The Phage Holin HolGH15 Exhibits Potential As an Antibacterial Agent to Control Listeria monocytogenes.

Listeria monocytogenes is an important foodborne pathogen that is a serious threat to public health security, and new strategies to control this bacterium in food are needed. HolGH15, derived from Staphylococcus aureus phage GH15, has shown antibacterial activity against several bacterial species. In this work, the antilisterial behavior and effectiveness of HolGH15 are further studied. To elucidate its antimicrobial modes against L. monocytogenes, cell integrity and membrane permeabilization assays were performed. When treated with HolGH15, the release of 260-nm-absorbing materials of L. monocytogenes was rapidly increased. HolGH15 triggered a significant increase in fluorescence intensity by flow cytometry. In membrane permeabilization assays, the cytoplasmic ß-galactosidase of L. monocytogenes treated with HolGH15 was released via an increase in the permeability of the membrane. HolGH15 caused changes in the structural properties of L. monocytogenes cells resulting in shrinkage, which evoked the release and removal of cellular contents and finally lead to cell death. Electron microscopy observations indicated that HolGH15 exhibited excellent bactericidal potency by permeabilizing the cell membrane, damaging membrane integrity, and inducing cellular content shrinkage or loss. Moreover, HolGH15 (at the final concentration of 240 µg/mL) reduced L. monocytogenes (at the initial concentration of 106 colony-forming unit/mL) to an undetectable level at 4°C. Collectively, HolGH15 has potential as a novel antimicrobial agent against L. monocytogenes in the manufacture and store of food by spraying or soaking, especially at refrigerated temperature.

Quantitative proteomic analysis reveals that the Rap1/MAPK/ERK pathway is inhibited through selenomethionine strengthening antioxidant activity.

To investigate the influence on the proteome of chicken skeletal muscles of Selenomethionine (SeMet) use, 36 chicks were fed with SeMet feeding for 35 days. A total of 72 1-day old broiler chicks were randomly allocated into two groups (n = 36/group): the control group (C group), the SeMet supplemented group (SeMet group). The Selenium (Se) concentrations of skeletal muscles from the chicks with basal diet (negative control group) and SeMet feeding were found to be 0.01 mg/kg and 0.40 mg/kg, respectively. The skeletal muscles from the two groups were investigated using isobaric Tags for Relative and Absolute Quantitation (iTRAQ), coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This proteomic analysis identified proteins that were differentially expressed between the two groups. A total of 3564 proteins from the SeMet and the control (C) groups at 35 days were analyzed. 86 proteins were found by iTRAQ to be differentially expressed in the SeMet group, including 38 up-regulated proteins and 48 down-regulated proteins. These differential proteins were later identified as being mainly involved in antioxidant and enzyme-regulating activities. Fluorescent quantitative PCR(qPCR) and Western blot analyse proved to be consistent with the results of iTRAQ identification. The differentially expressed proteins (DEPs) identified in our work could be specific biomarkers related to SeMet intake in chicks. SeMet intake may strengthen antioxidant activity through Rap1/mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) signal pathways.

Liver X receptor α participates in LPS-induced reduction of triglyceride synthesis in bovine mammary epithelial cells.

Lipopolysaccharides (LPS) could induce milk fat depression via regulating the body and blood fat metabolism. However, it is not completely clear how LPS might regulate triglyceride synthesis in dairy cow mammary epithelial cells (DCMECs). DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS. The level of triglyceride synthesis, the expression and activity of the liver X receptor α (LXRα), enzymes related to de novo fatty acid synthesis, and the expression of the fatty acid transporters were investigated. We found that LPS decreased the level of triglyceride synthesis via a down-regulation of the transcription, translation, and nuclear translocation level of the LXRα. The results also indicated that the transcription level of the LXRα target genes, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthetase (FAS), acetyl-CoA carboxylase-1 (ACC1), were significantly down-regulated in DCMECs after LPS treatment. Our data may provide new insight into the mechanisms of milk fat depression caused by LPS.

Niacin Alleviates Dairy Cow Mastitis by Regulating the GPR109A/AMPK/NRF2 Signaling Pathway.

Mastitis is one of three bovine diseases recognized as a cause of substantial economic losses every year throughout the world. Niacin is an important feed additive that is used extensively for dairy cow nutrition. However, the mechanism by which niacin acts on mastitis is not clear. The aim of this study is to investigate the mechanism of niacin in alleviating the inflammatory response of mammary epithelial cells and in anti-mastitis. Mammary glands, milk, and blood samples were collected from mastitis cows not treated with niacin (n = 3) and treated with niacin (30 g/d, n = 3) and healthy cows (n = 3). The expression of GPR109A, IL-6, IL-1ß, and TNF-α in the mammary glands of the dairy cows with mastitis was significantly higher than it was in the glands of the healthy dairy cows. We also conducted animal experiments in vivo by feeding rumen-bypassed niacin. Compared with those in the untreated mastitis group, the somatic cell counts (SCCs) and the expression of IL-6, IL-1ß, and TNF-α in the blood and milk were lower. In vitro, we isolated the primary bovine mammary epithelial cells (BMECs) from the mammary glands of the healthy cows. The mRNA levels of IL-6, IL-1ß, TNF-α, and autophagy-related genes were detected after adding niacin, shRNA, compound C, trans retinoic acid, 3-methyladenine to BMECs. Then GPR109A, AMPK, NRF-2, and autophagy-related proteins were detected by Western blot. We found that niacin can activate GPR109A and phosphorylate AMPK, and promote NRF-2 nuclear import and autophagy to alleviate LPS-induced inflammatory response in BMECs. In summary, we found that niacin can reduce the inflammatory response of BMECs through GPR109A/AMPK/NRF-2/autophagy. We also preliminarily explored the alleviative effect of niacin on mastitis in dairy cows.

Metabonomics analysis of Zi goose follicular granulosa cells using ENO1 gene expression interference.

The Zi goose is native to North-east China and is noted for its high egg production. Alpha enolase (ENO1) is a glycolytic enzyme which functions as a plasminogen receptor in follicular granulosa cells (FGCs), with several studies showing that FGCs can support follicular development. By transfecting the ENO1 interfering plasmid (shRNA) into FGCs, ENO1 expression in these cells was downregulated, suggesting the successful knock-down of ENO1 in these cells. In this knock-down model, we detected 13 metabolites from FGCs using LC/MS. When compared with the non-coding shRNA (NC) group, the lower level metabolites were (R)-(+)-citronellic acid, altretamine, 3-hydroxycaproic acid, heptadecanoic acid, cholecalciferol vitamin D3, indole, benzoic acid, capric acid, caffeic acid, azelaic acid, 3,4-dihydroxyhydrocinnamic acid and cholic acid, while oleic acid was detected at high levels. To further examine the results of metabolomics, six key metabolites were verified by gas chromatography-mass spectrometry (GC-MS). We found that vitamin D3, indole, benzoic acid, capric acid and cholic acid were significantly downregulated in the shRNA group, while oleic acid was significantly upregulated. This observation was consistent with the metabolomics data. Through these studies, we found that decreased ENO1 levels altered certain metabolite levels in FGCs.

A noncoding RNA LINC00504 interacts with c-Myc to regulate tumor metabolism in colon cancer.

Accumulating evidence has shown a critical role of long-non-coding RNAs (lncRNAs) during multiple tumor progression. However, the potential functions of LINC00504 in colon cancer as well as its mechanisms remain obscure. By lncRNA profiling, we identified LINC00504 as a novel oncogenic lncRNA in colon cancer. The lncRNA LINC00504 was markedly upregulated in colon cancer cell lines and specimens. LINC00504 increases viability and migration of colon cells in vitro. Furthermore, LINC00504 also enhances colon cancer xenograft tumors in vivo. We noted that LINC00504 regulates metabolism at a transcriptional level which influences multiple metabolic pathways, such as glucose metabolism, pentose phosphate pathway, and tricarboxylic acid cycle. Mechanistic study showed that LINC00504 could interact with c-Myc to promote chromatin recruitment of c-Myc and enhance its transactivation activity. Collectively, our results showed that LINC00504 serves as an important transcriptional regulator for c-Myc in colon cancer cells. LINC00504 can reprogram central metabolism in colon cancer cells implying that LINC00504 may serve as a potential target for therapeutic intervention.

HMGB1-mediated differential response on hippocampal neurotransmitter disorder and neuroinflammation in adolescent male and female mice following cold exposure.

Stress induces many different sex-specific physiological and psychological responses during adolescence. Although the impact of certain brain stressors has been reported in the literature, the influence of cold stress on the mechanisms underlying hippocampal neurotransmitter disorder and neuroinflammation remain unstudied. Adolescent male and female C57BL/6 mice were exposed to 4 °C temperatures, 3 h per day for 1 week. Serum CORT and blood gas analysis was then used to assess body status. Using western blotting, immunofluorescence and immunohistochemistry we also assessed glial cell number and microglial activation, as well as inflammatory cytokine levels and related protein expression levels. The phenomena of excessive CORT, microglial activation, increased acetylate-HMGB1 levels, NF-κB signaling pathway activation, pro-inflammatory cytokine release, neuronal apoptosis and neurotransmitter disorder were demonstrated in mouse hippocampal tissue following cold exposure. We believe that these phenomena are mediated by the HMGB1/TLR4/NFκB pathway. Finally, the male inflammatory response in hippocampal tissue was more severe and the influence of cold exposure on neurotransmitter was greater in females.

The Favored Mechanism for Coping with Acute Cold Stress: Upregulation of miR-210 in Rats.

BACKGROUND/AIMS: The main aim of this study was to determine the mechanisms by which rno-miR-210-3p affects changes in gene expression, metabolism, apoptosis and proliferation of cells under acute cold stress (ACS) conditions. METHODS: The treatment group (n=6, weight 340±20 g) was exposed to ACS (temperature 4±0.5°C, relative humidity 45±0.5%) and the control group (n=6, weight 340±20 g) to normal temperature (NT) (temperature 24±0.5°C, relative humidity 45±0.5%). Rat liver samples were collected for qRT-PCR and western blot analyses to detect relative expression of rno-miR-210-3p, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2. For cell experiments, 100 pmol/dish rno-miR-210-3p mimic and 150 pmol/dish rno-miR-210-3p inhibitor were used. Mitochondrial glucose flux and glycolysis were measured using the XFe24 Extracellular Flux Analyzer. Cells were collected for apoptosis analysis 24 h after transfection and proliferation was quantified using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China), according to the manufacturer's instructions. RESULTS: In the rat experiment, expression of rno-miR-210-3p under ACS was increased sharply while ISCU, E2F3, RAD52, and PSMB6 levels declined, along with protein expression of ISCU and PSMB6. In cell experiments, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes were downregulated while ISCU and PSMB6 protein expression decreased with upregulation of rno-miR-210-3p. Conversely, in response to decreased rno-miR-210-3p expression, ISCU, E2F3, RAD52, PSMB6 and GPD2 genes were upregulated, in addition to ISCU and PSMB6 proteins. Upregulation of miR-210 inhibited cell proliferation and induced cell death whereas its downregulation promoted cell proliferation. Upregulation or downregulation of miR-210 promoted glycolysis and mitochondrial respiration of BRL cells. However, downregulation of miR-210 caused acid production in cells. CONCLUSION: Expression of rno-miR-210-3p is significantly increased under ACS. Upregulation of rno-miR-210-3p inhibits the expression of ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes, promotes glycolysis of liver and enhances the mitochondrial respiratory capacity of cells, but may also cause cell death. Our findings collectively indicate that regulation of rno-miR-210-3p is a preferential mechanism of choice used by the body to cope with ACS.

The association between preoperative serum interleukin-6 levels and postoperative prognosis in patients with T2 gallbladder cancer.

BACKGROUND: Interleukin-6 (IL-6) is closely associated with tumor progression. Whether it can predict postoperative prognosis of patients with T2 gallbladder cancer (GBC) remains controversial. METHODS: We retrospectively collected the medical records of 125 patients with T2 GBC. Then, we analyzed the association between preoperative serum IL-6 levels and postoperative survival by multivariate Cox analyses and Kaplan-Meier curves in exploratory subgroups. RESULTS: Predictive effects of serum IL-6 levels on overall survival were similar across most of the evaluated subgroups, except in different tumor location subgroups. The independent odds ratio (OR) of serum IL-6 levels was 2.57 (95%CI 1.73-3.82) in the hepatic side subgroup, while it was 1.15 (95%CI 0.68-1.93) in the peritoneal side subgroup (P = 0.014 for interaction). When we categorized serum IL-6 levels by median value (4.2 pg/mL), the 5-year survival rate of patients with high serum IL-6 levels was significantly higher in the hepatic side subgroup (58.5% vs 14.8%, P < 0.001), but no such difference was found in the peritoneal side subgroup (62.2% vs 67.6%, P = 0.722). CONCLUSIONS: Preoperative serum IL-6 is significantly associated with prognostic implications in patients with hepatic side T2 GBC, not in those with peritoneal side tumors.

LPS-induced reduction of triglyceride synthesis and secretion in dairy cow mammary epithelial cells via decreased SREBP1 expression and activity.

Sterol regulatory element binding protein 1 (SREBP1) has a central regulatory effect on milk fat synthesis. Lipopolysaccharides (LPS) can induce mastitis and cause milk fat depression in cows. SREBP1 is also known to be associated with inflammatory regulation. Thus, in the current study, we hypothesized that LPS-induced milk fat depression in dairy cow mammary epithelial cells (DCMECs) operates via decreased SREBP1 expression and activity. To examine the hypothesis, DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS (10 µg/ml). LPS treatment of DCMECs suppressed lipid-metabolism-related transcription factor SREBP1 mRNA expression, nuclear translocation and protein expression, leading to reduced triglyceride content. The transcription levels of acetyl-CoA carboxylase-1 and fatty acid synthetase were significantly down-regulated in DCMECs after LPS treatment, suggesting that acetyl-CoA carboxylase-1 and fatty acid synthetase involved in de novo milk fat synthesis was regulated by SREBP1. In summary, these results suggest that LPS induces milk fat depression in dairy cow mammary epithelial cells via decreased expression of SREBP1 in a time-dependent manner.