Hepatitis C virus (HCV) RNA replication depends on viral protein association with intracellular membranes, but the influence of membrane composition on viral replication is unclear. We report that HCV RNA replication and assembly of the viral replication complex require geranylgeranylation of one or more host proteins. In cultured hepatoma cells, HCV RNA replication was disrupted by treatment with lovastatin, an inhibitor of 3-hydroxy-3-methyglutaryl CoA reductase, or with an inhibitor of protein geranylgeranyl transferase I, each of which induced the dissolution of the HCV replication complex. Viral replication was not affected by treatment of cells with an inhibitor of farnesyl transferase. When added to lovastatin-treated cells, geranylgeraniol, but not farnesol, restored replication complex assembly and viral replication. Inasmuch as the HCV genome does not encode a canonical geranylgeranylated protein, the data suggest the involvement of a geranylgeranylated host protein in HCV replication. Inhibition of its geranylgeranylation affords a therapeutic strategy for treatment of HCV infection.A pproximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV), and these individuals account for a majority of all cases of chronic liver disease (1). The public health impact of HCV is compounded by the overall low response rate to current IFN-based therapies for treating HCV infection, underscoring the need for new therapeutic strategies to combat the HCV pandemic. HCV is a singlestranded positive sense RNA virus and member of the Flaviviridae (2). The 9.6-kb HCV genome encodes a single polyprotein that is posttranslationally processed into at least 10 individual structural and nonstructural (NS) viral proteins, the latter of which are sufficient to support HCV RNA replication (3). Current studies support a model in which HCV infection results in assembly of the viral RNA and NS proteins into a replication complex that associates with the host cell endoplasmic reticulum (ER). Viral-directed processes convert the ER into a membranous web conducive to virus replication (4-6). The cellular cofactors and membrane constituents that contribute to assembly and maintenance of the HCV replication complex are not known.Cell membrane composition is subject to modification through the mevalonate pathway, which produces cholesterol and nonsterol isoprenoid products (7). Two of the mevalonate-derived isoprenoids, farnesyl (15 carbons) and geranylgeranyl (20 carbons), are attached to membrane proteins via formation of a cysteine thioether (7,8). This process, called protein prenylation, targets certain proteins to cell membranes where they regulate many cellular functions, ranging from vesicle budding and fusion to growth. Therapeutic control of the mevalonate pathway has proven effective for the clinical treatment of hypercholesterolemia and is achieved in part through the use of statin compounds (7, 9). Statins block mevalonate production by inhibiting 3-hydroxy-3-methylglutaryl CoA reductase (HMG...

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.

Hepatitis C virus (HCV), a single-stranded positive-sense RNA virus of the Flaviviridae family, infects more than 170 million people worldwide and is the leading cause of liver failure in the United States. A unique feature of HCV is that the viral life cycle depends on cholesterol metabolism in host cells. This review summarizes the cholesterol metabolic pathways that are required for the replication, secretion, and entry of HCV. The potential application of drugs that alter host cholesterol metabolism in treating HCV infection is also discussed.

Hepatic sinusoidal obstruction syndrome (HSOS) can be caused by pyrrolizidine alkaloids(PAs)-containing herbals. Since PAs exposure is obscure and clinical presentation of HSOS is unspecific, it is challenge to establish the diagnosis of PAs-induced HSOS. Gynura segetum is one of the most wide-use herbals containing PAs. The aim of our study is to describe the features of contrast-enhanced computed tomography (CT) in gynura segetum-induced HSOS, and then determine diagnostic performance of radiological signs. We retrospectively analyzed medical records and CT images of HSOS patients (71 cases) and the controls (222 cases) enrolled from January 1, 2008, to Oct 31, 2015. The common findings of contrast CT in PAs-induced HSOS included: ascites (100%), hepatomegaly (78.87%), gallbladder wall thickening (86.96%), pleural effusion (70.42%), hepatic vein narrowing (87.32%), patchy liver enhancement (92.96%), and heterogeneous hypoattenuation (100%); of these signs, patchy enhancement and heterogeneous hypoattenuation were valuable features. Then, the result of diagnostic performance demonstrated that contrast CT possessed better performance in diagnosing PAs-induced HSOS compared with various parameters of Seattle criteria. In conclusion, the patients with PAs-induced HSOS display distinct radiologic features at CT-scan, which reveals that contrast-enhanced CT provides an effective noninvasive method for diagnosing PAs-induced HSOS.

Introduction. An increasing number of studies are utilizing different magnetic resonance (MR) methods to quantify bone marrow fat due to its potential role in osteoporosis. Our aim is to compare the measurements of bone marrow fat among T1-weighted magnetic resonance imaging (MRI), modified Dixon method (also called fat fraction MRI (FFMRI)), and magnetic resonance spectroscopy (MRS). Methods. Contiguous MRI scans were acquired in 27 Caucasian postmenopausal women with a modified Dixon method (i.e., FFMRI). Bone marrow adipose tissue (BMAT) of T1-weighted MRI and bone marrow fat fraction of the L3 vertebra and femoral necks were quantified using SliceOmatic and Matlab. MRS was also acquired at the L3 vertebra. Results. Correlation among the three MR methods measured bone marrow fat fraction and BMAT ranges from 0.78 to 0.88 (P < 0.001) in the L3 vertebra. Correlation between BMAT measured by T1-weighted MRI and bone marrow fat fraction measured by modified FFMRI is 0.86 (P < 0.001) in femoral necks. Conclusion. There are good correlations among T1-weighted MRI, FFMRI, and MRS for bone marrow fat quantification. The inhomogeneous distribution of bone marrow fat, the threshold segmentation of the T1-weighted MRI, and the ambiguity of the FFMRI may partially explain the difference among the three methods.

Interleukin-1b (IL-1b) is released from activated microglia and involved in the neurodegeneration of acute and chronic brain disorders, such as stroke and Alzheimer's disease, in which extracellular acidification has been shown to occur. Here, we examined the extracellular acidic pH regulation of IL-1b production, especially focusing on TDAG8, a major proton-sensing G-protein-coupled receptor, in mouse microglia. Extracellular acidification inhibited lipopolysaccharide -induced IL-1b production, which was associated with the inhibition of IL-1b cytoplasmic precursor and mRNA expression. The IL-1b mRNA and protein responses were significantly, though not completely, attenuated in microglia derived from TDAG8-deficient mice compared with those from wildtype mice. The acidic pH also stimulated cellular cAMP accumulation, which was completely inhibited by TDAG8 deficiency. Forskolin and a cAMP derivative, which specifically stimulates protein kinase A (PKA), mimicked the proton actions, and PKA inhibitors reversed the acidic pH-induced IL-1b mRNA expression. The acidic pH-induced inhibitory IL-1b responses were accompanied by the inhibition of extracellular signal-related kinase and c-Jun N-terminal kinase activities. The inhibitory enzyme activities in response to acidic pH were reversed by the PKA inhibitor and TDAG8 deficiency. We conclude that extracellular acidic pH inhibits lipopolysaccharide-induced IL-1b production, at least partly, through the TDAG8/cAMP/PKA pathway, by inhibiting extracellular signalrelated kinase and c-Jun N-terminal kinase activities, in mouse microglia. Keywords: acidification, cAMP, interleukin-1b, microglia, protein kinase A, TDAG8. Microglia fulfill a central role in the innate and adaptive immune system in brain. The activation of microglia and subsequent increase in the synthesis and release of proinflammatory cytokines, such as IL-1, have been shown to occur under acute, e.g., stroke and traumatic brain injury, and chronic conditions, e.g., Alzheimer's disease and Parkinson's disease (Yenari et al. 2010). IL-1 is known to have a causal role in their neurodegeneration, although the cytokine is also thought to be involved in the recovery of the neuronal functions ( -benzoyladenosine-3′,5′-cyclic monophosphate; NF-jB, nuclear factor-jB; p38 MAPK, p38 mitogen-activated protein kinase; PKA, cAMP-dependent protein kinase; proIL-1b cytoplasmic precursor of IL-1b; TLR, toll-like receptor.

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p70 S6 kinase (S6K1) is a serine/threonine kinase that phosphorylates the insulin receptor substrate-1 (IRS-1) at serine 1101 and desensitizes insulin receptor signaling. S6K1 hyperactivation due to overnutrition leads to hyperglycemia and type 2 diabetes. Our recent study showed that A77 1726, the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide, is an inhibitor of S6K1. Whether leflunomide can control hyperglycemia and sensitize the insulin receptor has not been tested. Here we report that A77 1726 increased AKT and S6K1 phosphorylation but decreased S6 and IRS-1 phosphorylation in 3T3-L1 adipocytes, C2C12 and L6 myotubes. A77 1726 increased insulin receptor tyrosine phosphorylation and binding of the p85 subunit of the PI-3 kinase to IRS-1. A77 1726 enhanced insulin-stimulated glucose uptake in L6 myotubes and 3T3-L1 adipocytes, and enhanced insulin-stimulated glucose transporter type 4 (GLUT4) translocation to the plasma membrane of L6 cells. Finally, we investigated the anti-hyperglycemic effect of leflunomide on and high-fat diet (HFD)-induced diabetes mouse models. Leflunomide treatment normalized blood glucose levels and overcame insulin resistance in glucose and insulin tolerance tests in and HFD-fed mice but had no effect on mice fed a normal chow diet (NCD). Leflunomide treatment increased AKT phosphorylation in the fat and muscle of mice but not in normal mice. Our results suggest that leflunomide sensitizes the insulin receptor by inhibiting S6K1 activity, and that leflunomide could be potentially useful for treating patients with both RA and diabetes.