was basically smooth and complete after PG treatment for 8 weeks. Similarly, OARSI scores and MMP13 expression were apparently decreased, whereas Aggrecan expression was elevated in PG-treated mice 8 weeks after DMM surgery. In conclusion, PG can effectively ameliorate inflammatory reactions and cartilage degeneration via suppressing the NF-κB pathway, thereby restraining the OA progression.

Thiamethoxam adsorption by ZnCl2 modified cow manure biochar: Mechanism and quantitative prediction.

The overuse of thiamethoxam (THM) has threatened the survival of living organisms and it is necessary to find an environmentally friendly material to remove THM frequently detected in water. Biochar prepared from cow manure modified with ZnCl2 (Zn-CBC) was used to remove THM. Compared to the unmodified cow manure biochar (CBC), the removal ratio of THM by Zn-CBC was enhanced 35 times. In the mechanistic analysis, SEM and BET showed that Zn-CBC had a good pore structure and its specific surface area (166.502 m2 g-1) increased to 17 times that of CBC, indicating that Zn-CBC had good pore adsorption properties. The adsorption kinetic and isotherm implied that the main mechanism was chemisorption including π-π interaction and H-bonding. Furthermore, the stable graphitized structure of Zn-CBC allowed for efficient adsorption and reusability. In addition, this study constructed an intelligent prediction model using batch experiment data, and the high R2 (0.978) and low RMSE (0.057) implied that the model could accurately and quantitatively predict the adsorption efficiency. This paper provides a novel perspective to simultaneously remove the neonicotinoid insecticides and realize the resource utilization of cow manure.

Association of carotid atherosclerotic plaque and intima-media thickness with the monocyte to high-density lipoprotein cholesterol ratio among low-income residents of rural China: a population-based cross-sectional study.

BACKGROUND: The monocytes to high-density lipoprotein cholesterol ratio (MHR) has been identified as a potential biomarker for cardiovascular and cerebrovascular diseases. In this population-based cross-sectional study, we explored the relationships among carotid artery disease (CAD), including the presence of carotid atherosclerotic plaque (CAP) and carotid artery intima-media thickness (CIMT), the MHR, and related parameter changes. METHODS: This cross-sectional study, Conducted from April to June 2019 in a rural area of Tianjin, involved middle-aged and elderly participants. Based on carotid ultrasound examinations, participants were divided into CAP and non-CAP groups. Logistic regression and Receiver Operating Characteristic (ROC) curve analyses were utilized to assess MHR's predictive value for CAP. Gender-specific analyses were also performed to examine predictive variations. The relationship between CIMT and MHR was evaluated using linear regression. RESULTS: Of the 2109 participants meeting the inclusion criteria, 51.6% were identified with CAP. Multivariate analysis revealed a significant association between MHR and CAP prevalence, (OR, 9.670; 95% CI, 2.359-39.631; P = 0.002), particularly in females (OR, 5.921; 95% CI, 1.823-19.231; P = 0.003), after adjusting for covariates. However, no significant correlation was found between CIMT and MHR when adjusted for other factors. The ROC analysis showed the area under the curve for MHR and CAP to be 0.569 (95% CI: 0.544-0.593; P < 0.001). CONCLUSIONS: These findings suggested that it is crucial to enhance early screening and intervention for CAD, specifically focusing on the prevention and progression of CAP, to address the unique health challenges faced by low-income groups in rural settings. Emphasizing these preventive measures could significantly contribute to improving cardiovascular health outcomes in this vulnerable population.

Quantitative Identification Method for Glass Panel Defects Using Microwave Detection Based on the CSAPSO-BP Neural Network.

To address the problem of the quantitative identification of glass panel surface defects, a new method combining the chaotic simulated annealing particle swarm algorithm (CSAPSO) and the BP neural network is proposed for the quantitative evaluation of microwave detection signals of glass panel defects. First, the parameters of the particle swarm optimization (PSO) algorithm are dynamically assigned using chaos theory to improve the global search capability of the PSO. Then, the CSAPSO-BP neural network model is constructed, and the return loss and phase of the microwave detection echo signal of glass panel defects are extracted as the input feature quantity of the network, from which the intrinsic connection between input and output is found through network training and testing to achieve the prediction of the depth and width of glass panel surface defects. The results show that the CSAPSO-BP network model can more accurately characterize the defect geometry of glass panels than the PSO-BP network model.

Energy Transfer Efficiency Based Nonlinear Ultrasonic Testing Technique for Debonding Defects of Aluminum Alloy Foam Sandwich Panels.

In order to improve the accuracy of detection results of debonding defects of aluminum alloy thin plate, the nonlinear ultrasonic technology is used to detect the simulated defect samples, aiming at problems such as near surface blind region caused by the interaction of incident wave, reflected wave and even second harmonic wave in a short time due to the small thickness of thin plates. An integral method based on energy transfer efficiency is proposed to calculate the nonlinear ultrasonic coefficient to characterize the debonding defects of thin plates. A series of simulated debonding defects of different sizes were made using aluminum alloy plates with four thicknesses of 1 mm, 2 mm, 3 mm and 10 mm. By comparing the traditional nonlinear coefficient with the integral nonlinear coefficient proposed in this paper, it is verified that both methods can quantitatively characterize the size of debonding defects. The nonlinear ultrasonic testing technology based on energy transfer efficiency has higher testing accuracy for thin plates.

Internal and External Pipe Defect Characterization via High-Frequency Lamb Waves Generated by Unidirectional EMAT.

Periodic permanent magnet(PPM) electromagnetic acoustic transducers (EMATs) are commonly employed for axial defect inspection in pipelines. However, the lowest-order shear horizontal waves (SH0) guided waves have difficulties in distinctly differentiating internal and external defects. To enhance the signal-to-noise ratio and resolution, a unidirectional electromagnetic acoustic transducer (EMAT) based on Circumferential Lamb waves (CLamb waves) is developed. Through structural parameter optimization and excitation frequency adjustment, high-amplitude and low-dispersion CLamb waves are successfully generated in the high-frequency-thickness product region of the dispersion curve. Finite element simulations and experimental validation confirm the capability of this EMAT in exciting CLamb waves for the detection of crack-like defects. Experimental results demonstrate that the excitation efficiency of the CLamb EMAT exceeds that of the periodic permanent magnet electromagnetic acoustic transducer by more than tenfold. The defect reflection signal of the CLamb EMAT exhibits higher resolution and more significant amplitude compared to the PPM EMAT. The integration of this method with SH0 mode detection allows for the inspection of both internal and external defects in pipelines, offering a new avenue for EMAT applications in pipeline inspection.

Effects of Dietary Protein and Lipid Levels on Growth, Metabolism, Antioxidative Capacity, and Fillet Quality of Adult Triploid Rainbow Trout Farmed in Net Cage.

The research is aimed at investigating the effects of dietary protein and lipid levels on adult triploid rainbow trout growth performance, feed utilization, digestive and metabolic enzyme activities, antioxidative capacity, and fillet quality. Nine diets containing three dietary protein levels (DP) (300, 350, and 400 g kg-1) and three dietary lipid levels (DL) (200, 250, and 300 g kg-1) were prepared using a 3 × 3 factorial design. In freshwater cages, 13,500 adult female triploid rainbow trout (3.2 ± 0.1 kg) were cultured for 77 days. Triplicate cages (500 fish per cage) were used as repetitions of each experimental diet. The findings revealed that as DP increased to 400 g kg-1 and DL raised to 300 g kg-1, the weight gain ratio (WGR) elevated significantly (P < 0.05). However, when DP ≥ 350 g kg-1, WGR was similar in the DL250 and DL300 groups. As DP raised to 350 g kg-1, the feed conversion ratio (FCR) notably decreased (P < 0.05). In the DP350DL300 group, lipids had a protein-sparing impact. High DP diet (400 g kg-1) generally improved fish health status by increasing antioxidant capacity in the liver and intestine. A high DL diet (300 g kg-1) showed no harmful effect on hepatic health based on plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and antioxidant capacity in the liver. For fillet quality, a high DP diet could increase fillet yield, improve fillet hardness, springiness, and water-holding capacity values, and inhibit the production of off-flavors caused by n-6 fatty acids. A high DL diet could increase odor intensity, and EPA, DHA, and n-3 fatty acid concentrations decrease the thrombogenicity index value. The maximum fillet redness value was discovered in the DP400DL300 group. Overall, for adult triploid rainbow trout (≥3 kg), the minimum recommended DP and DL according to growth performance were 400 and 250 g kg-1, respectively; DP and DL based on feed utilization were 350 and 200 g kg-1, respectively; DP and DL based on fillet quality were 400 and 300 g kg-1, respectively.

[Mechanism of Gegen Qinlian Decoction in improving glucose metabolism in vitro and in vivo by alleviating hepatic endoplasmic reticulum stress].

This study investigated the mechanism of Gegen Qinlian Decoction(GQD) in improving glucose metabolism in vitro and in vivo by alleviating endoplasmic reticulum stress(ERS). Molecular docking was used to predict the binding affinity between the main effective plasma components of GQD and ERS-related targets. Liver tissue samples were obtained from normal rats, high-fat-induced diabetic rats, rats treated with metformin, and rats treated with GQD. RNA and protein were extracted. qPCR was used to measure the mRNA expression of ERS marker glucose-regulated protein 78(GRP78), and unfolded protein response(UPR) genes inositol requiring enzyme 1(Ire1), activating transcription factor 6(Atf6), Atf4, C/EBP-homologous protein(Chop), and caspase-12. Western blot was used to detect the protein expression of GRP78, IRE1, protein kinase R-like ER kinase(PERK), ATF6, X-box binding protein 1(XBP1), ATF4, CHOP, caspase-12, caspase-9, and caspase-3. The calcium ion content in liver tissues was determined by the colorimetric assay. The ERS-HepG2 cell model was established in vitro by inducing with tunicamycin for 6 hours, and 2.5%, 5%, and 10% GQD-containing serum were administered for 9 hours. The glucose oxidase method was used to measure extracellular glucose levels, flow cytometry to detect cell apoptosis, glycogen staining to measure cellular glycogen content, and immunofluorescence to detect the expression of GRP78. The intracellular calcium ion content was measured by the colorimetric assay. Whereas Western blot was used to detect GRP78 and ERS-induced IRE1, PERK, ATF6, and eukaryotic translation initiation factor 2α(eIF2α) phosphorylation. Additionally, the phosphorylation levels of insulin receptor substrate 1(IRS1), phosphatidylinositol 3-kinase regulatory subunit p85(PI3Kp85), and protein kinase B(Akt), which were involved in the insulin signaling pathway, were also measured. In addition, the phosphorylation levels of c-Jun N-terminal kinases(JNKs), which were involved in both the ERS and insulin signaling pathways, were measured by Western blot. Molecular docking results showed that GRP78, IRE1, PERK, ATF4, and various compounds such as baicalein, berberine, daidzein, jateorhizine, liquiritin, palmatine, puerarin and wogonoside had strong binding affinities, indicating that GQD might interfere with ERS-induced UPR. In vivo results showed that GQD down-regulated the mRNA transcription of Ire1, Atf6, Atf4, Grp78, caspase-12, and Chop in diabetic rats, and down-regulated GRP78, IRE1, PERK, as well as ERS-induced apoptotic factors ATF4 and CHOP, caspase-12, caspase-9, and caspase-3, while up-regulating XBP1 to enhance adaptive UPR. In addition, GQD increased the calcium ion content in liver tissues, which facilitated correct protein folding. In vitro results showed that GQD increased glucose consumption in ERS-induced HepG2 cells without significantly affecting cell viability, increased liver glycogen synthesis, down-regulated ATF6 and p-eIF2α(Ser51), and down-regulated IRE1, PERK, and GRP78, as well as p-IRS1(Ser312) and p-JNKs(Thr183/Tyr185), while up-regulating p-PI3Kp85(Tyr607) and p-Akt(Ser473). These findings suggested that GQD alleviates excessive ERS in the liver, reduces insulin resistance, and improves hepatic glucose metabolism in vivo and in vitro.

A New Design of the Dual-Mode and Pure Longitudinal EMAT by Using a Radial-Flux-Focusing Magnet.

An electromagnetic acoustic transducer (EMAT) is suitable for measuring the propagation time more accurately without causing abrasion to the transducer during testing due to the principle of its excitation. This work designs a flux-concentrating EMAT with a radial-flux-focusing permanent magnet to significantly enhance static magnetic field strength. Through theoretical analysis and finite element simulation, two kinds of coils are designed according to the concentration areas of the horizontal and vertical components of the magnetic field. One is used to generate pure longitudinal waves, and the other is used to generate both longitudinal waves and shear waves. The experimental comparison shows that the amplitudes of the pure longitudinal wave and the dual-mode wave excited by the two kinds of coils with the radial-flux-focusing magnet are more than two times higher than those with the ordinary magnet. Therefore, the flux-concentrating EMAT with the appropriate coil provides an insight into realizing more accurate detection where longitudinal wave detection is required.

[Anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol against H_2O_2-induced oxidative damage in pancreatic ß cells (INS-1 cells)].

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic ß-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 µmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 µmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 µmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 µmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 µmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 µmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic ß cells.PDX-1 is a key nuclear transcription factor of pancreatic ß cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic ß cells, and improve insulin synthesis and secretion.

Targeted Gene Insertion and Replacement in the Basidiomycete Ganoderma lucidum by Inactivation of Nonhomologous End Joining Using CRISPR/Cas9.

Targeted gene insertion or replacement is a promising genome-editing tool for molecular breeding and gene engineering. Although CRISPR/Cas9 works well for gene disruption and deletion in Ganoderma lucidum, targeted gene insertion and replacement remain a serious challenge due to the low efficiency of homologous recombination (HR) in this species. In this work, we demonstrate that the DNA double-strand breaks induced by Cas9 were mainly repaired via the nonhomologous end joining (NHEJ) pathway, at a frequency of 96.7%. To establish an efficient target gene insertion and replacement tool in Ganoderma, we first inactivated the NHEJ pathway via disruption of the Ku70 gene (ku70) using a dual single guide RNA (sgRNA)-directed gene deletion method. Disruption of the ku70 gene significantly decreased NHEJ activity in G. lucidum. Moreover, ku70 disruption strains exhibited 96.3% and 93.1% frequencies of targeted gene insertion and replacement, respectively, when target DNA with the orotidine 5'-monophosphate decarboxylase (ura3) gene and 1.5-kb homologous 5'- and 3'-flanking sequences was used as a donor template, compared to 3.3% and 0%, respectively, at these targeted sites for a control strain (Cas9 strain). Our results indicated that ku70 disruption strains were efficient recipients for targeted gene insertion and replacement. This tool will advance our understanding of functional genomics in G. lucidum. IMPORTANCE Functional genomic studies in Ganoderma have been hindered by the absence of adequate genome-engineering tools. Although CRISPR/Cas9 works well for gene disruption and deletion in G. lucidum, targeted gene insertion and replacement have remained a serious challenge due to the low efficiency of HR in these species, although such precise genome modifications, including site mutations, site-specific integrations, and allele or promoter replacements, would be incredibly valuable. In this work, we inactivated the NHEJ repair mechanism in G. lucidum by disrupting the ku70 gene using the CRISPR/Cas9 system. Moreover, we established a target gene insertion and replacement method in ku70-disrupted G. lucidum that possessed high-efficiency gene targeting. This technology will advance our understanding of the functional genomics of G. lucidum.

Alcohol Consumption and Stroke Risk in Men: A Population-Based Cohort Study in Rural Tianjin, China.

BACKGROUND: Although the protective effects of alcohol consumption against future cardiovascular disease have been published, the effects of alcohol on stroke risk remain controversial. METHOD: We assessed the effects of alcohol consumption on stroke risk in a poorly educated, low-income population in rural China. Between 1991 and 2018, a population-based cohort study was conducted in rural Tianjin, China, to examine stroke risk. All registered stroke events were clinically verified using available computed tomography or MRI scans. The stroke risk was analyzed, according to the extent of alcohol consumption, using Cox regression analyses. RESULTS: We identified 352 incident stroke events among male participants during the study period. The stroke incidences (per 100,000 person-years) were 965.3 overall, 575.9 for ischemic stroke events, 208.4 for hemorrhagic stroke events, and 181.0 for undefined stroke events. Overall, alcohol consumption provided a 32% reduction in the total stroke risk. Low-dose alcohol consumption (≤12 g/day) showed a negative association with total (hazard ratio [HR], 0.64; 95% confidence interval [CI], 0.46-0.88; p = 0.008) and ischemic (HR, 0.66; 95% CI, 0.44-0.98; p = 0.039) strokes. Alcohol consumption was not significantly associated with hemorrhagic strokes. After age stratification, alcohol consumption was protective against total and ischemic strokes in men aged ≥55 years old, with the risk of each stroke type decreasing by 46 and 49%, respectively. Low-dose alcohol consumption was inversely associated with both total and ischemic stroke risks, with the risks decreasing by 56 and 65%, respectively. Alcohol consumption was not significantly associated with strokes among men aged <55 years old. CONCLUSIONS: These findings suggest that low-dose alcohol consumption may decrease the risk of ischemic strokes among men. Even so, the adverse effects of alcohol on the liver and pancreas cannot be ignored. Additionally, the effects of alcohol consumption on stroke risk vary with age, protecting against ischemic and total strokes among males ≥55 years old. Nevertheless, recommending light drinking and its potential health benefits should not be generalized to men of all ages.

[Molecular mechanism of Gegen Qinlian Decoction in promoting differentiation of brown adipose tissue to improve glucose and lipid metabolism disorders in diabetic rats].

This study explored the molecular mechanism underlying the Gegen Qinlian Decoction(GQD) promoting the differentiation of brown adipose tissue(BAT) to improve glucose and lipid metabolism disorders in diabetic rats. After the hypoglycemic effect of GQD on diabetic rats induced by high-fat diet combined with a low dose of streptozotocin was confirmed, the total RNA of rat BAT around scapula was extracted. Nuclear transcription genes Prdm16, Pparγc1α, Pparα, Pparγ and Sirt1, BAT marker genes Ucp1, Cidea and Dio2, energy expenditure gene Ampkα2 as well as BAT secretion factors Adpn, Fndc5, Angptl8, IL-6 and Rbp4 were detected by qPCR, then were analyzed by IPA software. Afterward, the total protein from rat BAT was extracted, and PRDM16, PGC1α, PPARγ, PPARα, SIRT1, ChREBP, AMPKα, UCP1, ADPN, NRG4, GLUT1 and GLUT4 were detected by Western blot. The mRNA expression levels of Pparγc1α, Pparα, Pparγ, Ucp1, Cidea, Ampkα2, Dio2, Fndc5, Rbp4 and Angptl8 were significantly increased(P<0.05) and those of Adpn and IL-6 were significantly decreased(P<0.05) in the GQD group compared with the diabetic group. In addition, Sirt1 showed a downward trend(P=0.104), whereas Prdm16 tended to be up-regulated(P=0.182) in the GQD group. IPA canonical pathway analysis and diseases-and-functions analysis suggested that GQD activated PPARα/RXRα and SIRT1 signaling pathways to promote the differentiation of BAT and reduce the excessive lipid accumulation. Moreover, the protein expression levels of PRDM16, PGC1α, PPARα, PPARγ, SIRT1, ChREBP, AMPKα, UCP1, GLUT1, GLUT4 and NRG4 were significantly decreased in the diabetic group(P<0.01), which were elevated after GQD intervention(P<0.05). Unexpectedly, the expression of ADPN protein in the diabetic group was up-regulated(P<0.01) as compared with the control group, which was down-regulated after the administration with GQD(P<0.01). This study indicated that GQD promoted BAT differentiation and maturity to increase energy consumption, which reduced the glucose and lipid metabolism disorders and thereby improved diabetes symptoms.

Extracellular vesicle-enclosed miR-486-5p mediates wound healing with adipose-derived stem cells by promoting angiogenesis.

Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing.