Quantitative phosphoproteomics explain cryopreservation-induced reductions in ram sperm motility.

Sperm cryopreservation decreases motility, probably due to changes in protein phosphorylation. Our objective was to use quantitative phosphoproteomics for systematic comparative analyses of fresh versus frozen-thawed sperm to identify factors causing cryo-injury. Ejaculates were collected (artificial vagina) from six Dorper rams, pooled, extended, and frozen over liquid nitrogen. Overall, 915, 3382, and 6875 phosphorylated proteins, phosphorylated peptides, and phosphorylation sites, respectively, were identified. At least two modified sites were present in 57.94% of the 6875 phosphosites identified, of which AKAP4 protein contained up to 331 modified sites. There were 732 phosphorylated peptides significantly up-regulated and 909 significantly down-regulated in frozen-thawed versus fresh sperm. Moreover, the conserved motif [RxxS] was significantly down-regulated in frozen-thawed sperm. Phosphorylation of sperm-specific proteins, e.g., AKAP3/4, CABYR, FSIP2, GSK3A/B, GPI, and ODF1/2 make them potential biomarkers to assess the quality of frozen-thawed ram sperm. Furthermore, these differentially phosphorylated proteins and modification sites were implicated in cryopreservation-induced changes in sperm energy production, fiber sheath composition, and various biological processes. We concluded that abnormal protein phosphorylation modifications are key regulators of reduced sperm motility. These novel findings implicated specific protein phosphorylation modifications in sperm cryo-injury. SIGNIFICANCE: This study used phosphorylated TMT quantitative proteomics to explore regulation of epigenetic modifications in frozen-thawed ram sperm. This experiment demonstrated that ram sperm freezing affects phosphorylation site modifications of proteins, especially those related to functions such as sperm motility and energy production. Furthermore, it is important to link functions of phosphorylated proteins with changes in sperm quality after freezing and thawing, and to clarify intrinsic reasons for sperm quality changes, which is of great importance for elucidating mechanisms of sperm freezing damage. Based on these protein markers and combined with cryoprotectant design theory, it provides a theoretical basis and data reference to study sperm cryoprotectants.

Melatonin enhances the sensitivity of colorectal cancer cells to 5-fluorouracil through the regulation of the miR-532-3p/ß-catenin pathway.

This research aimed to investigate whether melatonin affected sensitivity to 5-fluorouracil (5-FU) in colorectal cancer (CRC) as well as to show the underlying molecular mechanism. Melatonin and 5-FU were added to CRC cells at varying doses. The effect of melatonin on sensitivity to 5-FU was investigated by measuring cell activity and apoptosis, and the potential underlying mechanism was further explored by detecting miR-532-3p expression and the associated pathway proteins. Melatonin could suppress cell malignancy in SW480 and HCT116 cells. Melatonin also significantly promoted sensitivity to 5-FU in CRC cells. miR-532-3p expression was downregulated in CRC and was also markedly enhanced when treated with 1 mmol/L melatonin. The inhibitory ability of the co-cultured melatonin, 5-FU, and miR-532-3p inhibitor on SW480 and HCT116 cells was markedly diminished, and the IC50 value was significantly enhanced. Relative to the melatonin group, melatonin+miR-532-3p inhibitor markedly declined apoptosis rate. Bioinformatics analysis predicted the target of miR-532-3p. ß-catenin level presented obvious downregulation in the melatonin group, while it was notably upregulated in the co-culture group in relative to with that in the melatonin group. Overall, melatonin promotes sensitivity to 5-FU in CRC cells by regulating the miR-532-3p/ß-catenin pathway.

Intramolecular cascade reaction sensing platform for rapid, specific and ultrasensitive detection of nitrite.

Nitrite is a carcinogenic substance in food. Excessive consumption of nitrite severely endangers human health. However, rapid and accurate quantification of nitrite by a simple tool is still very challenging. In this work, we designed a practical sensing platform based on 8-(o-phenylenediamine)-boron dipyrromethene (BDP-OPD) to determine nitrite in food. BDP-OPD can take a specific diazotization-cyclization cascade reaction with nitrite to form boron dipyrromethene (BODIPY), giving rise to a remarkable chromogenic reaction along with high contrast fluorescence turn-on response towards nitrite. BDP-OPD has high sensitivity, rapid response, and good selectivity. Furthermore, a portable smartphone-based fluorescence device integrated with a self-programmed Python program was fabricated, which has been successfully used to determine nitrite in food with the advantages of rapid response, low cost, ease of operation, portability, and satisfactory recoveries (92-112%). The good sensing performance rendered BDP-OPD a promising fluorescence platform for on-site visual detection of nitrite.

FTO Alleviates CdCl2-Induced Apoptosis and Oxidative Stress via the AKT/Nrf2 Pathway in Bovine Granulosa Cells.

Cadmium (Cd) is a common environmental heavy metal contaminant of reproduction toxicity. Cd accumulation in animals leads to the damage of granulosa cells. However, its mechanism needs to be elucidated. This research found that treating granulosa cells with Cd resulted in reduced cell viability. The flow cytometry results showed that Cd increased the degree of apoptosis and level of superoxide anion (O2-) in granulosa cells. Further analysis showed that Cd treatment resulted in reduced expression levels of nuclear factor erythroid 2-related factor-2 (Nrf2), superoxide dismutase (SOD), catalase (CAT) and NAD(P)H: quinone oxidoreductase 1 (NQO1), and an increased expression level of malondialdehyde (MDA); the expression levels of Bcl-2 associated X (Bax) and caspase-3 increased, whereas that of B-cell lymphoma 2 (Bcl-2) decreased. Changes in m6A methylation-related enzymes were noted with Cd-induced damage to granulosa cells. The results of transcriptome and MeRIP sequencing revealed that the AKT pathway participated in Cd-induced damage in granulosa cells, and the MAX network transcriptional repressor (MNT) may be a potential target gene of fat mass and obesity-associated protein (FTO). FTO and YTH domain family member 2 (YTHDF2) regulated MNT expression through m6A modification. FTO overexpression alleviated Cd-induced apoptosis and oxidative stress through the activation of the AKT/Nrf2 pathway; this process could be reversed using siMNT. Overall, these findings associated m6A with Cd-induced damage to granulosa cells and provided insights into Cd-induced granulosa cell cytotoxicity from a new perspective centered on m6A modification.

LncRNA SNHG6 Inhibits Apoptosis by Regulating EZH2 Expression via the Sponging of MiR-101-3p in Esophageal Squamous-Cell Carcinoma.

BACKGROUND: The long non-coding RNA (lncRNA) SNHG6 was significantly upregulated in esophageal squamous-cell carcinoma (ESCC), and it promoted ESCC cell proliferation, invasion, and migration. However, the effects of SNHG6 on cell apoptosis and the corresponding underlying mechanisms have not yet reported. METHODS: Apoptosis was detected by flow cytometric analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used for mRNA and protein quantification, respectively. A luciferase reporter assay was performed to verify downstream target genes for SNHG6 and miR-101-3p. RESULTS: Dysregulation of SNHG6 inhibited apoptosis in ESCC cells and regulated the expression of apoptosis-related proteins such as Bcl-2, Mcl-1, Bax and Caspase-3. Functionally, miR-101-3p could compete binding with 3'-untranslated region of SNHG6 and downregulation of miR-101-3p reversed its effect on cell apoptosis in SNHG6 knockdown cells. EZH2 was confirmed as a downstream target gene of miR-101-3p, silencing EZH2 expression had the same effect on apoptosis and protein expression as knocking down SNHG6. Overexpression of EZH2 reversed the effects of miR-101-3p overexpression on cell apoptosis in ESCC cells. CONCLUSION: In this study, we found that upregulation of the lncRNA SNHG6 inhibited apoptosis via miR-101-3p/EZH2 axis in ESCC. These findings may contribute to the diagnosis and treatment of ESCC.

Cold atmospheric plasma induces GSDME-dependent pyroptotic signaling pathway via ROS generation in tumor cells.

Cold atmospheric plasma (CAP) has been proposed as a novel promising anti-cancer treatment modality. Apoptosis and necrosis have been revealed in CAP-induced cell death, but whether CAP induces pyroptosis, another kind of programmed cell death is still unknown. In the present study, we first reported that CAP effectively induced pyroptosis in a dose-dependent manner in Gasdermin E (GSDME) high-expressed tumor cell lines. Interestingly, the basal level of GSDME protein was positively correlated with the sensitivity to CAP in three selected cancer cell lines, implying GSDME might be a potential biomarker of prognosis in the forthcoming cancer CAP treatment. Moreover, our study revealed that CAP-induced pyroptosis depended on the activation of mitochondrial pathways (JNK/cytochrome c/caspase-9/caspase-3) and the cleavage of GSDME but not Gasdermin D (GSDMD). ROS generation induced by CAP was identified to initiate the pyroptotic signaling. These results complemented our knowledge on CAP-induced cell death and provide a strategy to optimize the effect of CAP cancer treatment.

[Effect of propofol and operative trauma on neurodevelopment and cognitive function of developing brain in rats].

OBJECTIVE: To investigate the effect of propofol and operative trauma on the neurodevelopment and cognitive function of the developing brain and its mechanism. METHODS: A total of 104 postnatal day 13 Sprague-Dawley rats were randomly divided into 4 groups: control group (treated by 7.5 mL/kg saline and sham surgery), propofol group (treated by 75 mg/kg propofol), surgery group (with abdominal surgery under local anesthesia) and propofol+surgery group (with abdominal surgery under local anesthesia plus 75 mg/kg propofol anesthesia). Thirteen rats from each group were randomly selected for detecting the content of TNF-α in the hippocampus and the expression levels of caspase-3 and c-fos in the brain. Morris Water Maze test was used to detect the cognitive ability of the other rats at 60 days old, after which TNF-α content in the hippocampus and caspase-3 and c-fos expressions in the brain were detected. RESULTS: In 13 day-old rats, TNF-α level and caspase-3 and c-fos expressions differed significantly between the surgery group and the other 3 groups (P<0.05) and were similar among the control group, propofol group and propofol+surgery group (P>0.05). In 60-day-old rats, Morris water maze test results, TNF-α level or expressions of caspase-3 and c-fos showed no significant differences among the 4 groups. CONCLUSION: Abdominal surgery can induce inflammation in the hippocampus and neuroapoptosis in neonatal rats rather than adult rats. Single-dose propofol anesthesia does not significantly affect neurodevelopment of young rats, and can relieve central inflammatory reaction induced by surgical trauma.

Anti-hepatitis B virus activities of α-DDB-FNC, a novel nucleoside-biphenyldicarboxylate compound in cells and ducks, and its anti-immunological liver injury effect in mice.

Infection with hepatitis B virus (HBV) continues to be a major global cause of acute and chronic liver disease with high mortality. Herein, we examined both the anti-HBV and hepatoprotective activity of α-DDB-FNC. In human HBV-transfected liver cell line HepG2.2.15, α-DDB-FNC effectively suppressed the secretion of HBV antigens in a time and dose-dependent manner with 25.11% inhibition on HBeAg and 43.68% on HBsAg at 2.5 µM on day 9. Consistent with the HBV antigen reduction, α-DDB-FNC (2.5 µM) also reduced HBV DNA level by 77.74% extracellularly and 78.94% intracellularly on day 9. In the duck hepatitis B virus (DHBV) infected ducks, after α-DDB-FNC was given once daily for 10 days, the serum and liver DHBV DNA levels were reduced markedly with 96.81% and 97.21% at 10 mgkg(-1) on day 10, respectively. In Con A-induced immunological liver-injury mice, α-DDB-FNC significantly inhibited the elevation of serum ALT, AST, TBiL and liver MDA, NO levels. Furthermore, significant improvement of the liver was observed after α-DDB-FNC treatment both in ducks and mice, as evaluated by the histopathological analysis. In conclusion, our results demonstrated that α-DDB-FNC possesses both antiviral activity against HBV and hepatoprotective effect to Con A-induced liver-injury mice.

Antiviral activity of FNC, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine, against human and duck HBV replication.

BACKGROUND: New drugs are needed to combat HBV infection. We investigated the anti-HBV activity of the deoxycytidine analogue FNC, which has anticancer activity and has been found to inhibit HCV replication. METHODS: In this study, a human hepatoma HepG2.2.15 cell culture system and duck HBV (DHBV) infection model were used as the in vitro and in vivo models to evaluate the anti-HBV activity of FNC. RESULTS: In the cell model, FNC effectively suppressed the secretion of the HBV antigens in a dose-dependent manner, with 50% effective concentration values of 0.037 µM for hepatitis B surface antigen and 0.044 µM for hepatitis B e antigen on day 9. Consistent with the HBV antigen reduction, FNC also reduced the HBV DNA level by 92.31% and 93.90% intracellularly and extracellularly, respectively. DHBV DNA levels were markedly reduced after treatment with the FNC at 0.5, 1.0 and 2.0 mg/kg•day dosages. The inhibition rate of FNC at the dose of 2.0 mg/kg•day reached 91.68% and 81.96%, in duck serum and liver, respectively, on day 10. Furthermore, significant liver histology restoration after FNC treatment was observed, as evaluated by the histopathological analysis. CONCLUSIONS: FNC can evidently inhibit the replication of HBV in the HepG2.2.15 cell line in vitro and inhibits DHBV replication in ducks in vivo. It could be potentially developed into a new anti-HBV drug.