The discovery of new methods for the synthesis of classes of potentially bioactive molecules remains an important goal for synthetic chemists. Vinylsulfonium salts have been used for the synthesis of a wide variety of small heterocyclic motifs; however, further developments to this important class of reagents has been focused on reaction with new substrates rather than development of new vinylsulfonium salts. We herein report the synthesis of a range of ?-substituted vinylsulfonium tetraphenylborates (10 examples) in a 3 step procedure from commercially available styrenes. The important role of the tetraphenylborate counterion on the stability and accessibility of the vinylsulfonium salts is also detailed. The ?-substituted vinylsulfonium tetraphenylborates gave good to excellent yields in the epoxyannulation of ?-amino ketones (15 examples) and the cyclopropanation of allylic amines (4 examples). Hydrogenation of an epoxyannulation product proceeded with good diastereoselectivity.
Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ? 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ? 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.
Abstract Developing oat cultivars with partial resistance to crown rust would be beneficial and cost-effective for disease management. Two recombinant inbred line populations were generated by crossing the susceptible cultivar 'Provena' with two partially resistant sources, 'CDC Boyer' and breeding line 94197A1-9-2-2-2-5. A third mapping population was generated by crossing the partially resistant sources to validate the QTL results. The three populations were evaluated for crown rust severity in the field at Louisiana State University (LSU) in 2009 and 2010 and at the Cereal Disease Laboratory (CDL) in St. Paul, Minnesota in 2009, 2010, and 2011. An iSelect platform assays containing 5744 oat single nucleotide polymorphisms was used to genotype the populations. From the 2009 CDL test, linkage analyses revealed two QTL for partial resistance in the Provena/CDC Boyer population on chromosome 19A. One of the 19A QTL was also detected in the 2009 LSU test. Another QTL was detected in on chromosome 12D in the CDL 2009 test. In the Provena/94197A1-9-2-2-2-5 population, only one QTL was detected on chromosome 13A in the CDL 2011 test. The 13A QTL from the Provena/94197A1-9-2-2-2-5 population was validated in CDC Boyer /94197A1-9-2-2-2-5 population in the CDL 2010 and 2011 tests. Comparative analysis of the significant markers sequences with the rice genome database revealed 15 candidate genes for disease resistance on chromosomes 4 and 6 of rice. These genes could be potential targets for cloning from the two resistant parents.
Nonalcoholic fatty liver disease (NAFLD) is recognized as the most common cause of chronic liver disease in the United States. Nonalcoholic steatohepatitis (NASH) occurs in a subset of patients with NAFLD and is characterized by the presence of hepa-tocellular injury, which is progressive in a substantial proportion of cases and can lead to cirrhosis and all of its complications. Although the diagnosis of NAFLD can be made through imaging studies or liver biopsy, the diagnosis of NASH still requires histologic confirmation. Liver biopsy should be performed in the presence of risk factors for advanced disease. Measures aimed at promoting weight loss, a healthier lifestyle, and optimization of metabolic risk factors remain the cornerstone of management of NAFLD. Therapeutic agents that are presently considered the most promising in NAFLD are effective in less than 50% of patients. Among patients with biopsy-proven NASH, treatment with pharmacologic agents should be considered; however, the role of specific agents in NASH still needs further study. Despite a wealth of research over the past 15 years, many controversies remain with respect to the diagnosis and management of NAFLD and NASH as well as the influence of alcohol on liver disease progression in these patients.
Rapidly evolving pathogens, such as human immunodeficiency and influenza viruses, escape immune defenses provided by most vaccine-induced antibodies. Proposed strategies to elicit broadly neutralizing antibodies require a deeper understanding of antibody affinity maturation and evolution of the immune response to vaccination or infection. In HIV-infected individuals, viruses and B cells evolve together, creating a virus-antibody "arms race." Analysis of samples from an individual designated CH505 has illustrated the interplay between an antibody lineage, CH103, and autologous viruses at various time points. The CH103 antibodies, relatively broad in their neutralization spectrum, interact with the CD4 binding site of gp120, with a contact dominated by CDRH3. We show by analyzing structures of progenitor and intermediate antibodies and by correlating them with measurements of binding to various gp120s that there was a shift in the relative orientation of the light- and heavy-chain variable domains during evolution of the CH103 lineage. We further show that mutations leading to this conformational shift probably occurred in response to insertions in variable loop 5 (V5) of the HIV envelope. The shift displaced the tips of the light chain away from contact with V5, making room for the inserted residues, which had allowed escape from neutralization by the progenitor antibody. These results, which document the selective mechanism underlying this example of a virus-antibody arms race, illustrate the functional significance of affinity maturation by mutation outside the complementarity determining region surface of the antibody molecule.
Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.
In the 2006 Report of the National Lipid Association's Statin Safety Task Force, a panel of experts in hepatology published their findings on specific questions related to the liver blood testing during statin therapy. Among their recommendations was that regulatory agencies reconsider the statin-labeling recommendation at that time, which required post-statin liver enzyme testing. Since then, the Food and Drug Administration altered statin labeling such that unless clinically indicated for other reasons, after a pre-statin therapy baseline evaluation, follow-up liver enzyme testing was not uniformly required after statin initiation. This 2014 report provides an update on interim issues relevant to statins and liver safety. Some of the points discussed include the value of baseline liver enzymes before initiating statin therapy, safety of statin use in patients with nonalcoholic fatty liver disease, potential drug interactions between statins and drugs used to treat hepatitis, the use of statins in liver transplant recipients, and the use of statins in patients with autoimmune liver disease. Finally, this panel provides diagnostic and algorithmic approaches when evaluating statin-treated patients who experience elevations in liver enzymes.
Tocochromanols are recognized for nutritional content, plant stress response, and seed longevity. Here we present a systems biological approach to characterize and develop predictive assays for genes affecting tocochromanol variation in barley. Major QTL, detected in three regions of a SNP linkage map, affected multiple tocochromanol forms. Candidate genes were identified through barley/rice orthology and sequenced in genotypes with disparate tocochromanol profiles. Gene-specific markers, designed based on observed polymorphism, mapped to the originating QTL, increasing R2 values at the respective loci. Polymorphism within promoter regions corresponded to motifs known to influence gene expression. Quantitative PCR analysis revealed a trend of increased expression in tissues grown at cold temperatures. These results demonstrate utility of a novel method for rapid gene identification and characterization, and provide a resource for efficient development of barley lines with improved tocochromanol profiles.
One of the key goals of the current reforms in the English National Health Service (NHS) under the Health and Social Care Act, 2012, is to increase the accountability of those responsible for commissioning care for patients (clinical commissioning groups (CCGs)), while at the same time allowing them a greater autonomy. This study was set out to explore CCGs developing accountability relationships.
Nutrition has not been a primary focus of many medical conditions despite its importance in the development and the severity of these diseases. This is certainly the case with nutrition and end-stage liver disease despite the well-established association of nutritional deficiencies and increased rates of complications and mortality in cirrhosis. This review provides an overview of nutrition in chronic liver disease with an emphasis on its pathogenesis as well as ways to assess nutritional status and intervene in an effort to improve nutrition.
Nonalcoholic fatty liver disease (NAFLD) remains the most common chronic liver disease in the western world and its prevalence is rising elsewhere. Among patients with NAFLD, those with nonalcoholic steatohepatitis (NASH) represent a large potential public health concern with risk for development of cirrhosis and hepatocellular carcinoma. The ability to diagnose and treat NAFLD and NASH has improved and continues to improve as understanding of the pathogenesis of this disease develops. This article highlights the key features of NAFLD and NASH, as well as the available and future promising treatment options.
The current reorganisation of the English NHS is one of the most comprehensive ever seen. This study reports early evidence from the development of clinical commissioning groups (CCGs), a key element in the new structures.
Plasmodium falciparum is an intracellular protozoan parasite that infects erythrocytes and hepatocytes. The blood stage of its life cycle causes substantial morbidity and mortality associated with millions of infections each year, motivating an intensive search for potential components of a multi-subunit vaccine. In this study, we present data showing that antibodies from natural infections can recognize a recombinant form of the relatively conserved merozoite surface antigen, PfRH5. Furthermore, we performed invasion inhibition assays on clinical isolates and laboratory strains of P. falciparum in the presence of affinity purified antibodies to RH5 and show that these antibodies can inhibit invasion in vitro.
Patients infected with hepatitis C virus (HCV) with elevated low-density lipoprotein (LDL) levels achieve higher sustained virologic response (SVR) rates after peginterferon (PegIFN)/ribavirin treatment versus patients with lower LDL. Our aim was to determine whether SVR rates in patients with low/elevated LDL can be improved by dose intensification.
With expanding waistlines, the prevalence of NAFLD has burgeoned to become the leading cause of chronic liver disease in the USA. A subset of patients with NAFLD meet criteria for NASH with its inherent risk of progression to cirrhosis. Verma et al. addressed the utility of alanine aminotransferase levels for predicting NASH or advanced fibrosis to decide who would benefit from the definitive test of liver biopsy.
Nonalcoholic fatty liver disease (NAFLD) is now recognized as part of the metabolic syndrome, and is specifically related to obesity and insulin resistance. Lifestyle modification is advocated for the treatment of NAFLD, but few studies have evaluated its impact on liver histology. The purpose of this study was to investigate which, if any, specific diet and exercise recommendations are associated with histopathologic changes.
The purpose of this paper is to explore the practical daily work undertaken by middle-level managers in Primary Care Trusts (PCTs), focusing upon the micro-processes by which these managers enact sensemaking in their organisations.
Colonoscopy is a well-accepted colon cancer screening modality that is recommended by the United States Multi-Society Task Force on all individuals greater than 50 years of age. Chronic hepatitis C (CHC) is a common cause of chronic liver disease with notably increased rates of infection in individuals born between 1945 and 1965. The Centers for Disease Control recently recommended all individuals of this "Baby Boomer" cohort undergo one time screening for CHC. As gastroenterologists interface with these patients for screening colonoscopy, this represents a unique opportunity to complete this screening and identify CHC patients at risk for advanced liver disease.
The language used by National Health Service (NHS) "commissioning" managers when discussing their roles and responsibilities can be seen as a manifestation of "identity work", defined as a process of identifying. This paper aims to offer a novel approach to analysing "identity work" by triangulation of multiple analytical methods, combining analysis of the content of text with analysis of its form.
Historically, primary medical care in the UK has been delivered by general practitioners who are independent contractors, operating under a contract, which until 2004 was subject to little performance management. In keeping with the wider political impetus to introduce markets and competition into the NHS, reforms were introduced to allow new providers to bid for contracts to provide primary care services in England. These contracts known as Alternative Provider Medical Services, were encouraged by two centrally-driven rounds of procurement (2007/8 and 2008/9). This research investigated the commissioning and operation of such Alternative Providers of Primary Care (APPCs).
Objective. To define the concept of "organizational philosophy" through identification of elements within undergraduate pharmacy curricula in the United Kingdom that contribute to students learning of professionalism. Methods. A qualitative study using curriculum mapping was conducted to identify "intended," "taught," and "received" curriculum in 3 schools of pharmacy. The study involved review of course materials, interviews with teaching staff members, focus groups with final year students, and observation of classes. Results. "Organizational philosophy" (totality of all contributors) played a vital part in students professionalism learning. Key contributions were not restricted to the "taught" curriculum but extended to the wider academic environment. Setting of high standards appeared important; role models had particular significance. Importance of professionalism learning being grounded and longitudinal throughout the curriculum was highlighted. An "integrated" organizational philosophy appeared to be achieved where maximum overlap occurred between "intended," "taught," and "received" curricula. Conclusions. Professionalism learning goes beyond the "taught" curriculum in pharmacy schools. The concept of "organizational philosophy" acknowledges the importance of integration between "intended," "taught," and "received" curriculum in the context of overall organization.
Vitamin D is a secosteroid with known effects on calcium homeostasis that has recently been shown to have other significant functions regarding immune modulation, cell differentiation and proliferation, and the inflammatory response. As our understanding of the many functions of vitamin D has grown, the presence of vitamin D deficiency (VDD) has become more evident in Western populations. Concomitantly, nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease. NAFLD and VDD are often found together, and while this is not unexpected, given their similar associations with obesity and sedentary lifestyle, a growing body of evidence points to a closely linked and potentially causative relationship between VDD and NAFLD. The epidemiologic association between VDD and NAFLD as well as the role of VDD in the pathogenesis of NAFLD and the available evidence on the clinical utility of vitamin D replacement in NAFLD populations are discussed.
Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism. DOI:http://dx.doi.org/10.7554/eLife.00333.001.
Boceprevir (BOC) added to peginterferon alfa-2b (PegIFN) and ribavirin (RBV) significantly increases sustained virologic response (SVR) rates over PegIFN/RBV alone in previously untreated adults with chronic hepatitis C genotype 1. We evaluate the relationship of incident anemia with triple therapy. A total of 1,097 patients received a 4-week lead-in of PegIFN/RBV followed by: (1) placebo plus PegIFN/RBV for 44 weeks (PR48); (2) BOC plus PegIFN/RBV using response-guided therapy (BOC/RGT); and (3) BOC plus PegIFN/RBV for 44 weeks (BOC/PR48). The management of anemia (hemoglobin [Hb]<10 g/dL) included RBV dose reduction and/or erythropoietin (EPO) use. A total of 1,080 patients had ?1 Hb measurement during treatment. The incidence of anemia was 50% in the BOC arms combined (363/726) and 31% in the PR48 arm (108/354, P<0.001). Among BOC recipients, lower baseline Hb and creatinine clearance were associated with incident anemia. In the BOC-containing arms, anemia was managed by the site investigators as follows: EPO without RBV dose reduction, 38%; RBV dose reduction without EPO, 8%; EPO with RBV dose reduction, 40%; and neither RBV dose reduction nor EPO, 14%. SVR rates were not significantly affected by management strategy (70%-74%), and overall patients with anemia had higher rates of SVR than those who did not develop anemia (58%). Serious and life-threatening adverse events (AEs) and discontinuations due to AEs among BOC-treated patients did not differ by EPO use.
Accurate segregation of genetic material in eukaryotes relies on the kinetochore, a multiprotein complex that connects centromeric DNA with microtubules. In yeast and humans, two proteins-Mif2/CENP-C and Chl4/CNEP-N-interact with specialized centromeric nucleosomes and establish distinct but cross-connecting axes of chromatin-microtubule linkage. Proteins recruited by Chl4/CENP-N include a subset that regulates chromosome transmission fidelity. We show that Chl4 and a conserved member of this subset, Iml3, both from Saccharomyces cerevisiae, form a stable protein complex that interacts with Mif2 and Sgo1. We have determined the structures of an Iml3 homodimer and an Iml3-Chl4 heterodimer, which suggest a mechanism for regulating the assembly of this functional axis of the kinetochore. We propose that at the core centromere, the Chl4-Iml3 complex participates in recruiting factors, such as Sgo1, that influence sister chromatid cohesion and encourage sister kinetochore biorientation.
A physically anchored consensus map is foundational to modern genomics research; however, construction of such a map in oat (Avena sativa L., 2n?=?6x?=?42) has been hindered by the size and complexity of the genome, the scarcity of robust molecular markers, and the lack of aneuploid stocks. Resources developed in this study include a modified SNP discovery method for complex genomes, a diverse set of oat SNP markers, and a novel chromosome-deficient SNP anchoring strategy. These resources were applied to build the first complete, physically-anchored consensus map of hexaploid oat. Approximately 11,000 high-confidence in silico SNPs were discovered based on nine million inter-varietal sequence reads of genomic and cDNA origin. GoldenGate genotyping of 3,072 SNP assays yielded 1,311 robust markers, of which 985 were mapped in 390 recombinant-inbred lines from six bi-parental mapping populations ranging in size from 49 to 97 progeny. The consensus map included 985 SNPs and 68 previously-published markers, resolving 21 linkage groups with a total map distance of 1,838.8 cM. Consensus linkage groups were assigned to 21 chromosomes using SNP deletion analysis of chromosome-deficient monosomic hybrid stocks. Alignments with sequenced genomes of rice and Brachypodium provide evidence for extensive conservation of genomic regions, and renewed encouragement for orthology-based genomic discovery in this important hexaploid species. These results also provide a framework for high-resolution genetic analysis in oat, and a model for marker development and map construction in other species with complex genomes and limited resources.
Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3)(2)-(Ndc10)(2) recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 Å resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly.
Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of ?-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.
Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.
Medication combinations that improve the efficacy of thiazolidinediones or ameliorate weight-gain side effects of therapy represent an attractive potential treatment for (NASH). The aim of this randomized, open-label trial was to assess the efficacy of rosiglitazone and metformin in combination versus rosiglitazone and losartan, compared to rosiglitazone alone, after 48 weeks of therapy. A total of 137 subjects with biopsy-proven NASH were enrolled and randomly assigned to receive either 4 mg twice-daily of rosiglitazone, 4 mg of rosiglitazone and 500 mg of metformin twice-daily, or 4 mg of rosiglitazone twice-daily and 50 mg of losartan once-daily for 48 weeks. Patients were screened for other etiologies of chronic liver disease, including daily alcohol intake in excess of 20 g. Repeat liver biopsy was performed after 48 weeks of therapy and reviewed in a blinded fashion by a single expert hepatopathologist. The primary aim of the study was to assess for differences between treatment groups in the improvement of steatosis, hepatocellular inflammation, and fibrosis. In total, 108 subjects completed the trial. Primary outcome revealed no significant difference between treatment groups in all histologic parameters (steatosis, P = 0.137; hepatocellular inflammation, P = 0.320; fibrosis, P = 0.229). Overall improvement in steatosis, hepatocellular inflammation, ballooning degeneration, and fibrosis was observed (P ? 0.001). Serum aminotransferases were reduced in all three groups (P < 0.001 within treatment, P > 0.05 between groups). Metformin did not significantly mitigate weight gain (P = 0.051).
Background: Epidemiologic data suggest that colonic adenomas have an increased tendency to occur in patients who are obese, African American, or have a positive family history of colon cancer, or diabetes mellitus. Recent data suggest that impaired glucose tolerance, dyslipidemia, and metabolic syndrome are associated with a higher risk for colonic adenomas. Patients with nonalcoholic fatty liver disease (NAFLD) often share several of the aforementioned risk factors for colonic adenomas. However, data are lacking about the relationship between NAFLD and colonic adenomas. The aim of this study was to systematically evaluate whether NAFLD is an independent risk factor for colonic adenomas.Methods: We performed a retrospective cohort observational study on 233 patients who underwent screening colonoscopies at Brooke Army Medical Center from November 2007 to March 2010 to assess for the association between NAFLD and colonic adenomas. Patients who had previously been found to have biopsy-proven simple steatosis (n?=?65) or nonalcoholic steatohepatitis (NASH) (n?=?29) were compared with a control group without fatty liver disease on sonographic imaging (n?=?139). Patients were stratified based on gender, race, body mass index (BMI), and family history and adjusted for variables previously known to be associated with increased adenoma risk.Results: The mean age was 54.7?±?6.0 years (48.5% women). Racial demographics were: 62.7% White, 18.5% Hispanic, 13.7%, African American, and 5.2% other. The mean BMI was 29.7?±?5.8. The prevalence of colonic adenomas was 25.1% in the control group and 24.4% in the NAFLD group to include simple steatosis and NASH (p?=?1.00). Furthermore, when adjusting for known confounders to include race, BMI, and family history no significant differences were found (p?=?0.33). However, the ultrasound-negative patients ranked lower in the number of adenomas per person (p?=?0.016).Conclusions: There was no difference in the prevalence of colonic adenomas when comparing the NAFLD group who had undergone colonoscopy with a group of control patients without NAFLD who had undergone colonoscopy. However, patients with negative ultrasounds appeared to have a lower polyp burden.
Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or uncoupling of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.
A specialized nucleosome is a component of all eukaryotic kinetochores. The core of this nucleosome contains a centromere-specific histone, CENP-A (the Cse4 gene product in budding yeast), instead of the usual H3. Assembly of a centromeric nucleosome depends on a specific chaperone, called Scm3 in yeast and HJURP in higher eukaryotes. We describe here the structure of a complex formed by an N-terminal fragment of Scm3 with the histone-fold domains of Cse4, and H4, all prepared as recombinant proteins derived from the budding yeast Kluyveromyces lactis. The contacts of Scm3 with Cse4 explain its selectivity for the centromere-specific histone; key residues at the interface are conserved in HJURP, indicating a common mechanism for centromeric-histone deposition. We also report the structure of a (Cse4 : H4)(2) heterotetramer; comparison with the structure of the Scm3:Cse4:H4 complex shows that tetramer formation and DNA-binding require displacement of Scm3 from the nucleosome core. The two structures together suggest that specific contacts between the chaperone and Cse4, rather than an altered overall structure of the nucleosome core, determine the selective presence of Cse4 at centromeres.
Coffee caffeine consumption (CC) is associated with reduced hepatic fibrosis in patients with chronic liver diseases, such as hepatitis C. The association of CC with nonalcoholic fatty liver disease (NAFLD) has not been established. The aim of this study was to correlate CC with the prevalence and severity of NAFLD. Patients involved in a previously published NAFLD prevalence study, as well as additional NASH patients identified in the Brooke Army Medical Center Hepatology clinic, were queried about their caffeine intake. A validated questionnaire for CC was utilized to assess for a relationship between caffeine and four groups: ultrasound negative (controls), bland steatosis/not-NASH, NASH stage 0-1, and NASH stage 2-4. A total of 306 patients responded to the CC questionnaire. Average milligrams of total caffeine/coffee CC per day in controls, bland steatosis/not-NASH, NASH stage 0-1, and NASH stage 2-4 were 307/228, 229/160, 351/255, and 252/152, respectively. When comparing patients with bland steatosis/not-NASH to those with NASH stage 0-1, there was a significant difference in CC between the two groups (P = 0.005). Additionally, when comparing patients with NASH stage 0-1 to those with NASH stage 2-4, there was a significant difference in coffee CC (P = 0.016). Spearmans rank correlation analysis further supported a negative relationship between coffee CC and hepatic fibrosis (r = -0.215; P = 0.035).
BACKGROUND The authors aimed to determine US and UK doctors professional values and reported behaviours, and the extent to which these vary with the context of care. METHOD 1891 US and 1078 UK doctors completed the survey (64.4% and 40.3% response rate respectively). Multivariate logistic regression was used to compare responses to identical questions in the two surveys. RESULTS UK doctors were more likely to have developed practice guidelines (82.8% UK vs 49.6% US, p<0.001) and to have taken part in a formal medical error-reduction programme (70.9% UK vs 55.7% US, p<0.001). US doctors were more likely to agree about the need for periodic recertification (completely agree 23.4% UK vs 53.9% US, p<0.001). Nearly a fifth of doctors had direct experience of an impaired or incompetent colleague in the previous 3 years. Where the doctor had not reported the colleague to relevant authorities, reasons included thinking that someone else was taking care of the problem, believing that nothing would happen as a result, or fear of retribution. UK doctors were more likely than US doctors to agree that significant medical errors should always be disclosed to patients. More US doctors reported that they had not disclosed an error to a patient because they were afraid of being sued. DISCUSSION The context of care may influence both how professional values are expressed and the extent to which behaviours are in line with stated values. Doctors have an important responsibility to develop their healthcare systems in ways which will support good professional behaviour.
Since the discovery over 15 years ago of a protein transcription factor that possessed the ability to cross the plasma membrane, cell-penetrating peptides (CPPs) have been evaluated for the ability to transport diverse cargoes into cells, tissues, and organs. Certain CPPs have been used for the intracellular delivery of information-rich molecules to modulate protein-protein interactions and thereby inhibit key cellular mechanisms of disease. The ability to introduce drugs into cells allows the conventional biodistribution of drugs to be altered in order to favorably impact toxicity, patient compliance, and other treatment factors. In this monograph, we present the current status and future prospects for the application of CPPs to the development of human therapeutics. We discuss some of the advantages and disadvantages of using CPPs in the in vivo setting, and review the current status of a number of preclinical and human clinical studies of CPP-mediated delivery of therapeutics. These include CPP-conjugated moieties directed against a growing variety of targets and disease areas, including cancer, cardiology, pain, and stroke. Our discussion focuses on those therapeutics that have been tested in humans, including a CPP conjugate for the treatment of acute myocardial infarction. The promising results obtained in a number of these studies indicate that CPPs may have an important role in the development of novel therapeutics.
Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.
Nine different near-atomic resolution structures of icosahedral viruses, determined by electron cryo-microscopy and published between early 2008 and late 2010, fulfil predictions made 15 years ago that single-particle cryo-EM techniques could visualize molecular detail at 3-4? resolution. This review summarizes technical developments, both in instrumentation and in computation, that have led to the new structures, which advance our understanding of virus assembly and cell entry.
Insulin resistance and diabetes are inextricably linked to chronic hepatitis C. Our understanding of this relationship continues to improve. This review focuses on the molecular mechanisms relating insulin resistance to hepatitis C with a subsequent overview of the consequences of hepatitis C-associated insulin resistance and diabetes, as well as perspectives for future management.
A novel series of AMPAR positive modulators is described that were identified by high throughput screening. The molecules of the series have been optimized from a high quality starting point hit to afford excellent developability, tolerability, and efficacy profiles, leading to identification of a clinical candidate. Unusually for an ion channel target, this optimization was integrated with regular generation of ligand-bound crystal structures and uncovered a novel chemotype with a unique and highly conserved mode of interaction via a trifluoromethyl group.
HCV has been classified into no fewer than six major genotypes and a series of subtypes. Each HCV genotype is unique with respect to its nucleotide sequence, geographic distribution, and response to therapy. Genotypes 1, 2, and 3 are common throughout North America and Europe. HCV genotype 4 (HCV-4) is common in the Middle East and in Africa, where it is responsible for more than 80% of HCV infections. It has recently spread to several European countries. HCV-4 is considered a major cause of chronic hepatitis, cirrhosis, hepatocellular carcinoma, and liver transplantation in these regions. Although HCV-4 is the cause of approximately 20% of the 170 million cases of chronic hepatitis C in the world, it has not been the subject of widespread research. Therefore, this document, drafted by a panel of international experts, aimed to review current knowledge on the epidemiology, natural history, clinical, histological features, and treatment of HCV-4 infections.
Peptides derived from the "stem" of dengue virus (DV) type 2 (DV2) envelope (E) protein inhibit DV2 infectivity, targeting a late-stage fusion intermediate. We show here that stem peptides from all DV serotypes cross-inhibit DV1 to DV4 but that corresponding peptides derived from related flaviviruses do not. This failure to inhibit infection is not due to poor interaction with the E protein but rather to loss of association with the virion membrane. Residues 442 to 444 of the stem are determinants of inhibition; increasing hydrophobicity in this region increases inhibitory strength. These results support a two-step model of how stem-derived peptides inhibit viral entry.
Successful colonoscopies require good bowel preparations-poor bowel preparations can increase medical costs, rates of missed lesions, and procedure duration. The combination of polyethylene glycol (PEG) 3350 without electrolytes (MiraLAX; Schering-Plough Healthcare Products, Inc, Kenilworth, NJ) and 64 oz of Gatorade (PepsiCo, Inc, Purchase, NY) has gained popularity as a bowel preparation regimen. However, the efficacy and tolerability of this approach has not been compared with standard bowel preparations in clinical trials. We compared split-dose (PEG) 3350 with electrolytes (GoLytely; Braintree Laboratories, Inc, Braintree, MA) with split-dose MiraLAX alone and in combination with pretreatment medications (bisacodyl or lubiprostone) to determine the efficacy and patient tolerability of MiraLAX as an agent for bowel preparation.
Non-enveloped viruses of different types have evolved distinct mechanisms for penetrating a cellular membrane during infection. Rotavirus penetration appears to occur by a process resembling enveloped-virus fusion: membrane distortion linked to conformational changes in a viral protein. Evidence for such a mechanism comes from crystallographic analyses of fragments of VP4, the rotavirus-penetration protein, and infectivity analyses of structure-based VP4 mutants. We describe here the structure of an infectious rotavirus particle determined by electron cryomicroscopy (cryoEM) and single-particle analysis at about 4.3 Å resolution. The cryoEM image reconstruction permits a nearly complete trace of the VP4 polypeptide chain, including the positions of most side chains. It shows how the two subfragments of VP4 (VP8(*) and VP5(*)) retain their association after proteolytic cleavage, reveals multiple structural roles for the ?-barrel domain of VP5(*), and specifies interactions of VP4 with other capsid proteins. The virion model allows us to integrate structural and functional information into a coherent mechanism for rotavirus entry.
This paper is a report of a study conducted to explore how specialist heart failure nurses negotiate treatment advice with patients, in the context of an increasing expectation that clinical staff in the National Health Services will follow guidelines in their daily work.
Prevalence of nonalcoholic fatty liver disease (NAFLD) has not been well established. The purpose of this study was to prospectively define the prevalence of both NAFLD and nonalcoholic steatohepatitis (NASH).
Elevated low-density lipoprotein (LDL) levels and statin use have been associated with higher sustained virological response (SVR) rates in patients receiving chronic hepatitis C therapy. However, these relationships have not been well characterized in randomized controlled trials. Furthermore, little is known about the relationship between high-density lipoprotein (HDL) and virological response. To determine whether baseline LDL or HDL levels and statin use affect SVR rates, we retrospectively evaluated the IDEAL (Individualized Dosing Efficacy Versus Flat Dosing to Assess Optimal Pegylated Interferon Therapy) trial, in which 3070 treatment-naive, hepatitis C virus (HCV) genotype 1-infected patients were treated for up to 48 weeks in one of the following arms: (1) peginterferon (PEG-IFN) alfa-2b at 1.5 microg/kg/week with ribavirin (RBV) at 800 to 1400 mg/day, (2) PEG-IFN alfa-2b at 1.0 microg/kg/week with RBV at 800 to 1400 mg/day, or (3) PEG-IFN alfa-2a at 180 microg/week with RBV at 1000 to 1200 mg/day. Virological responses were assessed by pretreatment statin use and baseline elevated LDL levels (> or =130 mg/dL) or low HDL levels (<40 mg/dL for men and <50 mg/dL for women). In 1464 patients with baseline elevated LDL levels or low HDL levels, the SVR rate was significantly higher than that in patients with normal levels (44.9% versus 34.0%, P < 0.001). In 66 patients receiving a statin pretreatment, the SVR rate was higher than the rate of those not receiving it (53.0% versus 39.3%, P = 0.02). In a multivariate logistic regression analysis using the stepwise selection method with baseline characteristics, a high LDL level [odds ratio (OR) = 1.6, 95% confidence interval (CI) = 1.4-1.8, P < 0.001], a low HDL level (OR = 0.5, 95% CI = 0.3-0.8, P = 0.004), and statin use (OR = 2.0, 95% CI = 1.1-3.7, P = 0.02) were independently associated with SVR.
In 2003, the new General Medical Services Contract introduced a pay-for-performance programme know as the Quality and Outcomes Framework (QOF) into UK general practice, with payment for meeting a number of both clinical and organisational quality standards.
Wnt association with its receptor, Frizzled (Fz), and recruitment by the latter of an adaptor, Dishevelled (Dvl), initiates signaling through at least two distinct pathways ("canonical" and "noncanonical"). Endocytosis and compartmentalization help determine the signaling outcome. Our previous work has shown that Dvl2 links at least one Frizzled family member (Fz4) to clathrin-mediated endocytosis by interacting with the ?2 subunit of the AP-2 clathrin adaptor, through both a classical endocytic tyrosine motif and a so-called "DEP domain." We report here the crystal structure of a chimeric protein that mimics the Dvl2-?2 complex. The DEP domain binds at one end of the elongated, C-terminal domain of ?2. This domain:domain interface shows that parts of the ?2 surface distinct from the tyrosine-motif site can help recruit specific receptors or adaptors into a clathrin coated pit. Mutation of residues at the DEP-?2 contact or in the tyrosine motif reduce affinity of Dvl2 for ?2 and block efficient internalization of Fz4 in response to ligation by Wnt5a. The crystal structure has thus allowed us to identify the specific interaction that leads to Frizzled uptake and to downstream, noncanonical signaling events.
Patients infected with hepatitis C virus (HCV) genotype 1, body weight ?85 kg, and high baseline viral load respond poorly to standard doses of pegylated interferon (peginterferon) and ribavirin. We evaluated intensified therapy with peginterferon alfa-2a plus ribavirin.
The monopolin complex regulates different types of kinetochore-microtubule attachments in fungi, ensuring sister chromatid co-orientation in Saccharomyces cerevisiae meiosis I and inhibiting merotelic attachment in Schizosaccharomyces pombe mitosis. In addition, the monopolin complex maintains the integrity and silencing of ribosomal DNA (rDNA) repeats in the nucleolus. We show here that the S. cerevisiae Csm1/Lrs4 monopolin subcomplex has a distinctive V-shaped structure, with two pairs of protein-protein interaction domains positioned approximately 10 nm apart. Csm1 presents a conserved hydrophobic surface patch that binds two kinetochore proteins: Dsn1, a subunit of the outer-kinetochore MIND/Mis12 complex, and Mif2/CENP-C. Csm1 point-mutations that disrupt kinetochore-subunit binding also disrupt sister chromatid co-orientation in S. cerevisiae meiosis I. We further show that the same Csm1 point-mutations affect rDNA silencing, probably by disrupting binding to the rDNA-associated protein Tof2. We propose that Csm1/Lrs4 functions as a molecular clamp, crosslinking kinetochore components to enforce sister chromatid co-orientation in S. cerevisiae meiosis I and to suppress merotelic attachment in S. pombe mitosis, and crosslinking rDNA repeats to aid rDNA silencing.
Auxilin, a J-domain containing protein, recruits the Hsc70 uncoating ATPase to newly budded clathrin-coated vesicles. The timing of auxilin arrival determines that uncoating will commence only after the clathrin lattice has fully assembled and after membrane fission is complete. Auxilin has a region resembling PTEN, a PI3P phosphatase. We have determined the crystal structure of this region of bovine auxilin 1; it indeed resembles PTEN closely. A change in the structure of the P loop accounts for the lack of phosphatase activity. Inclusion of phosphatidylinositol phosphates substantially enhances liposome binding by wild-type auxilin, but not by various mutants bearing changes in loops of the C2 domain. Nearly all these mutations also prevent recruitment of auxilin to newly budded coated vesicles. We propose a specific geometry for auxilin association with a membrane bilayer and discuss implications of this model for the mechanism by which auxilin detects separation of a vesicle from its parent membrane.
Hepatocellular carcinoma (HCC) is a common and deadly malignancy that is increasing in incidence in developed countries. The emergence of hepatitis C virus (HCV) accounts for about half of this increase in HCC, although the etiology of HCC in 15%-50% of new HCC cases remains unclear. The most common form of chronic liver disease in developed countries is nonalcoholic fatty liver disease (NAFLD), which encompasses a broad spectrum of histopathology. The prevalence of NAFLD, including the more aggressive nonalcoholic steatohepatitis (NASH), is increasing with the growing epidemics of diabetes and obesity. NASH can progress to cirrhosis and its related complications. Growing evidence suggests that NASH accounts for a large proportion of idiopathic or cryptogenic cirrhosis, which is associated with the typical risk factors for NASH. HCC is a rare, although important complication of NAFLD. Diabetes and obesity have been established as independent risk factors for the development of HCC. New evidence also suggests that hepatic iron deposition increases the risk of HCC in NASH-derived cirrhosis. Multiple case reports and case reviews of HCC in the setting of NASH support the associations of diabetes and obesity with the risk of HCC, as well as suggest age and advanced fibrosis as significant risks. Insulin resistance and its subsequent inflammatory cascade that is associated with the development of NASH appear to play a significant role in the carcinogenesis of HCC. The complications of NASH, including cirrhosis and HCC, are expected to increase with the growing epidemic of diabetes and obesity.
Spatial regulation of microtubule (MT) dynamics contributes to cell polarity and cell division. MT rescue, in which a MT stops shrinking and reinitiates growth, is the least understood aspect of MT dynamics. Cytoplasmic Linker Associated Proteins (CLASPs) are a conserved class of MT-associated proteins that contribute to MT stabilization and rescue in vivo. We show here that the Schizosaccharomyces pombe CLASP, Cls1p, is a homodimer that binds an alphabeta-tubulin heterodimer through conserved TOG-like domains. In vitro, CLASP increases MT rescue frequency, decreases MT catastrophe frequency, and moderately decreases MT disassembly rate. CLASP binds stably to the MT lattice, recruits tubulin, and locally promotes rescues. Mutations in the CLASP TOG domains demonstrate that tubulin binding is critical for its rescue activity. We propose a mechanism for rescue in which CLASP-tubulin dimer complexes bind along the MT lattice and reverse MT depolymerization with their bound tubulin dimer.
Experiments in cell-free systems have demonstrated that the VP5 cleavage fragment of the rotavirus spike protein, VP4, undergoes a foldback rearrangement that translocates three clustered hydrophobic loops from one end of the molecule to the other. This conformational change resembles the foldback rearrangements of enveloped virus fusion proteins. By recoating rotavirus subviral particles with recombinant VP4 and VP7, we tested the effects on cell entry of substituting hydrophilic for hydrophobic residues in the clustered VP5 loops. Several of these mutations decreased the infectivity of recoated particles without preventing either recoating or folding back. In particular, the V391D mutant had a diminished capacity to interact with liposomes when triggered to fold back by serial protease digestion in solution, and particles recoated with this mutant VP4 were 10,000-fold less infectious than particles recoated with wild-type VP4. Particles with V391D mutant VP4 attached normally to cells and internalized efficiently, but they failed in the permeabilization step that allows coentry of the toxin alpha-sarcin. These findings indicate that the hydrophobicity of the VP5 apex is required for membrane disruption during rotavirus cell entry.
Papillomaviruses, members of a group of dsDNA viruses associated with epithelial growths and tumors, have compact capsids assembled from 72 pentamers of the protein L1. We have determined the structure of bovine papillomavirus by electron cryomicrosopy (cryoEM), at approximately 3.6 A resolution. The density map, obtained from single-particle analysis of approximately 4,000 particle images, shows the trace of the L1 polypeptide chain and reveals how the N- and C-terminal "arms" of a subunit (extensions from its beta-jelly-roll core) associate with a neighboring pentamer. Critical contacts come from the C-terminal arm, which loops out from the core of the subunit, forms contacts (including a disulfide) with two subunits in a neighboring pentamer, and reinserts into the pentamer from which it emanates. This trace corrects one feature of an earlier model. We discuss implications of the structure for virion assembly and for pathways of infectious viral entry. We suggest that it should be possible to obtain image reconstructions of comparable resolution from cryoEM images of asymmetric particles. From the work on papillomavirus described here, we estimate that such a reconstruction will require about 1.5 million images to achieve the same number of averaged asymmetric units; structural variability will increase this number substantially.
The mechanism of membrane fusion by "class II" viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein (E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The approximately 55-aa-residue, membrane proximal "stem" can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4 degrees C rather than 37 degrees C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.
Many biological and chemical processes proceed through one or more intermediate steps. Statistical analysis of dwell-time distributions from single molecule trajectories enables the study of intermediate steps that are not directly observable. Here, we discuss the application of the randomness parameter and model fitting in determining the number of steps in a stochastic process. Through simulated examples, we show some of the limitations of these techniques. We discuss how shot noise and heterogeneity among the transition rates of individual steps affect how accurately the number of steps can be determined. Finally, we explore dynamic disorder in multistep reactions and show that the phenomenon can obscure the presence of rate-limiting intermediate steps.
The rotavirus inner capsid particle, known as the "double-layered particle" (DLP), is the "payload" delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter approximately 700 A) in the asymmetric unit of the P2(1)2(1)2(1) unit cell of dimensions a=740 A, b=1198 A, and c=1345 A. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 A resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 A). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T=13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two "subdomain" boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: lambda1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a "5-fold hub" that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.
New World hemorrhagic fever arenaviruses are rodent-borne agents that cause severe human disease. The GP1 subunit of the surface glycoprotein mediates cell attachment through transferrin receptor 1 (TfR1). We report the structure of Machupo virus (MACV) GP1 bound with human TfR1. Atomic details of the GP1-TfR1 interface clarify the importance of TfR1 residues implicated in New World arenavirus host specificity. Analysis of sequence variation among New World arenavirus GP1s and their host-species receptors, in light of the molecular structure, indicates determinants of viral zoonotic transmission. Infectivities of pseudoviruses in cells expressing mutated TfR1 confirm that contacts at the tip of the TfR1 apical domain determine the capacity of human TfR1 to mediate infection by particular New World arenaviruses. We propose that New World arenaviruses that are pathogenic to humans fortuitously acquired affinity for human TfR1 during adaptation to TfR1 of their natural hosts.
Hepatitis C virus (HCV) treatment is frequently complicated by anemia from ribavirin (RBV)-related hemolysis and peginterferon-alfa (PEG-IFN)-related bone marrow suppression. We investigated the relationships among treatment outcomes, anemia, and their management with RBV dose reduction and/or erythropoiesis-stimulating agents (ESAs).
In addition to its effects in the liver, chronic hepatitis C virus (HCV) infection can have serious consequences for other organ systems. Extrahepatic manifestations include mixed cryoglobulinemia (MC) vasculitis, lymphoproliferative disorders, renal disease, insulin resistance, type 2 diabetes, sicca syndrome, rheumatoid arthritis-like polyarthritis, and autoantibody production; reductions in quality of life involve fatigue, depression, and cognitive impairment. MC vasculitis, certain types of lymphoma, insulin resistance, and cognitive function appear to respond to anti-HCV therapy. However, treatments for HCV and other biopsychosocial factors can reduce quality of life and complicate management. HCV treatment has a high overall cost that increases when extrahepatic manifestations are considered. HCV appears to have a role in the pathogenesis of MC vasculitis, certain types of lymphoma, and insulin resistance. Clinicians who treat patients with HCV infections should be aware of potential extrahepatic manifestations and how these can impact and alter management of their patients.
During rotavirus entry, a virion penetrates a host cell membrane, sheds its outer capsid proteins, and releases a transcriptionally active subviral particle into the cytoplasm. VP5, the rotavirus protein believed to interact with the membrane bilayer, is a tryptic cleavage product of the outer capsid spike protein, VP4. When a rotavirus particle uncoats, VP5 folds back, in a rearrangement that resembles the fusogenic conformational changes in enveloped-virus fusion proteins. We present direct experimental evidence that this rearrangement leads to membrane binding. VP5 does not associate with liposomes when mounted as part of the trypsin-primed spikes on intact virions, nor does it do so after it has folded back into a stably trimeric, low-energy state. But it does bind liposomes when they are added to virions before uncoating, and VP5 rearrangement is then triggered by addition of EDTA. The presence of liposomes during the rearrangement enhances the otherwise inefficient VP5 conformational change. A VP5 fragment, VP5CT, produced from monomeric recombinant VP4 by successive treatments with chymotrypsin and trypsin, also binds liposomes only when the proteolysis proceeds in their presence. A monoclonal antibody that neutralizes infectivity by blocking a postattachment entry event also blocks VP5 liposome association. We propose that VP5 binds lipid bilayers in an intermediate conformational state, analogous to the extended intermediate conformation of enveloped-virus fusion proteins.
Induction of effective antibody responses against HIV-1 infection remains an elusive goal for vaccine development. Progress may require in-depth understanding of the molecular mechanisms of neutralization by monoclonal antibodies. We have analyzed the molecular actions of two rare, broadly neutralizing, human monoclonal antibodies, 4E10 and 2F5, which target the transiently exposed epitopes in the membrane proximal external region (MPER) of HIV-1 gp41 envelope during viral entry. Both have long CDR H3 loops with a hydrophobic surface facing away from the peptide epitope. We find that the hydrophobic residues of 4E10 mediate a reversible attachment to the viral membrane and that they are essential for neutralization, but not for interaction with gp41. We propose that these antibodies associate with the viral membrane in a required first step and are thereby poised to capture the transient gp41 fusion intermediate. These results bear directly on strategies for rational design of HIV-1 envelope immunogens.
Primary liver cancer is the fifth most common malignancy worldwide and the third leading cause of cancer mortality. Non-alcoholic fatty liver disease is the most common cause of chronic liver disease in the United States encompassing a spectrum of entities marked by hepatic steatosis in the absence of significant alcohol consumption. Although simple steatosis follows a generally benign course, the more aggressive form, non-alcoholic steatohepatitis, can progress to cirrhosis and result in complications including hepatocellular carcinoma. A significant number of cases of hepatocellular carcinoma remain cryptogenic without known underlying chronic liver disease. It is increasingly recognized that non-alcoholic steatohepatitis likely accounts for a substantial portion of cryptogenic hepatocellular carcinoma.
Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support. Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 microm(2) area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles. Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop. From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.
Nonalcoholic fatty liver disease (NAFLD) has become one of the most common causes of liver disease worldwide and has grown proportionately with the rise in obesity. The prevalence of NAFLD is now thought to be around 20% to 40% of the entire population in industrialized Western countries. Insulin resistance, a product of obesity, is central to the pathogenesis of NAFLD, and is improved with weight loss, making this modality the primary goal of therapy. A combination of dietary modifications and increased physical activity, although hard to maintain, is thought to have significant long-term benefits, although further study is required to determine the best and most effective approaches to lifestyle modification. Alternatively, for those individuals who are unable to lose weight despite aggressive efforts, bariatric surgery, which has been shown rather convincingly to improve underlying fatty liver disease, may offer a solution. This review discusses dietary modification, exercise, weight loss pharmacotherapy, and surgical intervention as potential options for patients with NAFLD.
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