Animals ; Carrier Proteins ; genetics ; Immunohistochemistry ; Lacrimal Apparatus ; metabolism ; Lung ; metabolism ; Mice ; Mice, Inbred NOD ; Microfilament Proteins ; genetics ; RNA, Small Interfering ; genetics ; physiology ; Random Allocation ; Sjogren's Syndrome ; genetics ; immunology ; therapy

Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Animals ; Carrier Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; Heterogeneous-Nuclear Ribonucleoprotein K ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.

METHODSThe hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.

RESULTSDAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.

CONTROL0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.

CONTROL0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.

CONCLUSIONDown-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

Carrier Proteins ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Down-Regulation ; Gene Expression Regulation ; Hep G2 Cells ; Hepatitis B Surface Antigens ; isolation & purification ; Hepatitis B e Antigens ; isolation & purification ; Hepatitis B virus ; physiology ; Humans ; Interleukin-6 ; metabolism ; NF-kappa B ; metabolism ; Plasmids ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Virus Replication

BACKGROUND: We investigated the rates of fecal transmission of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and carbapenem-resistant Enterobacteriaceae (CRE) among patients admitted to intensive care units (ICUs). METHODS: From June to August 2012, rectal cultures were acquired from all patients at ICU admission. For patients not carrying ESBL-E or CRE at admission, follow-up cultures were performed to detect acquisition. A chromogenic assay was used to screen for ESBL-E and CRE. Bacterial species identification and antibiotic susceptibility tests were performed using the Vitek 2 system (bioMerieux, France). ESBL genotypes were determined by PCR, and clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis. RESULTS: Out of 347 ICU admissions, 98 patients were found to be carriers of ESBL-E (28.2%, 98/347). Follow-up cultures were acquired from 91 of the patients who tested negative for ESBL-E at admission; the acquisition rate in this group was 12.1% (11/91), although none was a nosocomial transmission. For CRE, the prevalence of fecal carriage was 0.3% (1/347), and the acquisition rate was 2.9% (4/140). None of the CRE isolates were carbapenemase-producers. CONCLUSIONS: The high prevalence of ESBL-E carriage on admission (28.2%), coupled with rare nosocomial transmission and the very low carriage rate of CRE (0.3%), challenge the routine use of active surveillance in non-epidemic settings. Nevertheless, passive surveillance measures, such as rapid and accurate screening of clinical specimens, will be critical for controlling the spread of CRE.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*metabolism ; Carbapenems/*pharmacology ; Carrier State/epidemiology ; Cross Infection/epidemiology/*transmission ; DNA, Bacterial/analysis ; Drug Resistance, Bacterial/drug effects ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae/enzymology/genetics/*physiology ; Enterobacteriaceae Infections/epidemiology/*transmission ; Feces/*microbiology ; Genotype ; Humans ; Intensive Care Units ; Polymerase Chain Reaction ; Prevalence ; Republic of Korea/epidemiology ; beta-Lactamases/*metabolism

Vitamin D3 up-regulated protein 1 (VDUP1) is a potent growth suppressor that inhibits tumor cell proliferation and cell cycle progression when overexpressed. In a previous study, we showed that VDUP1 knockout (KO) mice exhibited accelerated liver regeneration because such animals could effectively control the expression of cell cycle regulators that drive the G1-to-S phase progression. In the present study, we further investigated the role played by VDUP1 in initial priming of liver regeneration. To accomplish this, VDUP1 KO and wild-type (WT) mice were subjected to 70% partial hepatectomy (PH) and sacrificed at different times after surgery. The hepatic levels of TNF-alpha and IL-6 increased after PH, but there were no significant differences between VDUP1 KO and WT mice. Nuclear factor-kappaB (NF-kappaB), c-Jun-N-terminal kinase (JNK), and signal transducer and activator of transcription 3 (STAT-3) were activated much earlier and to a greater extent in VDUP1 KO mice after PH. A single injection of TNF-alpha or IL-6 caused rapid activation of JNK and STAT-3 expression in both mice, but the responses were stronger and more sustained in VDUP1 KO mice. In conclusion, our findings provide evidence that VDUP1 plays a role in initiation of liver regeneration.
Animals ; Blotting, Western ; Carrier Proteins/*genetics/metabolism ; Cell Proliferation ; *Gene Expression Regulation ; Hepatectomy ; Hepatocytes/*cytology/physiology ; JNK Mitogen-Activated Protein Kinases/genetics/metabolism ; Liver/*physiology ; Male ; Mice, Knockout ; NF-kappa B/genetics/metabolism ; Polymerase Chain Reaction ; *Regeneration ; STAT3 Transcription Factor/genetics/metabolism ; Thioredoxins/*genetics/metabolism

The p53 signaling pathway works as a potent barrier to tumor progression. Two single nucleotide polymorphisms (SNPs) in the gene loci of p53 pathway, p53 codon 72 Arg72Pro and MDM2 SNP309 (T > G), have been shown to cause perturbation of p53 function, but the effect of the two SNPs on the risk of hepatocellular carcinoma (HCC) remains inconsistent. This study investigated the influence of combined p53 Arg72Pro and MDM2 SNP309 on the risk of developing HCC in patients with chronic hepatitis B virus infection, and evaluated the significance of the two combined SNPs on patient prognosis. In total, 350 HCC patients, 230 non-HCC patients, and 96 healthy controls were genotyped for the p53 Arg72Pro and MDM2 SNP309. The combined p53 Pro/Pro and MDM2 G/G genotype was significantly associated with HCC risk (P = 0.047). Multivariate analysis indicated that combined p53 Pro/Pro and MDM2 G/G genotype was an independent factor affecting recurrence and survival (P < 0.05). Patients with combined p53 Pro/Pro and MDM2 G/G genotypes had a poorer prognosis than other genotypes, P < 0.01 for both disease-free survival (DFS) and overall survival (OS). DFS and OS rates also differed significantly between Barcelona Clinic Liver Cancer (BCLC) stage A patients with combined p53 Pro/Pro and MDM2 G/G and other genotypes (P < 0.05). Thus, the combined p53 Pro/Pro and MDM2 G/G genotype is associated with increased risk of developing HCC and is an independent adverse prognostic indicator in early stage HCC.
Carcinoma, Hepatocellular ; diagnosis ; genetics ; surgery ; virology ; Carrier State ; virology ; Cohort Studies ; Female ; Genetic Predisposition to Disease ; genetics ; Hepatitis B virus ; physiology ; Humans ; Liver Neoplasms ; diagnosis ; genetics ; surgery ; virology ; Male ; Middle Aged ; Neoplasm Staging ; Polymorphism, Single Nucleotide ; Prognosis ; Proto-Oncogene Proteins c-mdm2 ; genetics ; Tumor Suppressor Protein p53 ; genetics

Autophagy is a self-degradation system of cellular components through an autophagosomal-lysosomal pathway. Over the last 15 yr, yeast genetic screens led to the identification of a number of genes involved in the autophagic pathway. Most of these autophagy genes are present in higher eukaryotes and regulate autophagy process for cell survival and homeostasis. Significant progress has recently been made to better understand the molecular mechanisms of the autophagy machinery. Especially, autophagy process, including the regulation of autophagy induction through mTOR and the nucleation and elongation in autophagosome formation through class III phosphatidylinositol 3-kinase complex and ubiquitin-like conjugation systems, became evident. While many unanswered questions remain to be answered, here, we summarize the recent process of autophagy with emphasis on molecules and their protein complexes along with advanced molecular mechanisms that regulate the autophagy machinery.
Autophagy/genetics/*physiology ; Carrier Proteins/genetics/metabolism ; Class III Phosphatidylinositol 3-Kinases/genetics/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Membrane Proteins/genetics/metabolism ; Microtubule-Associated Proteins/genetics/metabolism ; Models, Biological ; Protein-Serine-Threonine Kinases/genetics/metabolism ; Small Ubiquitin-Related Modifier Proteins/genetics/metabolism

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism

Biotin is an important micronutrient that serves as an essential enzyme cofactor. Bacteria obtain biotin either through de novo synthesis or by active uptake from exogenous sources. Mycobacteria are unusual amongst bacteria in that their primary source of biotin is through de novo synthesis. Here we review the importance of biotin biosynthesis in the lifecycle of Mycobacteria. Genetic screens designed to identify key metabolic processes have highlighted a role for the biotin biosynthesis in bacilli growth, infection and survival during the latency phase. These studies help to establish the biotin biosynthetic pathway as a potential drug target for new anti-tuberculosis agents.
Biotin ; biosynthesis ; Carbon-Carbon Ligases ; metabolism ; Carrier Proteins ; metabolism ; Cell Membrane ; metabolism ; Coenzymes ; metabolism ; Fatty Acids ; biosynthesis ; Genes, Bacterial ; Genome, Bacterial ; Metabolic Networks and Pathways ; Molecular Structure ; Mycobacterium Infections ; microbiology ; Mycobacterium tuberculosis ; genetics ; metabolism ; pathogenicity ; physiology ; Virulence