Focal ischemic stroke modifies microglia-derived exosomal miRNAs: potential role of mir-212-5p in neuronal protection and functional recovery.

BACKGROUND: Ischemic stroke is a severe type of stroke with high disability and mortality rates. In recent years, microglial exosome-derived miRNAs have been shown to be promising candidates for the treatment of ischemic brain injury and exert neuroprotective effects. Mechanisms underlying miRNA dysregulation in ischemic stroke are still being explored. Here, we aimed to verify whether miRNAs derived from exosomes exert effects on functional recovery. METHODS: MiR-212-5p agomir was employed to upregulate miR-212-5p expression in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Western blot analysis, qRT-PCR and immunofluorescence staining and other methods were applied to explore the underlying mechanisms of action of miR-212-5p. RESULTS: The results of our study found that intervention with miR-212-5p agomir effectively decreased infarct volume and restored motor function in MCAO/R rats. Mechanistically, miR-212-5p agomir significantly reduced the expression of PlexinA2 (PLXNA2). Additionally, the results obtained in vitro were similar to those achieved in vivo. CONCLUSION: In conclusion, the present study indicated that PLXNA2 may be a target gene of miR-212-5p, and miR-212-5p has great potential as a target for the treatment and diagnosis of ischemic stroke.

Mucin1 induced trophoblast dysfunction in gestational diabetes mellitus via Wnt/ß-catenin pathway.

BACKGROUND: To elucidate the role of Mucin1 (MUC1) in the trophoblast function (glucose uptake and apoptosis) of gestational diabetes mellitus (GDM) women through the Wnt/ß-catenin pathway. METHODS: Glucose uptake was analyzed by plasma GLUT1 and GLUT4 levels with ELISA and measured by the expression of GLUT4 and INSR with immunofluorescence and Western blotting. Apoptosis was measured by the expression of Bcl-2 and Caspase3 by Western blotting and flow cytometry. Wnt/ß-catenin signaling measured by Western blotting. In vitro studies were performed using HTR-8/SVneo cells that were cultured and treated with high glucose (HG), sh-MUC1 and FH535 (inhibitor of Wnt/ß-catenin signaling). RESULTS: MUC1 was highly expressed in the placental trophoblasts of GDM, and the Wnt/ß-catenin pathway was activated, along with dysfunction of glucose uptake and apoptosis. MUC1 knockdown resulted in increased invasiveness and decreased apoptosis in trophoblast cells. The initial linkage between MUC1, the Wnt/ß-catenin pathway, and glucose uptake was confirmed by using an HG-exposed HTR-8/SVneo cell model with MUC1 knockdown. MUC1 knockdown inhibited the Wnt/ß-catenin signaling pathway and reversed glucose uptake dysfunction and apoptosis in HG-induced HTR-8/SVneo cells. Meanwhile, inhibition of Wnt/ß-catenin signaling could also reverse the dysfunction of glucose uptake and apoptosis. CONCLUSIONS: In summary, the increased level of MUC1 in GDM could abnormally activate the Wnt/ß-catenin signaling pathway, leading to trophoblast dysfunction, which may impair glucose uptake and induce apoptosis in placental tissues of GDM women.

Role and mechanism of miR-548-3p/ DAG1 in the occurrence and malignant transformation of laryngeal carcinoma / Papel y mecanismo de miR-548-3p/DAG1 en la aparición y transformación maligna del carcinoma de laringe

Abstract The AMC-HN-8 cell line and the primary human laryngeal epithelial cell lines were utilized in this work to explore the molecular mechanism of miR-548-3p regulating the gene DAG1 to induce the occurrence and malignant transformation of laryngeal carcinoma. Non-coding RNA miR-548- 3p overexpression plasmid, interference plasmid and blank plasmid were constructed, and the plasmids were transfected into AMC-HN-8 cells, respectively. Meanwhile, a non-transfected plasmid group and a human laryngeal epithelial primary cell group were set up. Five groups of cells were named as NC (Normal control), Model, Ov-miR-548-3p, Sh-miR-548-3p and Blank-plasmid group. The luciferase reporter experiment was used to analyze the regulation characteristics of hsa-miR-548-3p on dystrophin-associated glycoprotein 1 (DAG1). Immunofluorescence was used to analyze the relative expression characteristics of the protein DAG1. The cell cloning experiment was used to analyze the proliferation characteristics of AMC-HN-8. The scratch healing test was used to analyze the migration ability of AMC-HN-8. The transwell test was used to analyze the invasion ability of AMC-HN-8. The RT-PCR was used to analyze the expression level of miR-548-3p. Western blot experiments were used to analyze the expression of protein DAG1, laminin α2 (LAMA2) and utrophin (UTRN). The luciferase report experiment and immunofluorescence test found that the expression of DAG1 and miR-548-3p are positively correlated. Cell cloning, scratching and migration experiments identified that the activity of laryngeal cancer cells was positively correlated with the expression of DAG1. The results of Western blot analysis further strengthened the above conclusions. Through carrying out research on the cellular levels, our work has demonstrated that miR-548-3p regulated the content of protein DAG1, and then further induced malignant transformation of laryngeal carcinoma.

Resumen En este trabajo se utilizaron la línea celular AMC-HN-8 y la línea celular epitelial laríngea humana primaria, para explorar el mecanismo molecular regulador del miR-548-3p sobre el gen DAG1 para inducir la aparición y la transformación maligna del carcinoma laríngeo. Se construyeron el plásmido de sobreexpresión de miR-548-3p de ARN no codificante, el plásmido de interferencia y el plásmido en blanco, y los plásmidos se transfectaron en células AMCHN-8 respectivamente. Mientras tanto, se establecieron un grupo de plásmidos no transfectados y un grupo de células primarias epiteliales laríngeas humanas. Se nombraron cinco grupos de células como NC (control normal), modelo, OvmiR-548-3p, Sh-miR-548-3p y grupo de plásmido en blanco. El experimento indicador de luciferasa se utilizó para analizar las características de regulación de hsa-miR-548-3p en la glicoproteína 1 asociada a distrofina (DAG1). Se utilizó inmunofluorescencia para analizar las características de expresión relativa de la proteína DAG1. El experimento de clonación celular se utilizó para analizar las características de proliferación de AMC-HN-8. Se utilizó la prueba de cicatrización por rascado para analizar la capacidad de migración de AMC-HN-8. La prueba de transwell se utilizó para analizar la capacidad de invasión de AMCHN-8. Se utilizó RT-PCR para analizar el nivel de expresión de miR-548-3p. Se usó un experimento de transferencia Western (Western blot) para analizar las expresiones de la proteína DAG1, laminina α2 (LAMA2) y utrofina (UTRN). El experimento de reporte de luciferasa y la prueba de inmunofluorescencia encontraron que la expresión de DAG1 y miR-548-3p están positivamente correlacionadas. Los experimentos de clonación celular, rascado y migración, identificaron que la actividad de las células cancerosas de laringe se correlacionó positivamente con la expresión de DAG1. Los resultados del análisis de transferencia Western fortalecieron aún más las conclusiones anteriores. A través de la investigación a nivel celular, nuestro proyecto ha demostrado que miR-548-3p regula el contenido de la proteína DAG1 y luego induce la transformación maligna del carcinoma de laringe.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma.

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Does dexmedetomidine as a neuraxial adjuvant facilitate better anesthesia and analgesia? A systematic review and meta-analysis.

BACKGROUND: Neuraxial application of dexmedetomidine (DEX) as adjuvant analgesic has been invetigated in some randomized controlled trials (RCTs) but not been approved because of the inconsistency of efficacy and safety in these RCTs. We performed this meta-analysis to access the efficacy and safety of neuraxial DEX as local anaesthetic (LA) adjuvant. METHODS: We searched PubMed, PsycINFO, Scopus, EMBASE, and CENTRAL databases from inception to June 2013 for RCTs that investigated the analgesia efficacy and safety for neuraxial application DEX as LA adjuvant. Effects were summarized using standardized mean differences (SMDs), weighed mean differences (WMDs) or odds ratio (OR) with suitable effect model. The primary outcomes were postoperative pain intensity and analgesic duration, bradycardia and hypotension. RESULTS: Sixteen RCTs involving 1092 participants were included. Neuraxial DEX significantly decreased postoperative pain intensity (SMD, -1.29; 95% confidence interval (CI), -1.70 to -0.89; P<0.00001), prolonged analgesic duration (WMD, 6.93 hours; 95% CI, 5.23 to 8.62; P<0.00001) and increased the risk of bradycardia (OR, 2.68; 95% CI, 1.18 to 6.10; P = 0.02). No evidence showed that neuraxial DEX increased the risk of other adverse events, such as hypotension (OR, 1.54; 95% CI, 0.83 to 2.85; P = 0.17). Additionally, neuraxial DEX was associated with beneficial alterations in postoperative sedation scores and number of analgesic requirements, sensory and motor block characteristics, and intro-operative hemodynamics. CONCLUSION: Neuraxial DEX is a favorable LA adjuvant with better and longer analgesia. The greatest concern is bradycardia. Further large sample trials with strict design and focusing on long-term outcomes are needed.

Human testis-expressed sequence 101 is limitedly distributed in germinal epithelium of testis and disappears in seminoma

BACKGROUND: Testis-expressed sequence 101 (TEX101) was found to be highly expressed in testis and involved in acrosome reaction in previous studies. Recently, the metastasis suppressor function of TEX101 in cancer was disclosed, but the comprehensive investigation of its expression has rarely been reported. In this study, the expression features of TEX101 in normal human organs and seminoma were systematically analyzed. RESULTS: Immunohistochemistry demonstrated intense staining of TEX101 in human testis tissues; however, its expression in 27 other types of normal human organs, including the ovary, was negligible. Higher expression of TEX101 was observed in the spermatocytes and spermatids of the testis, but relatively lower staining was detected in spermatogonia. Western blotting showed a single TEX101 band of 38 kDa in human testis, but it did not correspond to the predicted molecular weight of its mature form at 21 KDa. Furthermore, we examined seminoma tissues by immunohistochemistry and found that none of the 36 samples expressed TEX101. CONCLUSIONS: Our data confirmed TEX101 to be a testis protein that could be related to the maturation process of male germ cells. The lack of TEX101 in seminoma indicated its potential role in tumor progression. This characteristic expression of TEX101 could provide a valuable reference for understanding its biological functions.

Association between AA-NAT gene polymorphism and reproductive performance in sheep

Arylalkylamine N-acetyltransferase (AA-NAT) is critical enzyme in Melatonin (MLT) biosynthesis for MLT regulating the animal seasonal breeding. In this study, DNA sequencing methods were applied to detect the polymorphisms of the AA-NAT gene in 179 Chinese sheep belonging to two non-seasonal reproduction breeds and two seasonal reproduction breeds. One mutation at exon 3 (NM_001009461:c.486A > G) was firstly described at the sheep AA-NAT locus. Hence, we described the SmaI PCR-RFLP method for detecting EX3 486A > G mutation, frequencies of the AA-NAT-G allele varied from 0.871 to 0.908 in two non-seasonal reproduction breeds and 0.517 to 0.578 in two seasonal reproduction breeds. The associations of SmaI polymorphism with estrus traits was analyzed in non-seasonal reproduction breeds sheep and seasonal reproduction breeds sheep, the significant statistical results were found between them, the GG genotype frequencies was higher in non-seasonal reproduction breeds (p < 0.001), while, the GA genotype frequencies was higher in seasonal reproduction breeds (p < 0.05). Hence, the EX3 486A > G mutation could facilitate association analysis and serve as a genetic marker for Chinese sheep breeding and genetics.