Inhibition of VDAC1 Rescues Aß 1-42-Induced Mitochondrial Dysfunction and Ferroptosis via Activation of AMPK and Wnt/ß-Catenin Pathways.

Beta-amyloid (Aß) accumulation in the brains of Alzheimer's disease (AD) patients leads to mitochondrial dysfunction and ferroptosis in neurons. Voltage-dependent anion channel 1 (VDAC1) is a major protein in the mitochondrial outer membrane. It has been reported that VDAC1 associated with mitochondrial dysfunction and ferroptosis. However, the mechanism by which VDAC1 regulates mitochondrial dysfunction and ferroptosis of neurons in AD remains unclear. This study is aimed at investigating the mechanism of action of VDAC1 in mitochondrial dysfunction and ferroptosis in neurons of the AD model. In this study, we determined cell viability after treatment with Aß 1-42 via the MTT assay. The SOD, MDA, ROS, and MMP production was measured via the SOD kit, MDA kit, DCFDA staining, and JC-1 staining. The memory abilities of mice were detected via the Morris water maze test. The expression of AMPK/mTOR, Wnt/ß-catenin, and GPX4 regulated by VDAC1 was detected via western blotting. Our present study showed that PC12 cells had decreased cell viability, increased LDH release, and decreased GPX4 expression after Aß 1-42 treatment. Meanwhile, Aß 1-42 induced MMP and SOD downregulation and increased MDA and ROS generation in PC12 cells. In addition, the expression of VDAC1 is increased in the brain tissue of AD mice and Aß 1-42-treated PC12 cells. Further investigation of the role of VDAC1 in regulating AD found that all effects induced by Aß 1-42 were reversed by inhibition of VDAC1. Additionally, inhibition of VDAC1 activates the AMPK/mTOR and Wnt/ß-catenin pathways. Taken together, these findings demonstrate that inhibition of VDAC1 alleviates mitochondrial dysfunction and ferroptosis in AD neurons by activating AMPK/mTOR and Wnt/ß-catenin.

Maternal separation affects anxiety-like behavior beginning in adolescence and continuing through adulthood and related to Dnmt3a expression.

Early life stress, including maternal separation, is among one of the main causes of anxiety in adolescents. DNA methyltransferase 3A (Dnmt3a) is a key molecule that regulates DNA methylation and is found to be associated with anxiety-like behavior. It is not clear whether maternal separation affects anxiety levels in mice at different developmental stages or whether Dnmt3a plays a role in this process. Here, by using the open field test to explore the effect of maternal separation on anxiety-like behavior in mice of different ages, it was found that maternal separation could successfully induce anxiety-like behavior in adolescent mice, which continued through adulthood. By using Western blot, we found that the levels of Dnmt3a in the hippocampus and cortex showed different trends in maternal separation mice on postnatal day (P)17. Furthermore, by using immunostaining, we found that the expression levels of Dnmt3a in the cortex and hippocampus were significantly different and decreased to varying degrees with the age of mice, which was the reason for different trends. Our results provide an experimental basis for further development of anxiety/depression treatment programs more suitable for adolescence.NEW & NOTEWORTHY Most anxiety disorders begin in adolescence and continue through adulthood, and research on adolescent anxiety's pathogenesis and treatment options is insufficient. In this research, our results show that maternal separation can successfully induce anxiety-like behavior in adolescent mice that continues through adulthood, further accompanied by abnormal expression of Dnmt3a, which provides an experimental basis for further development of anxiety/depression treatment programs more suitable for adolescence.

[Study on immunogenicity elicited by a recombinant vaccine of rBCG-Rv3133c to fight against dormancy Mycobacterium tuberculosis].

To obtain a vaccine to defend from dormancy Mycobacterium tuberculosis, we constructed the recombinant Bacilli Calmette-Guérin (BCG) vaccine with Rv3133c encoding dormancy-correlated transcriptional regulatory protein DosR in Mycobacterium tuberculosis as a target gene, and evaluated its immunogenicity in BALB/c mice. In this study, we constructed the recombinant plasmids of rpMV361-Rv3133c using gene colon technology. We then transformed BCG strains with above-mentioned plasmids to obtain recombinant vaccine of rBCG-Rv3133c. We used the rBCG strains successfully constructed to vaccinate in BALB/c mice. 30d and 180d after immunization, the specific antibody titers were determined to investigate humoral responses induced by recombinant vaccine. We detected changes of splenocyte subsets of CD4+T, CD8+ T cells and cytokine of IFN-gamma secreted by splenocytes for evaluation of cellular immune responses. The results showed that the rBCG-Rv3133c was able to induce higher levels of antibody titer, stronger proliferative responses and higher IFN-gamma production comparing with BCG vaccine. The results also suggested that this recombinant vaccine was a more efficacious tuberculosis vaccine for further study.

Neuronal activity in the dorsal dentate gyrus during extinction regulates fear memory extinction and renewal.

Memory extinction and renewal are major factors that limits the efficacy of exposure therapy. The dorsal dentate gyrus (dDG) plays a crucial role in spatial memory, and epigenetic modifications in the dDG play an important role in fear memory renewal. However, whether dDG activity regulates fear memory extinction and renewal remains unclear. In this study, we showed that an extinction procedure that prevents fear memory renewal (extinction within the reconsolidation window) leads to increased c-fos expression in the dDG. Chemicogenetic activation of dDG excitatory neurons during extinction training elevated fear memory extinction and prevented renewal, whereas inhibition of dDG excitatory neurons inhibited fear memory extinction. We also demonstrated that inhibiting fear engram cells (neurons active during fear acquisition) during extinction training inhibits fear memory extinction. Therefore, dDG activity during fear extinction plays an important role in fear memory extinction and renewal.

Meta analysis of aerobic exercise improving intelligence and cognitive function in patients with Alzheimer's disease.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disease. This study aims to explore the intervention and treatment effects of aerobic exercise and different exercise modes on AD through meta-analysis. METHODS: Using the set inclusion and exclusion criteria, retrieve the China national knowledge infrastructure (CNKI), Wanfang Data Knowledge Service Platform, China Science and Technology Journal Database, Cochrane Library, and PubMed were searched from January 1, 2012, to December 31, 2021. Cochrane risk bias assessment tool was used to evaluate the quality of the included articles, and ReMan5.4.1 was used for forest plot analysis of mini-mental state exam (MMSE) score indicators included in the included articles. RESULTS: Twelve randomized controlled trials and 795 samples were included. Meta analysis of all articles: I2 = 91%, P ≤ .00001, (MD = 2.95, 95%CI [2.49, 3.40], P ≤ .00001). Meta analysis of 5 fit aerobics groups: I2 = 4%, P = .38, (MD = 1.53, 95%CI [0.72, 2.33], P = .0002); meta-analysis of three spinning groups: I2 = 3%, P = .36, (MD = 1.79, 95%CI [0.29, 3.29], P = .02). CONCLUSION: Aerobic exercise can effectively improve intellectual and cognitive impairment in AD patients, and for different forms of aerobic exercise, the therapeutic effect of spinning aerobic exercise is better than that of fit aerobics.

EphA2 as a new target for breast cancer and its potential clinical application.

PURPOSE: The aim of this research was to study the expression of EphA2 to assess its suitability as a new breast cancer target. METHODS: Immunohistochemistry (IHC) was used to detect EphA2 protein expression in pathology tissue samples from 250 cases of breast cancer, and the expression of EphA2 mRNA was detected by in situ hybridization (ISH). Breast cancer cells were isolated and cultured. The expression of EphA2 in the cells was detected by the indirect immunofluorescence assay (IFA), and the expression of EphA2 in breast cancer was analysed. RESULTS: EphA2 protein and mRNA were mainly expressed in tumor cells and vascular endothelial cells. EphA2 protein was expressed in 187 cases, with a positive rate of 74.80%, whereas EphA2 mRNA was expressed in 209 cases, with a positive rate of 83.60%. EphA2 protein and mRNA expression were correlated with lymph node metastasis, clinical stage, and breast cancer histologic grade (P<0.05). In addition, the positive expression rates of EphA2 protein and EphA2 mRNA were correlated (P<0.05). EphA2 was barely expressed in normal breast cells but highly expressed in breast cancer cells. CONCLUSION: EphA2 is highly expressed in breast cancer tissues and has the potential to be a new breast cancer target, providing a preliminary basis for the development of new targeted drugs for breast cancer and the construction of fluorescent-targeted tracers for fluorescence-guided mastoscopic breast-conserving surgery.

Anatomical variations of the upper thoracic sympathetic chain.

The aim is to clearly delineate the upper thoracic sympathetic chains and neural connections between the chains and ventral rami of the thoracic nerves, and to provide an anatomical foundation for successful upper thoracic sympathicotomy for treating upper essential hyperhidrosis. The upper thoracic sympathetic chains, upper five intercostal nerves, and neural connections between them in 50 halves of 25 adult cadavers have been dissected, measured, and mapped. The stellate ganglion had an incidence of 80%. The second to the fourth thoracic sympathetic ganglia were commonly located in the corresponding intercostal spaces with the presence of 92%, 68%, and 50%, respectively. The incidence of the first and second intercostal rami was 40% and 6%, and that of the ascending or descending rami from the second, third and fourth ganglia was 54%, 24%, and 14%, respectively. Additional rami communicantes joined the ventral ramus of the 1st thoracic nerve proximal to the point where the latter gave a branch to the brachial plexus. The farthest horizontal distance from the sympathetic chain to the junction between the additional rami communicantes and the second to the fourth intercostal nerves was 29.1 mm. Only 16% of cadavers had similar anatomy bilaterally. Anatomical variations of the upper thoracic sympathetic trunk in relation to intercostal nerves, which may be one of the causes resulting in surgical failures and recurrences, were striking. Attention should be given to such anatomical variations when planning thoracic sympathicotomy.

Cytotoxic effect of Ad-ephrinA1-caspase-3-T on breast cancer cells in vitro.

BACKGROUND: Research whether the Ad-ephrinA1-caspase-3-T has a targeted cytotoxic effect on breast cancer cells in vitro. METHODS: First, the breast cancer cells were isolated, cultured and identified. The Ad-ephrinA1-caspase-3-T was used to infect human breast cells, and detect the effect that Ad-ephrinA1-caspase-3-T made on the viability of breast cancer cells with EphA2 positive by MTT. The effect of Ad-ephrinA1-caspase-3-T on the apoptosis of breast cancer cells was detected by flow cytometer. Finally, the specific cytotoxic effect of Ad-ephrinA1-caspase-3-T on target cells was examined by antibody neutralization experiments. RESULTS: Breast cancer cells showed adherent growth, and high levels of EphA2 receptor and ephrinA1 expression were seen in breast cancer cells. Ad-ephrinA1-caspase-3-T showed a strong cytotoxic effect on breast cancer cells, and the growth inhibition rate was 42.3%. After culture for 72 h, the apoptosis rates corresponding to the dilution ratios of 1, 2, 4, 8 and 16 were 36%, 24%, 18%, 8%, and 7%, respectively, which were significantly higher than those of the control group, whose apoptosis rate was 3.5%. Neutralizing antibody could effectively reduce the cytotoxic effect of Ad-ephrinA1-caspase-3-T on EphA2 positive breast cancer cells. CONCLUSIONS: The Ad-ephrinA1-caspase-3-T has a targeted cytotoxic effect on breast cancer cells in vitro.

Epidemiology and phylogeny of spike gene of porcine epidemic diarrhea virus from Yunnan, China.

Porcine epidemic diarrhea (PED) causes acute enteric disease and yellowish watery diarrhea, making piglets fast dehydration to death. PED threatens pig industry and leads to substantial economic losses. After the first reports, PED in Yunnan province, China was again identified in 2013 during an epidemiological survey, with follow-up data showing an overall positive rate of 17.47% during 2013-2017, lower than that in other provinces in China. The complete S gene of porcine epidemic diarrhea virus (PEDV) is 4149-4158 bp long. Phylogenetic analysis of S gene was performed using 9 new isolates from Yunnan province, China, together with 225 full-length S genes available in GenBank. The nine Yunnan isolates were clustered into classical G1b and pandemic G2a groups, indicating new variants have been emerging in Yunnan province. When taking the previously submitted 3 isolates from China into consideration, all the 12 isolates were clustered into 4 groups, i.e., G1a, G1b, G2a and G2b, suggesting that a highly diverse and complex clustering might result from co-infections in more than 13 provinces in China, as well in South Korea, Japan, Vietnam, Thai and USA. Identification of new types of PEDV strains would stimulate the development of effective vaccines for the prevention and control of PED in a more precise manner.

[Isolating and culturing rat marrow mesenchymal stem cells and studying their phenotypical and functional properties].

OBJECTIVE: To establish a method for isolating and cultivating mesenchymal stem cells (MSCs) from SD rat bone marrow and to study their phenotypical and functional properties. METHODS: MSCs from rat bone marrow were separated and purified by gradient centrifugation and adherence to the culture plastic; then the cells were expanded by subculture successively. The growth curves were drawn, and the morphology was observed. In an attempt to analyze immuno- and adhesive-phenotype and differentiation properties, the MSCs were evaluated with cytochemical and immunocytochemical methods. RESULTS: MSCs belonged to the mononuclear cells of marrow, they could be isolated and purified by gradient centrifugation and adherence to the culture plastic; their living behavior was quite stable in Dulbecco's Modified Eagle's Medium with low glucose (L-DMEM) containing 100 ml/L newborn bovine serum; the growth curves of passage 1, 3 and 5 were much similar, exhibiting a 2.2-fold increase in cell number after each passage. The cells were noted to have a large expansive potential and a typical fibroblast-like morphology, and they uniformly expressed CD44, CD54, fibronectin and collagen I. When incubated in medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, the MSCs underwent differentiation into osteoblasts, showing positive stain of alkaline phosphatase activity and mineralized nodules. CONCLUSION: The cells obtained in this experiment possess phenotypical and functional properties of mesenchymal stem cells, and the method for isolating and culturing rat marrow-derived MSCs has been established.

Evaluation of protective efficacy conferred by a recombinant Mycobacterium bovis BCG expressing a fusion protein of Ag85A-ESAT-6.

BACKGROUND: We previously constructed a recombinant bacille Calmette-Guérin (rBCG-AE) strain that could express a fused Ag85A-ESAT-6 protein. That study suggested that the rBCG-AE strain was able to induce a higher titer of antibody and elicit a more long-lived and stronger Th1-type cellular immune responses than the parental BCG strain, the rBCG-A strain (i.e., expressing Ag85A), or the rBCG-E strain (i.e., expressing ESAT-6). METHODS: In the current study, we further investigated the strain's protective efficacy against Mycobacterium tuberculosis H37Rv infection in BALB/c mice through evaluating organ bacterial loads, lung histopathology, lung immunohistochemistry, and net weight gain or loss by using conventional BCG, rBCG-A, and rBCG-E as the controls. RESULTS: From the 3rd to 9th weeks after the challenge infection, the bacterial counts were significantly lower in tissues (e.g., spleen and lung tissues) in the mice immunized with rBCG-AE than in the control group, but were higher than the counts in the BCG group. The pathological damage in the lung tissues of the rBCG-AE group gradually improved from the 6th to 9th weeks after being infected with M. tuberculosis H37Rv, but the score of pathological changes in the rBCG-AE group was obviously higher than the score in the BCG group. There was no difference in the percentage of IFN-γ and iNOS positive cells in the lung tissues of the rBCG-AE and BCG groups. CONCLUSION: The results suggest that rBCG-AE can not promote protective efficacy against M. tuberculosis H37Rv infection, compared to the BCG vaccine.