Chronic hepatitis C (HCV) is a difficult to treat disease affecting over 3 million Americans. Protease inhibitors increase the effectiveness of standard therapy but are costly. A genetic assay may identify patients most likely to benefit from this treatment advance.
Cost-effectiveness assessment of new protease inhibitors and an Interleukin-28B (IL- 28B) genotyping assay for treating chronic HCV
Decision-analytic Markov model
Published literature and expert opinion
Treatment-naïve patients with chronic, genotype 1 HCV mono-infection
Strategies are defined by the use of IL-28B genotyping and type of treatment (standard therapy: pegylated interferon with ribavirin; triple therapy: standard therapy and a protease inhibitor). IL-28B guided triple therapy stratifies CC genotype patients to standard therapy and non-CC types to triple therapy.
Discounted costs (2010 U.S. dollars) and quality-adjusted life years (QALYs); incremental cost effectiveness ratios
Results of Base-Case Analysis
For patients with mild and advanced fibrosis, universal triple therapy reduces life-time risk of hepatocellular-carcinoma by 39% and 29% and increases quality-adjusted life expectancy by 3% and 8% compared to standard therapy. Gains from IL- 28B guided triple therapy are smaller. If the protease inhibitor costs $1,100 per week, universal triple therapy costs $102,600 per QALY (mild fibrosis) or $51,500 per QALY (advanced fibrosis) compared to IL-28B guided triple therapy and $70,100 per QALY and $36,300 per QALY compared to standard therapy.
Results of Sensitivity Analysis
Results are sensitive to the cost of protease inhibitors and treatment adherence rates.
Lack of long-term comparative effectiveness data on the new protease inhibitors
Both universal triple therapy and IL-28B guided triple therapy are cost-effective with the least expensive protease inhibitor for patients with advanced fibrosis.
Primary Funding Source
Stanford Graduate Fellowship
Tumor-homing peptides that recognize specific markers on tumor cells have shown potential as drug carriers for targeted cancer therapy. Bombesin receptors are frequently overexpressed or ectopically expressed in a wide range of human tumors. Bombesin and its analogues have been widely used as drug carriers for tumor imaging and tumor therapy. However, the cargos used in previous studies, including radioactive and chemotherapeutic agents, are usually small molecules. Mitochondrial-disrupting peptides depolarize the mitochondria and trigger apoptosis after entering tumor cells. We are interested in whether the bombesin analogue, Bn(6–14), which contains a bombesin receptor-binding motif, can specifically deliver the mitochondria-disrupting peptide, B28, to tumor cells. To this end, we created a chimeric peptide, B28Bn(6–14), by conjugating B28 to Bn(6–14) at its N-terminus. The cytotoxicity of B28Bn(6–14) in tumor cells was much stronger than unconjugated B28. The IC50 values of B28Bn(6–14) in tumor cells (1.7–3.5 µM) were approximately 10 times lower than B28. However, conjugation of B28 to Bn(2–7), which lacks the bombesin receptor-binding motif, did not increase its cytotoxicity. In addition, the IC50 values of B28Bn(6–14) in tumor cells (1.7–3.5 µM) was 3–10 times lower than in normal cells (10.8–16.8 µM). We found that selective binding of B28Bn(6–14) to tumor cells is Bn(6–14)-dependent. Upon entering the tumor cell, B28Bn(6–14) accumulated in the mitochondria and triggered caspase-dependent apoptosis. Intratumoral and intraperitoneal administration of B28Bn(6–14) substantially suppressed the growth of DU145 tumor xenografts in mice. These results demonstrate that Bn(6–14) is able to deliver the mitochondria-disrupting peptide to tumor cells, and B28Bn(6–14) should be further developed as novel anti-cancer agent.
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.
The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous curvature in the outer leaflets of cell membranes. Our study provides novel insight into the understanding of how IFITM protein family restricts viral membrane fusion and infection.
Many pathogenic viruses contain an envelope that must fuse with the cell membrane in order to gain entry and initiate infection. This process is mediated by one or more glycoproteins present on the surface of the virions, known as viral fusion proteins. Recently, a family of interferon-inducible transmembrane (IFITM) protein has been shown to block viral infection, including those of highly pathogenic viruses. Here we provide evidence that these IFITM proteins potently suppress membrane fusion induced by representatives of all three classes of viral fusion proteins. Interestingly, we found that the block is not at the steps of receptor binding or low pH that triggers conformational changes of viral fusion proteins required for membrane fusion. Rather, we discovered that the creation of hemifusion, an intermediate in which the outer membranes of the two lipid bilayers have merged but the inner membranes still remain intact is blocked by IFITM proteins. We further demonstrated that overexpression of IFITM proteins rigidify the cell membrane, thereby reducing membrane fluidity and fusion potential. Our study provides novel insight into the understanding of how IFITM proteins restrict viral entry and infection.
Lung cancer is the most common cause of cancer-related death worldwide. A poor overall survival rate of 16% necessitates the need for novel treatment strategies. Mouse models of lung cancer are important tools for analyzing the significance of somatic mutations in the initiation and progression of lung cancer. Of additional importance, however, are animal models of virally induced cancers. JSRV is a simple betaretrovirus that causes contagious lung cancer in sheep known as ovine pulmonary adenocarcinoma and closely resembles human lung adenocarcinoma. Previously we showed that expression of the JSRV envelope (Env) from an AAV vector induced lung tumors in immunodeficient mice, but not in immunocompetent mice. Because of the importance of studying lung cancer in the context of an intact immune system we sought to improve our mouse model. In this report, we employed the use of a strong JSRV enhancer-promoter combination to express Env at high levels and demonstrate for the first time, lung tumor induction in immunocompetent mice. This occurred despite a robust Env-specific antibody-mediated immune response. The PI3K/Akt and MAPK pathways were activated in both immunocompetent and immunodeficient mice, however, differential activation of PTEN, GSKα, p70S6K, p38MAPK, ATF2 and STAT5 was observed. A JSRV Env lung tumor-derived cell line was shown to have a similar signal transduction activation profile as Env-induced lung tumors in C57BL/6 mice. Given the similarities between our model and pulmonary adenocarcinomas in humans, and the ease with which tumors can be induced in any transgenic mouse, this system can be used to uncover novel mechanisms involved lung tumorigenesis.
Connective tissue growth factor (CTGF; also known as CCN2) is an inflammatory mediator, and shows elevated levels in regions of severe injury and inflammatory diseases. CTGF is abundantly expressed in osteoarthritis (OA). However, the relationship between CTGF and IL-6 in OA synovial fibroblasts (OASFs) is mostly unknown.
OASFs showed significant expression of CTGF, and expression was higher than in normal SFs. OASFs stimulation with CTGF induced concentration-dependent increases in IL-6 expression. CTGF mediated IL-6 production was attenuated by αvβ5 integrin neutralized antibody and apoptosis signal-regulating kinase 1 (ASK1) shRNA. Pretreatment with p38 inhibitor (SB203580), JNK inhibitor (SP600125), AP-1 inhibitors (Curcumin and Tanshinone IIA), and NF-κB inhibitors (PDTC and TPCK) also inhibited the potentiating action of CTGF. CTGF-mediated increase of NF-κB and AP-1 luciferase activity was inhibited by SB203580 and SP600125 or ASK1 shRNA or p38 and JNK mutant.
Our results suggest that CTGF increased IL-6 production in OASFs via the αvβ5 integrin, ASK1, p38/JNK, and AP-1/NF-κB signaling pathways.
Food restriction (FR) decreases BDNF expression in hypothalamic and hindbrain regions that regulate feeding and metabolic efficiency, while increasing expression in hippocampal and neocortical regions. Drugs of abuse alter BDNF expression within the mesocorticolimbic dopamine (DA) pathway, and modifications of BDNF expression within this pathway alter drug-directed behavior. Although FR produces a variety of striatal neuroadaptations and potentiates the rewarding effects of abused drugs, the effects of FR on BDNF expression and function within the DA pathway are unknown. The primary purpose of the present study was to examine the effect of FR on protein levels of BDNF and its TrkB receptor in component structures of the mesocorticolimbic pathway. Three to four weeks of FR, with stabilization of rats at 80% of initial body weight, did not alter BDNF or TrkB levels in nucleus accumbens, caudate-putamen, or medial prefrontal cortex. However, FR decreased TrkB levels in the ventral tegmental area (VTA), without change in levels of BDNF protein or mRNA. The finding that FR also decreased TrkB levels in substantia nigra, with elevation of BDNF protein, suggests that decreased TrkB in VTA could be a residual effect of increased BDNF during an earlier phase of FR. Voltage-clamp recordings in VTA DA neurons indicated decreased glutamate receptor transmission. These data might predict lower average firing rates in FR relative to ad libitum fed subjects, which would be consistent with previous evidence of decreased striatal DA transmission and upregulation of postsynaptic DA receptor signaling. However, FR subjects also displayed elevated VTA levels of phospho-ERK1/2, which is an established mediator of synaptic plasticity. Because VTA neurons are heterogeneous with regard to neurochemistry, function and target projections, the relationship(s) between the three changes observed in VTA, and their involvement in the augmented striatal and behavioral responsiveness of FR subjects to drugs of abuse, remains speculative.
food restriction; BDNF; TrkB; ventral tegmental area; reward; ERK 1/2
The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.
In the title molecule, C13H21ClO2, there is an intramolecular C—H⋯Cl hydrogen bond. The conformation about the C=C bond is E and the six-membered ring has a chair conformation. In the crystal, molecules are linked by pairs of O—H⋯O hydrogen bonds, forming inversion dimers, which are consolidated by C—H⋯O hydrogen bonds. The dimers are linked via C—H.·O hydrogen bonds, forming chains along .
The intra- and postoperative complications resulting from surgery for giant thyroid gland tumors (diameter greater than 10 cm) present serious challenges to patient recovery. Although there are a number of methods, all have limitations. In this study, we present our experience with several complications of surgical treatment of giant thyroid gland tumors to increase the awareness and aid the prevention of these complications. A total of 137 consecutive patients who underwent surgical treatment in Henan Tumor Hospital were retrospectively analyzed. Statistics pertaining to the patients’ clinical factors were gathered. We found that the most common surgical complications were recurrent laryngeal nerve (RLN) injury and symptomatic hypoparathyroidism. Other complications included incision site infections, bleeding, infection and chyle fistula, the incidence of which increased significantly with increasing extent of surgery from group I (near-total thyroidectomy) to group V (total thyroidectomy plus lateral neck dissection). Low complication rates may be achieved with more accurate knowledge of the surgical anatomy, skilled surgical treatment and experience. More extensive surgery results in a greater number of complications.
complications; giant thyroid gland; recurrent laryngeal nerve; hypoparathyroidism
Recently, an infant and child feeding index (ICFI) constructed on brief recalls of breastfeeding, feeding frequency and food diversification was assumed to provide long-term prediction about child feeding practices. The aim of this study was to investigate the association between the cross-sectional ICFI (CS-ICFI) or longitudinal ICFI (L-ICFI) and child anthropometric indices in downtown Shanghai, China.
The prospective cohort study included 180 infants aged 5-7 mo with their main caregivers who were visited 3 times every 6 months over 12 months. A CS-ICFI was constructed for each visit by using data on feeding practices based on 24-h and 7-d recalls. An L-ICFI was constructed with use of the 3 CS-ICFIs. The associations between ICFI and length-for-age z score (LAZ), weight-for-age z score (WAZ), and weight-for-length z score (WLZ) were examined. The stability of the CS-ICFI was assessed by using repeatability coefficient (RC).
The L-ICFI was positively associated with LAZ and WAZ at Visit 3(beta = 0.151, P = 0.040 and beta = 0.173, P = 0.024, respectively). Moreover, the CS-ICFI at Visit 1 was positively associated with LAZ, WAZ and WLZ (beta = 0.160, P = 0.029; beta = 0.191, P = 0.009; beta = 0.176, P = 0.020) at Visit 3, and the CS-ICFI at Visit 3 was also positively associated with LAZ (beta = 0.176, P = 0.016). Stability of the CS-ICFI was shown by the value of 0.14 (95% CI: 0.07, 0.31) of the RC, which differed significantly from 0 (P < 0.05).
The ICFI constructed on brief recalls based on cross-sectional studies can be used to evaluate the effects of child feeding practice on child growth.
Child feeding practice; Brief recalls; Anthropometric indices; Repeatability coefficient
Previously, a learning-free measure was used to demonstrate that chronic food restriction (FR) increases the reward magnitude of a wide range of abused drugs. Moreover, a variety of striatal neuroadaptations were detected in FR subjects, some of which are known to be involved in synaptic plasticity but have been ruled out as modulators of acute drug reward magnitude. Little is known about effects of FR on drug-conditioned place preference (CPP) and brain regional mechanisms that may enhance CPP in FR subjects. The purpose of the present study was to compare the expression and persistence of a conditioned place preference (CPP) induced by a relatively low dose of cocaine (7.0 mg/kg, i.p.) in ad libitum fed (AL) and FR rats and take several brain regional biochemical measures following the first CPP conditioning session to probe candidate mechanisms that may underlie the more robust CPP observed in FR subjects. Behaviorally, AL subjects displayed a CPP upon initial testing which extinguished rapidly over the course of subsequent test sessions while CPP in FR subjects persisted. Despite previous reports of elevated BDNF protein in forebrain regions of FR rats, the FR protocol used in the present study did not alter BDNF levels in dorsal hippocampus, nucleus accumbens or medial prefrontal cortex. On the other hand, FR rats, whether injected with cocaine or vehicle, displayed elevated p-ERK1/2 and p-Ser845-GluA1 in dorsal hippocampus. FR rats also displayed elevated p-ERK1/2 in medial prefrontal cortex and elevated p-ERK1 in nucleus accumbens, with further increases produced by cocaine. The one effect observed exclusively in cocaine-treated FR rats was increased p-Ser845-GluA1 in nucleus accumbens. These findings suggest a number of avenues for continuing investigation with potential translational significance.
food restriction; cocaine; conditioned place preference; synaptic plasticity; addiction
Adiponectin exerts multiple regulatory functions in the body and in the hypothalamus primarily through activation of its two receptors, adiponectin receptor1 and adiponectin receptor 2. Recent studies have shown that adiponectin receptors are widely expressed in other areas of the brain including the hippocampus. However, the functions of adiponectin in brain regions other than the hypothalamus are not clear. Here, we report that adiponectin can protect cultured hippocampal neurons against kainic acid-induced (KA) cytotoxicity. Adiponectin reduced the level of reactive oxygen species, attenuated apoptotic cell death, and also suppressed activation of caspase-3 induced by KA. Pretreatment of hippocampal primary neurons with an AMPK inhibitor, compound C, abolished adiponectin-induced neuronal protection. The AMPK activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, attenuated KA-induced caspase-3 activity. These findings suggest that the AMPK pathway is critically involved in adiponectin-induced neuroprotection and may mediate the antioxidative and anti-apoptotic properties of adiponectin.
Adiponectin; Neuroprotection; Hippocampus; Kainic acid; AMPK
In the present study we identified a novel gene, Homo Sapiens Chromosome 1 ORF109 (c1orf109, GenBank ID: NM_017850.1), which encodes a substrate of CK2. We analyzed the regulation mode of the gene, the expression pattern and subcellular localization of the predicted protein in the cell, and its role involving in cell proliferation and cell cycle control.
Dual-luciferase reporter assay, chromatin immunoprecipitation and EMSA were used to analysis the basal transcriptional requirements of the predicted promoter regions. C1ORF109 expression was assessed by western blot analysis. The subcellular localization of C1ORF109 was detected by immunofluorescence and immune colloidal gold technique. Cell proliferation was evaluated using MTT assay and colony-forming assay.
We found that two cis-acting elements within the crucial region of the c1orf109 promoter, one TATA box and one CAAT box, are required for maximal transcription of the c1orf109 gene. The 5′ flanking region of the c1orf109 gene could bind specific transcription factors and Sp1 may be one of them. Employing western blot analysis, we detected upregulated expression of c1orf109 in multiple cancer cell lines. The protein C1ORF109 was mainly located in the nucleus and cytoplasm. Moreover, we also found that C1ORF109 was a phosphoprotein in vivo and could be phosphorylated by the protein kinase CK2 in vitro. Exogenous expression of C1ORF109 in breast cancer Hs578T cells induced an increase in colony number and cell proliferation. A concomitant rise in levels of PCNA (proliferating cell nuclear antigen) and cyclinD1 expression was observed. Meanwhile, knockdown of c1orf109 by siRNA in breast cancer MDA-MB-231 cells confirmed the role of c1orf109 in proliferation.
Taken together, our findings suggest that C1ORF109 may be the downstream target of protein kinase CK2 and involved in the regulation of cancer cell proliferation.
Promoter; Transcription; CK2 kinase; c1orf109; Proliferation
Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus that was originally identified from human prostate cancer patients and subsequently linked to chronic fatigue syndrome. Recent studies showed that XMRV is a recombinant mouse retrovirus; hence, its association with human diseases has become questionable. Here, we demonstrated that XMRV envelope (Env)-mediated pseudoviral infection is not blocked by lysosomotropic agents and cellular protease inhibitors, suggesting that XMRV entry is not pH-dependent. The full length XMRV Env was unable to induce syncytia formation and cell-cell fusion, even in cells overexpressing the viral receptor, XPR1. However, truncation of the C-terminal 21 or 33 amino acid residues in the cytoplasmic tail (CT) of XMRV Env induced substantial membrane fusion, not only in the permissive 293 cells but also in the nonpermissive CHO cells that lack a functional XPR1 receptor. The increased fusion activities of these truncations correlated with their enhanced SU shedding into culture media, suggesting conformational changes in the ectodomain of XMRV Env. Noticeably, further truncation of the CT of XMRV Env proximal to the membrane-spanning domain severely impaired the Env fusogenicity, as well as dramatically decreased the Env incorporations into MoMLV oncoretroviral and HIV-1 lentiviral vectors resulting in greatly reduced viral transductions. Collectively, our studies reveal that XMRV entry does not require a low pH or low pH-dependent host proteases, and that the cytoplasmic tail of XMRV Env critically modulates membrane fusion and cell entry. Our data also imply that additional cellular factors besides XPR1 are likely to be involved in XMRV entry.
In the crystal structure of the title compound, C5H8N2O, molecules are linked by weak C—H⋯O hydrogen bonds, forming a three-dimensional network.
Background and Aims
Chronic hepatitis C (HCV) is a liver disease affecting over 3 million Americans. Liver biopsy is the gold standard for assessing liver fibrosis and is used as a benchmark for initiating treatment, though it is expensive and carries risks of complications. FibroTest is a non-invasive biomarker assay for fibrosis, proposed as a screening alternative to biopsy.
We assessed the cost-effectiveness of FibroTest and liver biopsy used alone or sequentially for six strategies followed by treatment of eligible U.S. patients: FibroTest only; FibroTest with liver biopsy for ambiguous results; FibroTest followed by biopsy to rule in; or to rule out significant fibrosis; biopsy only (recommended practice); and treatment without screening. We developed a Markov model of chronic HCV that tracks fibrosis progression. Outcomes were expressed as expected lifetime costs (2009 USD), quality-adjusted life-years (QALYs), and incremental cost-effectiveness ratios (ICER).
Treatment of chronic HCV without fibrosis screening is preferred for both men and women. For genotype 1 patients treated with pegylated interferon and ribavirin, the ICERs are $5,400/QALY (men) and $6,300/QALY (women) compared to FibroTest only; the ICERs increase to $27,200/QALY (men) and $30,000/QALY (women) with the addition of telaprevir. For genotypes 2 and 3, treatment is more effective and less costly than all alternatives. In clinical settings where testing is required prior to treatment, FibroTest only is more effective and less costly than liver biopsy. These results are robust to multi-way and probabilistic sensitivity analyses.
Early treatment of chronic HCV is superior to the other fibrosis screening strategies. In clinical settings where testing is required, FibroTest screening is a cost-effective alternative to liver biopsy.
We report key mechanistic differences between the reverse transcriptases (RT) of human immunodeficiency virus type-1 (HIV-1) and of xenotropic murine leukemia virus-related virus (XMRV), a gammaretrovirus that can infect human cells. Steady and pre-steady state kinetics demonstrated that XMRV RT is significantly less efficient in DNA synthesis and in unblocking chain-terminated primers. Surface plasmon resonance experiments showed that the gammaretroviral enzyme has a remarkably higher dissociation rate (koff) from DNA, which also results in lower processivity than HIV-1 RT. Transient kinetics of mismatch incorporation revealed that XMRV RT has higher fidelity than HIV-1 RT. We identified RNA aptamers that potently inhibit XMRV, but not HIV-1 RT. XMRV RT is highly susceptible to some nucleoside RT inhibitors, including Translocation Deficient RT inhibitors, but not to non-nucleoside RT inhibitors. We demonstrated that XMRV RT mutants K103R and Q190M, which are equivalent to HIV-1 mutants that are resistant to tenofovir (K65R) and AZT (Q151M), are also resistant to the respective drugs, suggesting that XMRV can acquire resistance to these compounds through the decreased incorporation mechanism reported in HIV-1.
Type I interferon protects cells from virus infection through the induction of a group of genes collectively named interferon-stimulated genes (ISGs). In this study, we utilized short hairpin RNA (shRNA) to deplete ISGs in SupT1 cells in order to identify ISGs that suppress the production of human immunodeficiency virus type 1 (HIV-1). Among the ISG candidates thus identified were interferon-induced transmembrane (IFITM) proteins, including IFITM1, IFITM2, and IFITM3, that potently inhibit HIV-1 replication at least partially through interfering with virus entry. Further mutagenesis analysis shows that the intracellular region, rather than the N- and C-terminal extracellular domains, is essential for the antiviral activity of IFITM1. Altogether, these data suggest that the IFITM proteins serve as important components of the innate immune system to restrict HIV-1 infection.
Rab GTPases function as modulators in intracellular transport. Rab5a, a member of the Rab subfamily of small GTPases, is an important regulator of vesicle traffic from the plasma membrane to early endosomes. Recent findings have reported that Rab5a gene was involved in the progression of cancer. In the present study, we investigated the effect of Rab5a on cervical cancer invasion and metastasis and the molecular mechanism underlying the involvement of Rab5a.
Rab5a expression was assessed by immunohistochemical analysis on a cervical cancer tissue microarray. RNA interference (RNAi) was performed to knock down the endogenous expression of Rab5a gene in HeLa and SiHa cells. Cell motility was evaluated using invasion assay and wound migration assay in vitro. The expression levels of integrin-associated molecules were detected by Western blot and immunofluorescence.
We found that Rab5a was expressed at a high level in cervical cancer tissues. Silencing of Rab5a expression significantly decreased cancer cell motility and invasiveness. The down-regulation of integrin-associated focal adhesion signaling molecules was further detected in Rab5a knockdown cells. Meanwhile, active GTP-bound Rac1, Cdc42, and RhoA were also down-regulated, accompanied with the reduction in the number and size of filopodia and lamellipodia.
Taken together, these data suggest that Rab5a functions in regulating the invasion phenotype, and we propose that this regulation may be via integrin-mediated signaling pathway in cervical cancer cells.
Osteoporotic hip fracture (HF) is a serious global public health problem associated with high morbidity and mortality. Hip bone size (BS) has been identified as one of key measurable risk factors for HF, independent of bone mineral density (BMD). Hip BS is highly genetically determined, but genetic factors underlying BS variation are still poorly defined. Here, we performed an initial genome-wide copy number variation (CNV) association analysis for hip BS in 1,627 Chinese Han subjects using Affymetrix GeneChip Human Mapping SNP 6.0 Array and a follow-up replicate study in 2,286 unrelated US Caucasians sample. We found that a copy number polymorphism (CNP267) located at chromosome 2q12.2 was significantly associated with hip BS in both initial Chinese and replicate Caucasian samples with p values of 4.73E-03 and 5.66E-03, respectively. An important candidate gene, four and a half LIM domains 2 (FHL2), was detected at the downstream of CNP267, which plays important roles in bone metabolism by binding to several bone formation regulator, such as insulin-like growth factor-binding protein 5 (IGFBP-5) and androgen receptor (AR). Our findings suggest that CNP267 region may be associated with hip BS which might influence the FHL2 gene downstream.
Two heterologous expression systems using thioredoxin (trxA) as a gene fusion part in Escherichia coli were developed to produce recombinant pediocin PA-1. Pediocin PA-1 structural gene pedA was isolated from Pediococcus acidilactici PA003 by the method of polymerase chain reaction (PCR), then cloned into vector pET32a(+), and expressed as thioredoxin-PedA fusion protein in the host strain E. coli BL21 (DE3). The fusion protein was in the form of inclusion body and was refolded before purification by nickel-iminodiacetic acid (Ni-IDA) agarose resin column. Biological activity of recombinant pediocin PA-1 was analyzed after cleavage of the fusion protein by enterokinase. Agar diffusion test revealed that 512-arbitrary unit (AU) recombinant pediocin PA-1 was obtained from 1 ml culture medium of E. coli (pPA003PED1) using Listeria monocytogenes as the indicator strain. Thioredoxin-PedA fusion gene was further cloned into pET20b(+). Thioredoxin-PedA fusion protein was detected in both the periplasmic and cytoplasmic spaces. The recombinant pediocin PA-1 from the soluble fraction attained 384 AU from 1 ml culture medium of E. coli (pPA003PED2). Therefore, biologically active pediocin PA-1 could be obtained by these two hybrid gene expression methods.
Bacteriocin; Fusion expression; Inclusion body; Pediocin PA-1; Thioredoxin
The alpha-2,6-sialyltransferase, ST6Gal-I, is a key enzyme that regulates the distribution of sialic acid-containing molecules on mammalian cell surfaces. However, the fact that its native form is membrane-bound and glycosylated has made structural characterization by X-ray crystallography of this eukaryotic protein difficult. Its large size (~40 KDa for just the catalytic domain) also poses a challenge for complete structure determination by nuclear magnetic resonance (NMR). However, even without complete structure determination, there are NMR strategies that can return targeted information about select regions of the protein, including information about the active site as seen from the perspective of its bound ligands. Here, in a continuation of a previous study, a spin-labeled mimic of a glycan acceptor ligand is used to identify additional amino acids located in the protein active site. In addition, the spin-labeled donor is used to characterize the relative placement of the two bound ligands. The ligand conformation and protein-ligand contact surfaces are studied by transferred nuclear Overhauser effects (trNOEs) and saturation transfer difference (STD) experiments. The data afforded by the above methods lead to a geometric model of the bound substrates that in many ways carries an imprint of the ST6Gal-I binding site.
In the title compound, C14H13NO, the two six-membered rings make a dihedral angle of 52.8 (3)°. An intramolecular N—H⋯O hydrogen bond involving an amine H atom and the adjacent carbonyl O atom occurs. In the crystal, N—H⋯O and C—H⋯N intermolecular hydrogen bonds are observed, which may be effective in stabilizing the structure.