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Find video protocols related to scientific articles indexed in Pubmed.
Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe haemophilia A, 2000-2011.
Blood
PUBLISHED: 10-24-2014
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The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe haemophilia A previously untreated patients born in the UK between 1/January/2000 and 31/December/2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Since exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titre, after a median (interquartile range) 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate Nexgen 45 (35.2%, 95% CI 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95%CI 18.6-31.4%) with Advate (P=0.04). The adjusted hazard ratio (95% CI) for Kogenate Bayer/Helixate Nexgen compared to Advate was 2.14 (1.12-4.10), P=0.02 for high titre and 1.75 (1.11-2.76), P=0.02 for all inhibitors. When excluding UK-RODIN patients the adjusted HR (95% CI) for high titre inhibitors was 2.00 (0.93-4.34), P=0.08. ReFacto AF was associated with a higher incidence of all, but not high titre, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands.
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Recombinant long-acting glycoPEGylated factor IX in hemophilia B: a multinational randomized phase 3 trial.
Blood
PUBLISHED: 09-28-2014
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This multinational, randomized, single-blind trial (NCT01333111 [www.clinicaltrials.gov]) investigated safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in 74 previously treated hemophilia B patients (FIX activity ?2 IU/dL). Patients received prophylaxis for 52 weeks, randomized to 10 IU/kg or 40 IU/kg once weekly, or on-demand treatment for 28 weeks. No patients developed inhibitors and no safety concerns were identified. Three-hundred and forty-five bleeding episodes were treated with an estimated success rate of 92.2%. The median annualized bleeding rates (ABRs) were 1.04 in the 40 IU/kg prophylaxis arm, 2.93 in the 10 IU/kg prophylaxis arm, and 15.58 in the on-demand treatment arm. In the 40 IU/kg arm, 10 of 15 patients (66.7%) experienced no bleeding episodes into target joints, compared with 1 of 13 patients (7.7%) in the 10 IU/kg arm. Health-related quality of life (HR-QoL) assessed with the EQ-5D VAS score improved from median 75 to 90 in the 40 IU/kg prophylaxis arm. Nonacog beta pegol was well tolerated and efficacious for treatment of bleeding episodes, and associated with low ABRs in patients on prophylaxis. Once-weekly prophylaxis with 40 IU/kg resolved target joint bleeds in 66.7% of the affected patients and improved HR-QoL.
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International society of neuropathology-haarlem consensus guidelines for nervous system tumor classification and grading.
Brain Pathol.
PUBLISHED: 09-10-2014
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Major discoveries in the biology of nervous system tumors have raised the question of how non-histological data such as molecular information can be incorporated into the next World Health Organization (WHO) classification of central nervous system tumors. To address this question, a meeting of neuropathologists with expertise in molecular diagnosis was held in Haarlem, the Netherlands, under the sponsorship of the International Society of Neuropathology (ISN). Prior to the meeting, participants solicited input from clinical colleagues in diverse neuro-oncological specialties. The present "white paper" catalogs the recommendations of the meeting, at which a consensus was reached that incorporation of molecular information into the next WHO classification should follow a set of provided "ISN-Haarlem" guidelines. Salient recommendations include that (i) diagnostic entities should be defined as narrowly as possible to optimize interobserver reproducibility, clinicopathological predictions and therapeutic planning; (ii) diagnoses should be "layered" with histologic classification, WHO grade and molecular information listed below an "integrated diagnosis"; (iii) determinations should be made for each tumor entity as to whether molecular information is required, suggested or not needed for its definition; (iv) some pediatric entities should be separated from their adult counterparts; (v) input for guiding decisions regarding tumor classification should be solicited from experts in complementary disciplines of neuro-oncology; and (iv) entity-specific molecular testing and reporting formats should be followed in diagnostic reports. It is hoped that these guidelines will facilitate the forthcoming update of the fourth edition of the WHO classification of central nervous system tumors.
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Attenuation of human respiratory syncytial virus by genome-scale codon-pair deoptimization.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 08-25-2014
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Human respiratory syncytial virus (RSV) is the most important viral agent of serious pediatric respiratory-tract disease worldwide. A vaccine or generally effective antiviral drug is not yet available. We designed new live attenuated RSV vaccine candidates by codon-pair deoptimization (CPD). Specifically, viral ORFs were recoded by rearranging existing synonymous codons to increase the content of underrepresented codon pairs. Amino acid coding was completely unchanged. Four CPD RSV genomes were designed in which the indicated ORFs were recoded: Min A (NS1, NS2, N, P, M, and SH), Min B (G and F), Min L (L), and Min FLC (all ORFs except M2-1 and M2-2). Surprisingly, the recombinant CPD viruses were temperature-sensitive for replication in vitro (level of sensitivity: Min FLC > Min L > Min B > Min A). All of the CPD mutants grew less efficiently in vitro than recombinant wild-type (WT) RSV, even at the typically permissive temperature of 32 °C (growth efficiency: WT > Min L > Min A > Min FLC > Min B). CPD of the ORFs for the G and F surface glycoproteins provided the greatest restrictive effect. The CPD viruses exhibited a range of restriction in mice and African green monkeys comparable with that of two attenuated RSV strains presently in clinical trials. This study provided a new type of attenuated RSV and showed that CPD can rapidly generate vaccine candidates against nonsegmented negative-strand RNA viruses, a large and expanding group that includes numerous pathogens of humans and animals.
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A Comprehensive Proteomic View of Responses of A549 Cells to Human Respiratory Syncytial Virus Infection.
Mol. Cell Proteomics
PUBLISHED: 08-08-2014
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Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II and III interferon, TNF-? and non-canonical NF-?B2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine- / key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious agents on host cells.
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Nonstatistical dynamics on the caldera.
J Chem Phys
PUBLISHED: 07-24-2014
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We explore both classical and quantum dynamics of a model potential exhibiting a caldera: that is, a shallow potential well with two pairs of symmetry related index one saddles associated with entrance/exit channels. Classical trajectory simulations at several different energies confirm the existence of the "dynamical matching" phenomenon originally proposed by Carpenter, where the momentum direction associated with an incoming trajectory initiated at a high energy saddle point determines to a considerable extent the outcome of the reaction (passage through the diametrically opposing exit channel). By studying a "stretched" version of the caldera model, we have uncovered a generalized dynamical matching: bundles of trajectories can reflect off a hard potential wall so as to end up exiting predominantly through the transition state opposite the reflection point. We also investigate the effects of dissipation on the classical dynamics. In addition to classical trajectory studies, we examine the dynamics of quantum wave packets on the caldera potential (stretched and unstretched). These computations reveal a quantum mechanical analogue of the "dynamical matching" phenomenon, where the initial expectation value of the momentum direction for the wave packet determines the exit channel through which most of the probability density passes to product.
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Fibrin-based clot formation as an early and rapid biomarker for progression of postpartum hemorrhage: a prospective study.
Blood
PUBLISHED: 07-14-2014
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This prospective, observational study investigated the utility of Fibtem A5 and Clauss fibrinogen as predictors of progression of postpartum hemorrhage (PPH). A consecutive cohort of 356 women experiencing 1000 to 1500 mL PPH was recruited. Fibtem and fibrinogen were measured and subsequent transfusions, invasive procedures, and bleed volume recorded. Women progressing to 8 U blood products (red blood cells [RBCs] + fresh frozen plasma [FFP] + platelets) had a median (interquartile range) fibrinogen and Fibtem A5 of 2.1 (1.8-3.4) g/L and 12 (7-17) mm, respectively, compared with 3.9 (3.2-4.5) and 19 (17-23) for those not progressing. On multivariate analysis, Fibtem was an independent predictor for progression to bleeds >2500 mL (95% confidence interval [CI], 0.85 [0.77-0.95]). Receiver operating characteristic area under the curve (95% CI) for progression to RBC transfusion was 0.67 (0.60-0.74) for fibrinogen and 0.61 (0.54-0.68) for Fibtem, and progression to >2500 mL was 0.71 (0.61-0.81) and 0.75 (0.66-0.85) for fibrinogen and Fibtem, respectively. Fibtem A5 <10 mm was associated with more prolonged bleeds (median [95% CI], 127 [44-210] compared with 65 [59-71] minutes; P = .018) and longer stay in the high-dependency unit (23.5 [18.4-28.5] compared with 10.8 [9.7-11.8] hours). Fibtem is a rapidly available early biomarker for progression of PPH.
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NOAC in acute coronary syndrome and AF?
Cardiovasc Hematol Disord Drug Targets
PUBLISHED: 06-24-2014
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Cardiovascular disease remains a major cause of morbidity and mortality in developed countries. New treatments, in the form of novel oral anticoagulants (NOAC) that reduce thrombotic risk are now available for patients with atrial fibrillation (AF) or acute coronary syndrome (ACS). Warfarin has been the cornerstone of thromboprophylaxis in patients with AF, but treatment is cumbersome, inconvenient and often unreliable, with fluctuating time in therapeutic range. Thrombotic events also continue to occur in a significant number of ACS patients despite antiplatelet therapy. Thus there is an unfilled need to reduce thrombotic events in ACS and AF patients. NOAC comprise direct factor Xa inhibitors (apixaban, rivaroxaban, darexaban, edoxaban), direct thrombin inhibitors (dabigatran) and PAR-1 antagonists (vorapaxar, atopaxar). In this review, we compare and contrast NOACs and review their individual and specific clinical trial database in ACS and AF. In the setting of ACS, the role of NOAC is unclear, as any reduction in ischemic events appears to be offset by hemorrhagic risk. However, NOAC have a definite place in the treatment of patients with non-valvular AF, where they are at least as effective, if not superior to warfarin.
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Evaluation of the contributions of individual viral genes to newcastle disease virus virulence and pathogenesis.
J. Virol.
PUBLISHED: 05-21-2014
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Naturally occurring Newcastle disease virus (NDV) strains vary greatly in virulence. The presence of multibasic residues at the proteolytic cleavage site of the fusion (F) protein has been shown to be a primary determinant differentiating virulent versus avirulent strains. However, there is wide variation in virulence among virulent strains. There also are examples of incongruity between cleavage site sequence and virulence. These observations suggest that additional viral factors contribute to virulence. In this study, we evaluated the contribution of each viral gene to virulence individually and in different combinations by exchanging genes between velogenic (highly virulent) strain GB Texas (GBT) and mesogenic (moderately virulent) strain Beaudette C (BC). These two strains are phylogenetically closely related, and their F proteins contain identical cleavage site sequences, (112)RRQKR?F(117). A total of 20 chimeric viruses were constructed and evaluated in vitro, in 1-day-old chicks, and in 2-week-old chickens. The results showed that both the envelope-associated and polymerase-associated proteins contribute to the difference in virulence between rBC and rGBT, with the envelope-associated proteins playing the greater role. The F protein was the major individual contributor and was sometimes augmented by the homologous M and HN proteins. The dramatic effect of F was independent of its cleavage site sequence since that was identical in the two strains. The polymerase L protein was the next major individual contributor and was sometimes augmented by the homologous N and P proteins. The leader and trailer regions did not appear to contribute to the difference in virulence between BC and GBT.
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Tracking the Commute Home From School Utilising GPS and Heart Rate Monitoring: Establishing the Contribution to Free-Living Physical Activity.
J Phys Act Health
PUBLISHED: 04-26-2014
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Active school commuting is widely regarded as a key opportunity for youth to participate in physical activity (PA). However, the accurate measurement of the commute journey home from school and its contribution to total free-living moderate-to-vigorous PA (MVPA) is relatively unexplored.
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Roaming dynamics in ion-molecule reactions: phase space reaction pathways and geometrical interpretation.
J Chem Phys
PUBLISHED: 04-10-2014
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A model Hamiltonian for the reaction CH4(+) -> CH3(+) + H, parametrized to exhibit either early or late inner transition states, is employed to investigate the dynamical characteristics of the roaming mechanism. Tight/loose transition states and conventional/roaming reaction pathways are identified in terms of time-invariant objects in phase space. These are dividing surfaces associated with normally hyperbolic invariant manifolds (NHIMs). For systems with two degrees of freedom NHIMS are unstable periodic orbits which, in conjunction with their stable and unstable manifolds, unambiguously define the (locally) non-recrossing dividing surfaces assumed in statistical theories of reaction rates. By constructing periodic orbit continuation/bifurcation diagrams for two values of the potential function parameter corresponding to late and early transition states, respectively, and using the total energy as another parameter, we dynamically assign different regions of phase space to reactants and products as well as to conventional and roaming reaction pathways. The classical dynamics of the system are investigated by uniformly sampling trajectory initial conditions on the dividing surfaces. Trajectories are classified into four different categories: direct reactive and non-reactive trajectories, which lead to the formation of molecular and radical products respectively, and roaming reactive and non-reactive orbiting trajectories, which represent alternative pathways to form molecular and radical products. By analysing gap time distributions at several energies, we demonstrate that the phase space structure of the roaming region, which is strongly influenced by nonlinear resonances between the two degrees of freedom, results in nonexponential (nonstatistical) decay.
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Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens.
Vaccine
PUBLISHED: 04-09-2014
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Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271-330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans.
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Origins and functional consequences of somatic mitochondrial DNA mutations in human cancer.
Elife
PUBLISHED: 03-28-2014
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Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.
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Human metapneumovirus SH and G glycoproteins inhibit macropinocytosis-mediated entry into human dendritic cells and reduce CD4+ T cell activation.
J. Virol.
PUBLISHED: 03-26-2014
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Human metapneumovirus (HMPV) is a major etiologic agent of respiratory disease worldwide. HMPV reinfections are common in healthy adults and children, suggesting that the protective immune response to HMPV is incomplete and short-lived. We used gene-deletion viruses to evaluate the role of the attachment G and small hydrophobic SH glycoproteins on virus uptake by primary human monocyte-derived dendritic cells (MDDC) in vitro and on subsequent MDDC maturation and activation of autologous T cells. HMPV with deletion of G and SH (?SHG) exhibited increased infectivity but had little effect on MDDC maturation. However, MDDC stimulated with ?SHG induced increased proliferation of autologous Th1-polarized CD4(+) T cells. This effect was independent of virus replication. Increased T cell proliferation was strictly dependent on contact between virus-stimulated MDDC and CD4(+) T cells. Confocal microscopy revealed that deletion of SH and G was associated with an increased number of immunological synapses between memory CD4(+) T cells and virus-stimulated MDDC. Uptake of HMPV by MDDC was found to be primarily by macropinocytosis. Uptake of wild-type (WT) virus was reduced compared to that of ?SHG, indicative of inhibition by the SH and G glycoproteins. In addition, DC-SIGN-mediated endocytosis provided a minor alternative pathway that depended on SH and/or G and thus operated only for WT. Altogether, our results show that SH and G glycoproteins reduce the ability of HMPV to be internalized by MDDC, resulting in a reduced ability of the HMPV-stimulated MDDC to activate CD4(+) T cells. This study describes a previously unknown mechanism of virus immune evasion.
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Animal model of respiratory syncytial virus: CD8+ T cells cause a cytokine storm that is chemically tractable by sphingosine-1-phosphate 1 receptor agonist therapy.
J. Virol.
PUBLISHED: 03-26-2014
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The cytokine storm is an intensified, dysregulated, tissue-injurious inflammatory response driven by cytokine and immune cell components. The cytokine storm during influenza virus infection, whereby the amplified innate immune response is primarily responsible for pulmonary damage, has been well characterized. Now we describe a novel event where virus-specific T cells induce a cytokine storm. The paramyxovirus pneumonia virus of mice (PVM) is a model of human respiratory syncytial virus (hRSV). Unexpectedly, when C57BL/6 mice were infected with PVM, the innate inflammatory response was undetectable until day 5 postinfection, at which time CD8(+) T cells infiltrated into the lung, initiating a cytokine storm by their production of gamma interferon (IFN-?) and tumor necrosis factor alpha (TNF-?). Administration of an immunomodulatory sphingosine-1-phosphate (S1P) receptor 1 (S1P1R) agonist significantly inhibited PVM-elicited cytokine storm by blunting the PVM-specific CD8(+) T cell response, resulting in diminished pulmonary disease and enhanced survival.
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Amplification of 2p as a genomic marker for transformation in lymphoma.
Genes Chromosomes Cancer
PUBLISHED: 02-16-2014
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To outline further genetic mechanisms of transformation from follicular lymphoma (FL) to diffuse large B-cell lymphoma (DLBCL), we have performed whole genome array-CGH in 81 tumors from 60 patients [29 de novo DLBCL (dnDLBCL), 31 transformed DLBCL (tDLBCL), and 21 antecedent FL]. In 15 patients, paired tumor samples (primary FL and a subsequent tDLBCL) were available, among which three possessed more than two subsequent tumors, allowing us to follow specific genetic alterations acquired before, during, and after the transformation. Gain of 2p15-16.1 encompassing, among others, the REL, BCL11A, USP34, COMMD1, and OTX1 genes was found to be more common in the tDLBCL compared with dnDLBCL (P?
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RSV-encoded NS2 promotes epithelial cell shedding and distal airway obstruction.
J. Clin. Invest.
PUBLISHED: 02-13-2014
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Respiratory syncytial virus (RSV) infection is the major cause of bronchiolitis in young children. The factors that contribute to the increased propensity of RSV-induced distal airway disease compared with other commonly encountered respiratory viruses remain unclear. Here, we identified the RSV-encoded nonstructural 2 (NS2) protein as a viral genetic determinant for initiating RSV-induced distal airway obstruction. Infection of human cartilaginous airway epithelium (HAE) and a hamster model of disease with recombinant respiratory viruses revealed that NS2 promotes shedding of infected epithelial cells, resulting in two consequences of virus infection. First, epithelial cell shedding accelerated the reduction of virus titers, presumably by clearing virus-infected cells from airway mucosa. Second, epithelial cells shedding into the narrow-diameter bronchiolar airway lumens resulted in rapid accumulation of detached, pleomorphic epithelial cells, leading to acute distal airway obstruction. Together, these data indicate that RSV infection of the airway epithelium, via the action of NS2, promotes epithelial cell shedding, which not only accelerates viral clearance but also contributes to acute obstruction of the distal airways. Our results identify RSV NS2 as a contributing factor for the enhanced propensity of RSV to cause severe airway disease in young children and suggest NS2 as a potential therapeutic target for reducing the severity of distal airway disease.
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Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.
Cancer Cell
PUBLISHED: 02-13-2014
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Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
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Chimeric bovine/human parainfluenza virus type 3 expressing respiratory syncytial virus (RSV) F glycoprotein: effect of insert position on expression, replication, immunogenicity, stability, and protection against RSV infection.
J. Virol.
PUBLISHED: 01-29-2014
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A recombinant chimeric bovine/human parainfluenza type 3 virus (rB/HPIV3) vector expressing the respiratory syncytial virus (RSV) fusion F glycoprotein previously exhibited disappointing levels of RSV F immunogenicity and genetic stability in children (D. Bernstein et al., Pediatr. Infect. Dis. J. 31:109-114, 2012; C.-F. Yang et al., Vaccine 31:2822-2827, 2013). To investigate parameters that might affect vaccine performance and stability, we constructed and characterized rB/HPIV3 viruses expressing RSV F from the first (pre-N), second (N-P), third (P-M), and sixth (HN-L) genome positions. There was a 30- to 69-fold gradient in RSV F expression from the first to the sixth position. The inserts moderately attenuated vector replication in vitro and in the upper and lower respiratory tracts of hamsters: this was not influenced by the level of RSV F expression and syncytium formation. Surprisingly, inserts in the second, third, and sixth positions conferred increased temperature sensitivity: this was greatest for the third position and was the most attenuating in vivo. Each rB/HPIV3 vector induced a high titer of neutralizing antibodies in hamsters against RSV and HPIV3. Protection against RSV challenge was greater for position 2 than for position 6. Evaluation of insert stability suggested that RSV F is under selective pressure to be silenced during vector replication in vivo, but this was not exacerbated by a high level of RSV F expression and generally involved a small percentage of recovered vector. Vector passaged in vitro accumulated mutations in the HN open reading frame, causing a dramatic increase in plaque size that may have implications for vaccine production and immunogenicity.
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Anthropometric and lifestyle characteristics of active and inactive Saudi and British adolescents.
Am. J. Hum. Biol.
PUBLISHED: 01-21-2014
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To compare the anthropometric and lifestyle characteristics of active and inactive adolescents in Saudi Arabia and Britain.
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Effects of naturally occurring six- and twelve-nucleotide inserts on Newcastle disease virus replication and pathogenesis.
PLoS ONE
PUBLISHED: 01-01-2014
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Newcastle disease virus (NDV) isolates contain genomes of 15,186, 15,192 or 15,198 nucleotides (nt). The length differences reflect a 6-nt insert in the 5' (downstream) non-translated region (NTR) of the N gene (15,192-nt genome) or a 12-nt insert in the ORF encoding the P and V proteins (causing a 4-amino acid insert; 15,198-nt genome). We evaluated the role of these inserts in the N and P genes on viral replication and pathogenicity by inserting them into genomes of two NDV strains that have natural genome lengths of 15,186 nt and represent two different pathotypes, namely the mesogenic strain Beaudette C (BC) and the velogenic strain GB Texas (GBT). Our results showed that the 6-nt and 12-nt inserts did not detectably affect N gene expression or P protein function. The inserts had no effect on the replication or virulence of the highly virulent GBT strain but showed modest degree of attenuation in mesogenic strain BC. We also deleted a naturally-occurring 6-nt insertion in the N gene from a highly virulent 15,192-nt genome-length virus, strain Banjarmasin. This resulted in reduced replication in vitro and reduced virulence in vivo. Thus, although these inserts had no evident effect on gene expression, protein function, or replication in vivo, they did affect virulence in two of the three tested strains.
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Live-attenuated respiratory syncytial virus vaccines.
Curr. Top. Microbiol. Immunol.
PUBLISHED: 12-24-2013
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Live-attenuated respiratory syncytial virus (RSV) vaccines offer several advantages for immunization of infants and young children: (1) they do not cause vaccine-associated enhanced RSV disease; (2) they broadly stimulate innate, humoral, and cellular immunity, both systemically and locally in the respiratory tract; (3) they are delivered intranasally; and (4) they replicate in the upper respiratory tract of young infants despite the presence of passively acquired maternally derived RSV neutralizing antibody. This chapter describes early efforts to develop vaccines through the classic methods of serial cold-passage and chemical mutagenesis, and recent efforts using reverse genetics to derive attenuated derivatives of wild-type (WT) RSV and to develop parainfluenza vaccine vectors that express RSV surface glycoproteins.
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Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease.
Curr. Top. Microbiol. Immunol.
PUBLISHED: 12-24-2013
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Human respiratory syncytial virus (RSV) is an enveloped, nonsegmented negative-strand RNA virus of family Paramyxoviridae. RSV is the most complex member of the family in terms of the number of genes and proteins. It is also relatively divergent and distinct from the prototype members of the family. In the past 30 years, we have seen a tremendous increase in our understanding of the molecular biology of RSV based on a succession of advances involving molecular cloning, reverse genetics, and detailed studies of protein function and structure. Much remains to be learned. RSV disease is complex and variable, and the host and viral factors that determine tropism and disease are poorly understood. RSV is notable for a historic vaccine failure in the 1960s involving a formalin-inactivated vaccine that primed for enhanced disease in RSV naïve recipients. Live vaccine candidates have been shown to be free of this complication. However, development of subunit or other protein-based vaccines for pediatric use is hampered by the possibility of enhanced disease and the difficulty of reliably demonstrating its absence in preclinical studies.
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Identification and quantification of aminophospholipid molecular species on the surface of apoptotic and activated cells.
Nat Protoc
PUBLISHED: 12-12-2013
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This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a cell-impermeable reagent (sulfo-NHS-biotin), and then they are isolated via a liquid:liquid extraction and quantified by reverse-phase liquid chromatography tandem MS (LC-MS/MS) against in-house-generated standards. This protocol describes a complementary method to existing assays that are not quantitative (e.g., annexin V flow cytometry), and it is applicable to the study of membrane reorganization in all cell types during apoptosis (e.g., during development, cancer, psychiatric disorders and other conditions, aging, vesiculation and cell division). The protocol takes ?2-4 d, including the generation of standards.
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Drug therapies in liver disease.
Clin Med
PUBLISHED: 12-04-2013
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The likelihood of a general physician encountering a patient with compensated and decompensated liver disease is increasing. This article provides an overview of pharmaceutical agents currently used in the management of cirrhosis and is designed to allow a better understanding of the rationale for using certain drugs in patients with often complex pathology.
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The effectiveness of a cognitive-behavioral intervention for pathological gambling: a country-wide study.
Ann Clin Psychiatry
PUBLISHED: 11-08-2013
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Clinicians lack adequate data on the effectiveness of treatment for pathological gambling in low- and middle-income countries.
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Role of C596 in the C-Terminal Extension of the Hemagglutinin-Neuraminidase Protein in Replication and Pathogenicity of a Highly Virulent Indonesian Strain of Newcastle Disease Virus.
J. Gen. Virol.
PUBLISHED: 11-06-2013
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We modified the hemagglutinin-neuraminidase (HN) glycoprotein of the virulent Newcastle disease virus (NDV) strain Banjarmasin/010/10 (Ban/010) by adding C-terminal extensions similar to those found in certain avirulent NDV strains. Extension of the 571-amino acid (aa) wild-type Ban/010 HN protein to 577 aa and 616 aa by removal of one or two translational stop codons moderately reduced HN function and viral pathogenicity in 1-day-old and 3-week-old chickens. Substantially, greater reductions were achieved by altering the 616-aa form by introducing a R596C mutation or by replacing the C-terminal extension with that of avirulent strain Ulster, which naturally contains the 596C assignment. These results show that extension of the C-terminus of HN reduces NDV pathogenicity, and that this effect is substantially increased by the presence of the 596C. These results indicate that this attenuating mechanism in avirulent strains such as Ulster can be directly imported into a highly virulent strain recently in circulation.?
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Nonstatistical dynamics on potentials exhibiting reaction path bifurcations and valley-ridge inflection points.
J Chem Phys
PUBLISHED: 10-29-2013
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We study reaction dynamics on a model potential energy surface exhibiting post-transition state bifurcation in the vicinity of a valley ridge inflection (VRI) point. We compute fractional yields of products reached after the VRI region is traversed, both with and without dissipation. It is found that apparently minor variations in the potential lead to significant changes in the reaction dynamics. Moreover, when dissipative effects are incorporated, the product ratio depends in a complicated and highly non-monotonic fashion on the dissipation parameter. Dynamics in the vicinity of the VRI point itself play essentially no role in determining the product ratio, except in the highly dissipative regime.
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Enrichment of FLI1 and RUNX1 mutations in families with excessive bleeding and platelet dense granule secretion defects.
Blood
PUBLISHED: 10-07-2013
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We analyzed candidate platelet function disorder genes in 13 index cases with a history of excessive bleeding in association with a significant reduction in dense granule secretion and impaired aggregation to a panel of platelet agonists. Five of the index cases also had mild thrombocytopenia. Heterozygous alterations in FLI1 and RUNX1, encoding Friend leukemia integration 1 and RUNT-related transcription factor 1, respectively, which have a fundamental role in megakaryocytopoeisis, were identified in 6 patients, 4 of whom had mild thrombocytopenia. Two FLI1 alterations predicting p.Arg337Trp and p.Tyr343Cys substitutions in the FLI1 DNA-binding domain abolished transcriptional activity of FLI1. A 4-bp deletion in FLI1, and 2 splicing alterations and a nonsense variation in RUNX1, which were predicted to cause haploinsufficiency of either FLI1 or RUNX1, were also identified. Our findings suggest that alterations in FLI1 and RUNX1 may be common in patients with platelet dense granule secretion defects and mild thrombocytopenia.
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Bruising in children who are assessed for suspected physical abuse.
Arch. Dis. Child.
PUBLISHED: 09-16-2013
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To describe the characteristics of bruising and mode of presentation of children referred to the paediatric child protection team with suspected physical abuse (PA), and the extent to which these differ between the children where abuse was confirmed and those where it was excluded.
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AKT1E17K mutations cluster with meningothelial and transitional meningiomas and can be detected by SFRP1 immunohistochemistry.
Acta Neuropathol.
PUBLISHED: 08-30-2013
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The activating E17K mutation in the AKT1 gene has been detected in several tumor entities. Currently several clinical studies with specific AKT1 inhibitors are under way. To determine whether AKT1 mutations are involved in human tumors of the nervous system, we examined a series of 1,437 tumors including 391 primary intracranial brain tumors and 1,046 tumors of the coverings of the central and peripheral nervous system. AKT1E17K mutations were exclusively seen in meningiomas and occurred in 65 of 958 of these tumors. A strong preponderance was seen in the variant of meningothelial meningioma WHO grade I of basal and spinal localization. In contrast, AKT1E17K mutations were rare in WHO grade II and absent in WHO grade III meningiomas. In order to more effectively detect this mutation, we tested for immunohistochemical markers associated with this alteration. We observed strong up-regulation of SFRP1 expression in all meningiomas with AKT1E17K mutation and in HEK293 cells after transfection with mutant AKT1E17K, but not in meningiomas and HEK293 cells lacking this mutation.
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A Cross-Cultural Comparison of Health Behaviors between Saudi and British Adolescents Living in Urban Areas: Gender by Country Analyses.
Int J Environ Res Public Health
PUBLISHED: 08-26-2013
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This study investigated the cross-cultural differences and similarity in health behaviors between Saudi and British adolescents. A school-based cross-sectional study was conducted at four cities in Saudi Arabia (Riyadh and Al-Khobar; N = 1,648) and Britain (Birmingham and Coventry; N = 1,158). The participants (14-18 year-olds) were randomly selected using a multistage stratified cluster sampling technique. Measurements included anthropometric, screen time, validated physical activity (PA) questionnaire and dietary habits. The overweight/obesity prevalence among Saudi adolescents (38.3%) was significantly (p < 0.001) higher than that found among British adolescents (24.1%). The British adolescents demonstrated higher total PA energy expenditure than Saudi adolescents (means ± SE = 3,804.8 ± 81.5 vs. 2,219.9 ± 65.5 METs-min/week). Inactivity prevalence was significantly (p < 0.001) higher among Saudi adolescents (64%) compared with that of British adolescents (25.5%). The proportions of adolescents exceeding 2 h of daily screen time were high (88.0% and 90.8% among Saudis and British, respectively). The majority of Saudi and British adolescents did not have daily intakes of breakfast, fruit, vegetables and milk. MANCOVA showed significant (p < 0.05) gender by country interactions in several lifestyle factors. There was a significant (p < 0.001) gender differences in the ratio of physical activity to sedentary behaviors. In conclusion, Saudi and British adolescents demonstrated some similarities and differences in their PA levels, sedentary behaviors and dietary habits. Unhealthy lifestyle behaviors among adolescents appear to be a cross-cultural phenomenon.
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In situ evolution of virus-specific cytotoxic T cell responses in the lung.
J. Virol.
PUBLISHED: 08-14-2013
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Cytotoxic T cells (CTL) play a critical role in the clearance of respiratory viral infections, but they also contribute to disease manifestations. In this study, we infected mice with a genetically modified pneumonia virus of mice (PVM) that allowed visualization of virus-specific CTL and infected cells in situ. The first virus-specific T cells entered the lung via blood vessels in the scattered foci of PVM-infected cells, which densely clustered around the bronchi at day 7 after infection. At this time, overall pulmonary virus load was maximal, but the mice showed no overt signs of disease. On days 8 to 9, T cells gained access to the infected bronchial epithelium and to the lung interstitium, which was associated with a reduction in the number of virus-infected cells within the initial clusters but could not prevent further virus spread throughout the lung tissue. Interestingly, recruitment of virus-specific CTL throughout the parenchyma was still ongoing on day 10, when the virus infection was already largely controlled. This also represented the peak of clinical disease. Thus, disease was associated with an exuberant T cell infiltration late in the course of the infection, which may be required to completely eliminate virus at residual foci of infection. PVM-induced immunopathology may thus result from the need to generate widespread T cell infiltrates to complete the elimination of virus-infected cells in a large organ like the lung. This experimental model provides the first insights into the spatiotemporal evolution of pulmonary antiviral T cell immunity in vivo.
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Experimental infection of adults with recombinant wild-type human metapneumovirus.
J. Infect. Dis.
PUBLISHED: 08-01-2013
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Human metapneumovirus (HMPV) causes lower respiratory tract infections in young children. rHMPV-SHs is a recombinant HMPV (rHMPV) based on a biologically derived wild-type HMPV strain. We characterized its infectivity and immunogenicity in healthy adults to determine whether it would be suitable for use as the parent virus for the development of live attenuated rHMPV vaccines.
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Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation.
J. Lipid Res.
PUBLISHED: 07-24-2013
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Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE? and D? attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA?, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 10? platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE?/D? into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.
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The respiratory syncytial virus fusion protein and neutrophils mediate the airway mucin response to pathogenic respiratory syncytial virus infection.
J. Virol.
PUBLISHED: 07-10-2013
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Respiratory syncytial virus (RSV) is the leading cause of death due to a viral etiology in infants. RSV disease is characterized by epithelial desquamation, neutrophilic bronchiolitis and pneumonia, and obstructive pulmonary mucus. It has been shown that infection of BALB/cJ mice with RSV clinical isolate A2001/2-20 (2-20) results in a higher early viral load, greater airway necrosis, and higher levels of interleukin-13 (IL-13) and airway mucin expression than infection with RSV laboratory strain A2. We hypothesized that the fusion (F) protein of RSV 2-20 is a mucus-inducing viral factor. In vitro, the fusion activity of 2-20 F but not that of A2 F was enhanced by expression of RSV G. We generated a recombinant F-chimeric RSV by replacing the F gene of A2 with the F gene of 2-20, generating A2-2-20F. Similar to the results obtained with the parent 2-20 strain, infection of BALB/cJ mice with A2-2-20F resulted in a higher early viral load and higher levels of subsequent pulmonary mucin expression than infection with the A2 strain. A2-2-20F infection induced greater necrotic airway damage and neutrophil infiltration than A2 infection. We hypothesized that the neutrophil response to A2-2-20F infection is involved in mucin expression. Antibody-mediated depletion of neutrophils in RSV-infected mice resulted in lower tumor necrosis factor alpha levels, fewer IL-13-expressing CD4 T cells, and less airway mucin production in the lung. Our data are consistent with a model in which the F and attachment (G) glycoprotein functional interaction leads to enhanced fusion and F is a key factor in airway epithelium infection, pathogenesis, and subsequent airway mucin expression.
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Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.
J. Virol.
PUBLISHED: 07-10-2013
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The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (r?2 and r?4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.
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Is liver biopsy necessary in the management of alcoholic hepatitis?
World J. Gastroenterol.
PUBLISHED: 06-28-2013
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Acute alcoholic hepatitis (AAH) is characterised by deep jaundice in patients with a history of heavy alcohol use, which can progress to liver failure. A clinical diagnosis of AAH can be challenging to make in patients without a clear alcohol history or in the presence of risk factors for other causes of acute liver failure. Other causes of acute on chronic liver failure such as sepsis or variceal haemorrhage should be considered. Liver biopsy remains the only reliable method to make an accurate diagnosis. However, there is controversy surrounding the use of liver biopsy in patients with AAH because of the risks of performing a percutaneous biopsy and limitations in access to transjugular biopsy. We review the existing literature and find there are few studies directly comparing clinical and histological diagnosis of AAH. In the small number of studies that have been conducted the correlation between a clinical and histological diagnosis of AAH is poor. Due to this lack of agreement together with difficulties in accessing transjugular liver biopsy outside tertiary referral centres and research institutions, we cannot advocate universal biopsy for AAH but there remains a definite role for liver biopsy where there is clinical diagnostic doubt or dual pathology. It also adds value in a clinical trial context to ensure a homogeneous trial population and to further our understanding of the disease pathology. Further prospective studies are required to determine whether non-invasive markers can be used to accurately diagnose AAH.
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Safety and infectivity of two doses of live-attenuated recombinant cold-passaged human parainfluenza type 3 virus vaccine rHPIV3cp45 in HPIV3-seronegative young children.
Vaccine
PUBLISHED: 06-10-2013
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Human parainfluenza virus type 3 (HPIV3) is a common cause of upper and lower respiratory tract illness in infants and young children. Live-attenuated cold-adapted HPIV3 vaccines have been evaluated in infants but a suitable interval for administration of a second dose of vaccine has not been defined.
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The interactive effects of physical fitness and acute aerobic exercise on electrophysiological coherence and cognitive performance in adolescents.
Exp Brain Res
PUBLISHED: 05-23-2013
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The current study examined the effects of physical fitness and aerobic exercise on cognitive functioning and coherence of the electroencephalogram in 30 adolescents between the ages of 13 and 14 years. Participants were first classified as fit or unfit and then performed a modified Eriksen flanker task after a bout of acute exercise and after a period of relaxation. Analysis of behavioural differences between the fit and unfit groups revealed an interaction between fitness levels and acute physical exercise. Specifically, fit participants had significantly faster reaction times in the exercise condition in comparison with the rest condition; unfit, but not fit, participants had higher error rates for NoGo relative to Go trials in the rest condition. Furthermore, unfit participants had higher levels of lower alpha, upper alpha, and beta coherence in the resting condition for NoGo trials, possibly indicating a greater allocation of cognitive resources to the task demands. The higher levels of alpha coherence are of particular interest in light of its reported role in inhibition and effortful attention. The results suggest that physical fitness and acute exercise may enhance cognition by increasing the efficacy of the attentional system.
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A fast liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QToF-MS) method for the identification of organic explosives and propellants.
Forensic Sci. Int.
PUBLISHED: 05-22-2013
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A fast, highly specific and sensitive method for the detection of an extensive list of organic explosives and propellants using an optimised Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QToF-MS) procedure has been developed. Analyte specific information including retention time, adduct accurate mass and fragmentation data was firstly collected using both positive and negative ion Atmospheric Pressure Chemical Ionisation (APCI) and entered into a Personal Compound Database/Library (PCDL). The custom PCDL can then be used to screen any sample for the presence of organic explosives and propellants, generating a match score for any identified compounds. To date over 50 organic explosives and propellants have been characterised and entered into the database representing those likely to be encountered in forensic and environmental samples and also a range of specialist explosives.
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The eEF2 kinase confers resistance to nutrient deprivation by blocking translation elongation.
Cell
PUBLISHED: 04-30-2013
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Metabolic adaptation is essential for cell survival during nutrient deprivation. We report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers cell survival under acute nutrient depletion by blocking translation elongation. Tumor cells exploit this pathway to adapt to nutrient deprivation by reactivating the AMPK-eEF2K axis. Adaptation of transformed cells to nutrient withdrawal is severely compromised in cells lacking eEF2K. Moreover, eEF2K knockdown restored sensitivity to acute nutrient deprivation in highly resistant human tumor cell lines. In vivo, overexpression of eEF2K rendered murine tumors remarkably resistant to caloric restriction. Expression of eEF2K strongly correlated with overall survival in human medulloblastoma and glioblastoma multiforme. Finally, C. elegans strains deficient in efk-1, the eEF2K ortholog, were severely compromised in their response to nutrient depletion. Our data highlight a conserved role for eEF2K in protecting cells from nutrient deprivation and in conferring tumor cell adaptation to metabolic stress. PAPERCLIP:
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Reduced H3K27me3 and DNA hypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas.
Cancer Cell
PUBLISHED: 03-29-2013
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Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in ?50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.
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Upregulating mutations in the TERT promoter commonly occur in adult malignant gliomas and are strongly associated with total 1p19q loss.
Acta Neuropathol.
PUBLISHED: 03-29-2013
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Telomere lengthening is one of the key events in most cancers, and depends largely on telomerase activation. Telomerase activation is a well-known phenomenon in gliomas; however, its mechanism remains obscure. In this study, we investigated the presence of mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in a series of 546 gliomas. We found a high incidence of mutually exclusive mutations located at two hot spots, C228T and C250T, in all subtypes of gliomas (55 %). The frequency of mutation was particularly high among primary glioblastomas (70 %) and pure oligodendroglial tumors (74 %), while relatively low in diffuse astrocytomas and anaplastic astrocytomas (19 and 25 %, respectively). The expression level of TERT in tumors carrying those mutations was on average 6.1 times higher than that of wild-type tumors, indicating that the mutated promoter leads to upregulation of TERT. TERT promoter mutations were observed in almost all tumors harboring concurrent total 1p19q loss and IDH1/2 mutations (98 %). Otherwise TERT promoter mutations were mostly observed among IDH wild-type tumors. Most EGFR amplifications (92 %) were also associated with TERT promoter mutations. Our data indicate that mutation of the TERT promoter is one of the major mechanisms of telomerase activation in gliomas. The unique pattern of TERT promoter mutations in relation to other genetic alterations suggests that they play distinct roles in the pathogenesis of oligodendroglial and astrocytic tumors. Our results shed a new light on the role of telomerase activation in the development of adult gliomas.
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Recurrent somatic alterations of FGFR1 and NTRK2 in pilocytic astrocytoma.
Nat. Genet.
PUBLISHED: 03-26-2013
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Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.
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Characterization of platelet aminophospholipid externalization reveals fatty acids as molecular determinants that regulate coagulation.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-25-2013
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Aminophospholipid (APL) trafficking across the plasma membrane is a key event in cell activation, apoptosis, and aging and is required for clearance of dying cells and coagulation. Currently the phospholipid molecular species externalized are unknown. Using a lipidomic method, we show that thrombin, collagen, or ionophore-activated human platelets externalize two phosphatidylserines (PSs) and five phosphatidylethanolamines (PEs). Four percent of the total cellular PE/PS pool (?300 ng/2 × 10(8) cells, thrombin), is externalized via calcium mobilization and protease-activated receptors-1 and -4, and 48% is contained in microparticles. Apoptosis and energy depletion (aging) externalized the same APLs in a calcium-dependent manner, and all stimuli externalized oxidized phospholipids, termed hydroxyeicosatetraenoic acid-PEs. Transmembrane protein-16F (TMEM-16F), the protein mutated in Scott syndrome, was required for PE/PS externalization during thrombin activation and energy depletion, but not apoptosis. Platelet-specific APLs optimally supported tissue factor-dependent coagulation in human plasma, vs. APL with longer or shorter fatty acyl chains. This finding demonstrates fatty acids as molecular determinants of APL that regulate hemostasis. Thus, the molecular species of externalized APL during platelet activation, apoptosis, and energy depletion were characterized, and their ability to support coagulation revealed. The findings have therapeutic implications for bleeding disorders and transfusion therapy. The assay could be applied to other cell events characterized by APL externalization, including cell division and vesiculation.
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Dual mechanism of integrin ?IIb?3 closure in procoagulant platelets.
J. Biol. Chem.
PUBLISHED: 03-21-2013
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Inactivation of integrin ?IIb?3 reverses platelet aggregate formation upon coagulation.
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Neonatal antibody responses are attenuated by interferon-? produced by NK and T cells during RSV infection.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 03-18-2013
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Respiratory syncytial virus (RSV) infects most children in the first year of life and is a major single cause of hospitalization in infants and young children. There is no effective vaccine, and antibody generated by primary neonatal infection is poorly protective against reinfection even with antigenically homologous viral strains. Studying the immunological basis of these observations in neonatal mice, we found that antibody responses to infection were low and unaffected by CD4 depletion, in contrast with adult mice, which had stronger CD4-dependent antibody responses. Natural killer cell depletion or codepletion of CD4(+) and CD8(+) cells during neonatal RSV infection caused a striking increase in anti-RSV antibody titer. These cells are major sources of the cytokine IFN-?, and blocking IFN-? also enhanced RSV-specific antibody responses in neonates. In addition, infection with a recombinant RSV engineered to produce IFN-? reduced antibody titer, confirming that IFN-? plays a pivotal role in inhibition of antibody responses after neonatal infection. These unexpected findings show that the induction of a strong cellular immune response may limit antibody responses in early life and that vaccines that induce IFN-?-secreting cells might, in some situations, be less protective than those that do not.
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Attenuation of live respiratory syncytial virus vaccines is associated with reductions in levels of nasal cytokines.
J. Infect. Dis.
PUBLISHED: 03-12-2013
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Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract illness (LRTI) in children. Several promising live-attenuated RSV vaccines are in development. Defining additional markers of attenuation could enhance clinical trials.
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Is diabetes mellitus a heart disease equivalent in women? Results from an international study of postmenopausal women in the Raloxifene Use for the Heart (RUTH) Trial.
Circ Cardiovasc Qual Outcomes
PUBLISHED: 03-12-2013
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Several studies have concluded that diabetes mellitus and heart disease carry similar risk for future cardiovascular disease (CVD). Most of these studies were too small to quantify independent risks specific to women. The purpose of this study was to determine whether diabetes mellitus is a coronary heart disease (CHD) risk equivalent for prediction of future CHD and CVD events in women.
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Differential expression and methylation of brain developmental genes define location-specific subsets of pilocytic astrocytoma.
Acta Neuropathol.
PUBLISHED: 03-05-2013
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Pilocytic astrocytomas (PAs) are the most common brain tumors in pediatric patients and can cause significant morbidity, including chronic neurological deficiencies. They are characterized by activating alterations in the mitogen-activated protein kinase pathway, but little else is known about their development. To map the global DNA methylation profiles of these tumors, we analyzed 62 PAs and 7 normal cerebellum samples using Illumina 450K microarrays. These data revealed two subgroups of PA that separate according to tumor location (infratentorial versus supratentorial), and identified key neural developmental genes that are differentially methylated between the two groups, including NR2E1 and EN2. Integration with transcriptome microarray data highlighted significant expression differences, which were unexpectedly associated with a strong positive correlation between methylation and expression. Differentially methylated probes were often identified within the gene body and/or regions up- or downstream of the gene, rather than at the transcription start site. We also identified a large number of differentially methylated genes between cerebellar PAs and normal cerebellum, which were again enriched for developmental genes. In addition, we found a significant association between differentially methylated genes and SUZ12 binding sites, indicating potential disruption of the polycomb repressor complex 2 (PRC2). Taken together, these data suggest that PA from different locations in the brain may arise from region-specific cells of origin, and highlight the potential disruption of key developmental regulators during tumorigenesis. These findings have implications for future basic research and clinical trials, as therapeutic targets and drug sensitivity may differ according to tumor location.
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Alix serves as an adaptor that allows human parainfluenza virus type 1 to interact with the host cell ESCRT system.
PLoS ONE
PUBLISHED: 02-18-2013
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The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 in vivo, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with AlixBro1 protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.
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Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-14-2013
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Glioblastoma (GB) is the most common and aggressive primary brain malignancy, with poor prognosis and a lack of effective therapeutic options. Accumulating evidence suggests that intratumor heterogeneity likely is the key to understanding treatment failure. However, the extent of intratumor heterogeneity as a result of tumor evolution is still poorly understood. To address this, we developed a unique surgical multisampling scheme to collect spatially distinct tumor fragments from 11 GB patients. We present an integrated genomic analysis that uncovers extensive intratumor heterogeneity, with most patients displaying different GB subtypes within the same tumor. Moreover, we reconstructed the phylogeny of the fragments for each patient, identifying copy number alterations in EGFR and CDKN2A/B/p14ARF as early events, and aberrations in PDGFRA and PTEN as later events during cancer progression. We also characterized the clonal organization of each tumor fragment at the single-molecule level, detecting multiple coexisting cell lineages. Our results reveal the genome-wide architecture of intratumor variability in GB across multiple spatial scales and patient-specific patterns of cancer evolution, with consequences for treatment design.
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A genome-wide association study of resistance to HIV infection in highly exposed uninfected individuals with hemophilia A.
Hum. Mol. Genet.
PUBLISHED: 01-30-2013
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Human genetic variation contributes to differences in susceptibility to HIV-1 infection. To search for novel host resistance factors, we performed a genome-wide association study (GWAS) in hemophilia patients highly exposed to potentially contaminated factor VIII infusions. Individuals with hemophilia A and a documented history of factor VIII infusions before the introduction of viral inactivation procedures (1979-1984) were recruited from 36 hemophilia treatment centers (HTCs), and their genome-wide genetic variants were compared with those from matched HIV-infected individuals. Homozygous carriers of known CCR5 resistance mutations were excluded. Single nucleotide polymorphisms (SNPs) and inferred copy number variants (CNVs) were tested using logistic regression. In addition, we performed a pathway enrichment analysis, a heritability analysis, and a search for epistatic interactions with CCR5 ?32 heterozygosity. A total of 560 HIV-uninfected cases were recruited: 36 (6.4%) were homozygous for CCR5 ?32 or m303. After quality control and SNP imputation, we tested 1 081 435 SNPs and 3686 CNVs for association with HIV-1 serostatus in 431 cases and 765 HIV-infected controls. No SNP or CNV reached genome-wide significance. The additional analyses did not reveal any strong genetic effect. Highly exposed, yet uninfected hemophiliacs form an ideal study group to investigate host resistance factors. Using a genome-wide approach, we did not detect any significant associations between SNPs and HIV-1 susceptibility, indicating that common genetic variants of major effect are unlikely to explain the observed resistance phenotype in this population.
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Secretory meningiomas are defined by combined KLF4 K409Q and TRAF7 mutations.
Acta Neuropathol.
PUBLISHED: 01-28-2013
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Meningiomas are among the most frequent intracranial tumors. The secretory variant of meningioma is characterized by glandular differentiation, formation of intracellular lumina and pseudopsammoma bodies, expression of a distinct pattern of cytokeratins and clinically by pronounced perifocal brain edema. Here we describe whole-exome sequencing analysis of DNA from 16 secretory meningiomas and corresponding constitutional tissues. All secretory meningiomas invariably harbored a mutation in both KLF4 and TRAF7. Validation in an independent cohort of 14 secretory meningiomas by Sanger sequencing or derived cleaved amplified polymorphic sequence (dCAPS) assay detected the same pattern, with KLF4 mutations observed in a total of 30/30 and TRAF7 mutations in 29/30 of these tumors. All KLF4 mutations were identical, affected codon 409 and resulted in a lysine to glutamine exchange (K409Q). KLF4 mutations were not found in 89 non-secretory meningiomas, 267 other intracranial tumors including gliomas, glioneuronal tumors, pituitary adenomas and metastases, 59 peripheral nerve sheath tumors and 52 pancreatic tumors. TRAF7 mutations were restricted to the WD40 domains. While KLF4 mutations were exclusively seen in secretory meningiomas, TRAF7 mutations were also observed in 7/89 (8 %) of non-secretory meningiomas. KLF4 and TRAF7 mutations were mutually exclusive with NF2 mutations. In conclusion, our findings suggest an essential contribution of combined KLF4 K409Q and TRAF7 mutations in the genesis of secretory meningioma and demonstrate a role for TRAF7 alterations in other non-NF2 meningiomas.
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Both TMEM16F-dependent and TMEM16F-independent pathways contribute to phosphatidylserine exposure in platelet apoptosis and platelet activation.
Blood
PUBLISHED: 01-09-2013
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Scott syndrome, a bleeding disorder caused by defective phospholipid scrambling, has been associated with mutations in the TMEM16F gene. The role of TMEM16F in apoptosis- or agonist-induced phosphatidylserine (PS) exposure was studied in platelets from a Scott syndrome patient and control subjects. Whereas stimulation of control platelets with the BH3-mimetic ABT737 resulted in 2 distinct fractions with moderate and high PS exposure, the high PS-exposing fraction was markedly delayed in Scott platelets. High, but not moderate, PS exposure in platelets was suppressed by chelation of intracellular Ca(2+), whereas caspase inhibition completely abolished ABT737-induced PS exposure in both Scott and control platelets. On the other hand, high PS exposure induced by the Ca(2+)-mobilizing agonists convulxin/thrombin fully relied on mitochondrial depolarization and was virtually absent in Scott platelets. Finally, PS exposure induced by collagen/thrombin was partly affected in Scott platelets, and the residual PS positive fraction was insensitive to inhibition of caspases or mitochondrial depolarization. In conclusion, TMEM16F is not required for, but enhances, caspase-dependent PS exposure; convulxin-/thrombin-induced PS exposure is entirely dependent on TMEM16F, whereas collagen/thrombin-induced PS exposure results from 2 distinct pathways, one of which involves mitochondrial depolarization and is mediated by TMEM16F.
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Evaluation of the replication, pathogenicity, and immunogenicity of avian paramyxovirus (APMV) serotypes 2, 3, 4, 5, 7, and 9 in rhesus macaques.
PLoS ONE
PUBLISHED: 01-01-2013
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Avian paramyxoviruses (APMV) serotypes 1-9 are frequently isolated from domestic and wild birds worldwide. APMV-1 (also called Newcastle disease virus, NDV) is attenuated in non-human primates and is being developed as a candidate human vaccine vector. The vector potential of the other serotypes was unknown. In the present study, we evaluated nine different biologically- or recombinantly-derived APMV strains for the ability to replicate and cause disease in rhesus macaque model. Five of the viruses were: biologically-derived wild type (wt) APMV-2, -3, -5, -7 and -9. Another virus was a recombinant (r) version of wt APMV-4. The remaining three viruses were versions of wt rAPMV-2, -4 and -7 in which the F cleavage site had been modified to be multi-basic. Rhesus macaques were inoculated intranasally and intratracheally and monitored for clinical disease, virus shedding from the upper and lower respiratory tract, and seroconversion. Virus shedding was not detected for wt APMV-5. Very limited shedding was detected for wt rAPMV-4 and modified rAPMV-4, and only in a subset of animals. Shedding by the other viruses was detected in every infected animal, and usually from both the upper and lower respiratory tract. In particular, shedding over a number of days in every animal was observed for modified rAPMV-2, wt APMV-7, and modified rAPMV-7. Modification of the F protein cleavage site appeared to increase shedding by wt rAPMV-2 and marginally by wt rAPMV-4. All APMVs except wt APMV-5 induced a virus-specific serum antibody response in all infected animals. None of the animals exhibited any clinical disease signs. These results indicate that APMVs 2, 3, 4, 7, and 9 are competent to infect non-human primates, but are moderately-to-highly restricted, depending on the serotype. This suggests that they are not likely to significantly infect primates in nature, and represent promising attenuated candidates for vector development.
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Comparative immunogenicity of HIV-1 gp160, gp140 and gp120 expressed by live attenuated newcastle disease virus vector.
PLoS ONE
PUBLISHED: 01-01-2013
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The development of a vaccine against human immunodeficiency virus-1 (HIV-1) capable of inducing broad humoral and cellular responses at both the systemic and mucosal levels will be critical for combating the global AIDS epidemic. We previously demonstrated the ability of Newcastle disease virus (NDV) as a vaccine vector to express oligomeric Env protein gp160 and induce potent humoral and mucosal immune responses. In the present study, we used NDV vaccine strain LaSota as a vector to compare the biochemical and immunogenic properties of vector-expressed gp160, gp120, and two versions of gp140 (a derivative of gp160 made by deleting the transmembrane and cytoplasmic domains), namely: gp140L, which contained the complete membrane-proximal external region (MPER), and gp140S, which lacks the distal half of MPER. We show that, similar to gp160, NDV-expressed gp140S and gp120, but not gp140L, formed higher-order oligomers that retained recognition by conformationally sensitive monoclonal antibodies. Immunization of guinea pigs by the intranasal route with rLaSota/gp140S resulted in significantly greater systemic and mucosal antibody responses compared to the other recombinants. Immunization with rLaSota/140S, rLaSota/140L rLaSota/120 resulted in mixed Th1/Th2 immune responses as compared to Th1-biased immune responses induced by rLaSota/160. Importantly, rLaSota/gp140S induced neutralizing antibody responses to homologous HIV-1 strain BaL.26 and laboratory adapted HIV-1 strain MN.3 that were stronger than those elicited by the other NDV recombinants. Additionally, rLaSota/gp140S induced greater CD4+ and CD8+ T-cell responses in mice. These studies illustrate that rLaSota/gp140S is a promising vaccine candidate to elicit potent mucosal, humoral and cellular immune responses to the HIV-1 Env protein.
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Newcastle disease virus fusion protein is the major contributor to protective immunity of genotype-matched vaccine.
PLoS ONE
PUBLISHED: 01-01-2013
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Virulent strains of Newcastle disease virus (NDV) can cause devastating disease in chickens worldwide. Although the current vaccines are substantially effective, they do not completely prevent infection, virus shedding and disease. To produce genotype-matched vaccines, a full-genome reverse genetics system has been used to generate a recombinant virus in which the F protein cleavage site has been changed to that of avirulent vaccine virus. In the other strategy, the vaccines have been generated by replacing the F and HN genes of a commercial vaccine strain with those from a genotype-matched virus. However, the protective efficacy of a chimeric virus vaccine has not been directly compared with that of a full-genome virus vaccine developed by reverse genetics. Therefore, in this study, we evaluated the protective efficacy of genotype VII matched chimeric vaccines by generating three recombinant viruses based on avirulent LaSota (genotype II) strain in which the open reading frames (ORFs) encoding the F and HN proteins were replaced, individually or together, with those of the circulating and highly virulent Indonesian NDV strain Ban/010. The cleavage site of the Ban/010 F protein was mutated to the avirulent motif found in strain LaSota. In vitro growth characteristics and a pathogenicity test indicated that all three chimeric viruses retained the highly attenuated phenotype of the parental viruses. Immunization of chickens with chimeric and full-length genome VII vaccines followed by challenge with virulent Ban/010 or Texas GB (genotype II) virus demonstrated protection against clinical disease and death. However, only those chickens immunized with chimeric rLaSota expressing the F or F plus HN proteins of the Indonesian strain were efficiently protected against shedding of Ban/010 virus. Our findings showed that genotype-matched vaccines can provide protection to chickens by efficiently preventing spread of virus, primarily due to the F protein.
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Coordinate deletion of N-glycans from the heptad repeats of the fusion F protein of Newcastle disease virus yields a hyperfusogenic virus with increased replication, virulence, and immunogenicity.
J. Virol.
PUBLISHED: 12-28-2011
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The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence.
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Diagnosis and management of von Willebrand disease in the United Kingdom.
Semin. Thromb. Hemost.
PUBLISHED: 11-18-2011
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The UK treatment strategy for von Willebrand disease (VWD) is based on consensus guidelines produced by the United Kingdom Haemophilia Centre Doctors Organization (UKHCDO) relating to the diagnosis and management of VWD. Selection of therapeutic products suitable for treatment of this complex inherited bleeding disorder is based on the observed response. Desmopressin (DDAVP), an analog of vasopressin, is the recommended treatment in individuals who respond to this drug on trial infusion. DDAVP clearly has no effect in type 3 VWD but may have variable clinical effect in individuals with other subtypes or may be contraindicated in some cases. In patients where DDAVP treatment is unsuitable, replacement factor concentrate containing von Willebrand factor (VWF) is the recommended alternative. Relevant concentrates are available for all patients in the United Kingdom, and treatment is administered by a network of 67 hemophilia treatment centers that also provide specialist care for individuals diagnosed with VWD. Patients diagnosed with the condition are registered on a national inherited bleeding disorder database administered by the UKHCDO on behalf of the Department of Health to aid in service planning and commissioning. Genetic testing is employed in the United Kingdom in certain situations, which is also performed in accordance with current UKHCDO guidelines.
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Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells.
J. Virol.
PUBLISHED: 11-16-2011
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Respiratory syncytial virus (RSV) is one of the major causes of respiratory infections in children, and it is the main pathogen causing bronchiolitis in infants. The binding and entry mechanism by which RSV infects respiratory epithelial cells has not yet been determined. In this study, the earliest stages of RSV infection in normal human bronchial epithelial cells were probed by tracking virions with fluorescent lipophilic dyes in their membranes. Virions colocalized with cholesterol-containing plasma membrane microdomains, identified by their ability to bind cholera toxin subunit B. Consistent with an important role for cholesterol in RSV infection, cholesterol depletion profoundly inhibited RSV infection, while cholesterol repletion reversed this inhibition. Merger of the outer leaflets of the viral envelope and the cell membrane appeared to be triggered at these sites. Using small-molecule inhibitors, RSV infection was found to be sensitive to Pak1 inhibition, suggesting the requirement of a subsequent step of cytoskeletal reorganization that could involve plasma membrane rearrangements or endocytosis. It appears that RSV entry depends on its ability to dock to cholesterol-rich microdomains (lipid rafts) in the plasma membrane where hemifusion events begin, assisted by a Pak1-dependent process.
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Population pharmacokinetics of recombinant factor VIII: the relationships of pharmacokinetics to age and body weight.
Blood
PUBLISHED: 10-31-2011
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Comparison of the pharmacokinetics (PK) of a coagulation factor between groups of patients can be biased by differences in study protocols, in particular between blood sampling schedules. This could affect clinical dose tailoring, especially in children. The aim of this study was to describe the relationships of the PK of factor VIII (FVIII) with age and body weight by a population PK model. The potential to reduce blood sampling was also explored. A model was built for FVIII PK from 236 infusions of recombinant FVIII in 152 patients (1-65 years of age) with severe hemophilia A. The PK of FVIII over the entire age range was well described by a 2-compartment model and a previously reported problem, resulting from differences in blood sampling, to compare findings from children and adults was practically abolished. The decline in FVIII clearance and increase in half-life with age could be described as continuous functions. Retrospective reduction of blood sampling from 11 to 5 samples made no important difference to the estimates of PK parameters. The obtained findings can be used as a basis for PK-based dose tailoring of FVIII in clinical practice, in all age groups, with minimal blood sampling.
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Evaluation of two chimeric bovine-human parainfluenza virus type 3 vaccines in infants and young children.
Vaccine
PUBLISHED: 09-22-2011
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Human parainfluenza virus type 3 (HPIV3) is an important cause of lower respiratory tract illness in children, yet a licensed vaccine or antiviral drug is not available. We evaluated the safety, tolerability, infectivity, and immunogenicity of two intranasal, live-attenuated HPIV3 vaccines, designated rHPIV3-N(B) and rB/HPIV3, that were cDNA-derived chimeras of HPIV3 and bovine PIV3 (BPIV3). These were evaluated in adults, HPIV3 seropositive children, and HPIV3 seronegative children. A total of 112 subjects participated in these studies. Both rB/HPIV3 and rHPIV3-N(B) were highly restricted in replication in adults and seropositive children but readily infected seronegative children, who shed mean peak virus titers of 10(2.8) vs. 10(3.7)pfu/mL, respectively. Although rB/HPIV3 was more restricted in replication in seronegative children than rHPIV3-N(B), it induced significantly higher titers of hemagglutination inhibition (HAI) antibodies against HPIV3. Taken together, these data suggest that the rB/HPIV3 vaccine is the preferred candidate for further clinical development.
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Progress in the development of human parainfluenza virus vaccines.
Expert Rev Respir Med
PUBLISHED: 08-24-2011
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In children under 5 years of age, human parainfluenza viruses (HPIVs) as a group are the second most common etiology of acute respiratory illness leading to hospitalization, surpassed only by respiratory syncytial virus but ahead of influenza viruses. Using reverse genetics systems for HPIV serotypes 1, 2 and 3 (HPIV1, 2 and 3), several live-attenuated HPIVs have been generated and evaluated as intranasal vaccines in adults and in children. Two vaccines against HPIV3 were found to be well tolerated, infectious and immunogenic in Phase I trials in HPIV3-seronegative infants and children and should progress to proof-of-concept trials. Vaccines against HPIV1 and HPIV2 are less advanced and have just entered pediatric trials.
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Newcastle disease virus expressing human immunodeficiency virus type 1 envelope glycoprotein induces strong mucosal and serum antibody responses in Guinea pigs.
J. Virol.
PUBLISHED: 08-17-2011
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Human immunodeficiency virus type 1 (HIV-1) is transmitted mainly through mucosal sites. Optimum strategies to elicit both systemic and mucosal immunity are critical for the development of vaccines against HIV-1. We therefore sought to evaluate the induction of systemic and mucosal immune responses by the use of Newcastle disease virus (NDV) as a vaccine vector. We generated a recombinant NDV, designated rLaSota/gp160, expressing the gp160 envelope (Env) protein of HIV-1 from an added gene. The gp160 protein expressed by rLaSota/gp160 virus was detected on an infected cell surface and was incorporated into the NDV virion. Biochemical studies showed that gp160 present in infected cells and in the virion formed a higher-order oligomer that retained recognition by conformationally sensitive monoclonal antibodies. Expression of gp160 did not increase the virulence of recombinant NDV (rNDV) strain LaSota. Guinea pigs were administered rLaSota/gp160 via the intranasal (i.n.) or intramuscular (i.m.) route in different prime-boost combinations. Systemic and mucosal antibody responses specific to the HIV-1 envelope protein were assessed in serum and vaginal washes, respectively. Two or three immunizations via the i.n. or i.m. route induced a more potent systemic and mucosal immune response than a single immunization by either route. Priming by the i.n. route was more immunogenic than by the i.m. route, and the same was true for the boosts. Furthermore, immunization with rLaSota/gp160 by any route or combination of routes induced a Th1-type response, as reflected by the induction of stronger antigen-specific IgG2a than IgG1 antibody responses. Additionally, i.n. immunization elicited a stronger neutralizing serum antibody response to laboratory-adapted HIV-1 strain MN.3. These data illustrate that it is feasible to use NDV as a vaccine vector to elicit potent humoral and mucosal responses to the HIV-1 envelope protein.
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Progress in understanding and controlling respiratory syncytial virus: still crazy after all these years.
Virus Res.
PUBLISHED: 08-14-2011
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Human respiratory syncytial virus (RSV) is a ubiquitous pathogen that infects everyone worldwide early in life and is a leading cause of severe lower respiratory tract disease in the pediatric population as well as in the elderly and in profoundly immunosuppressed individuals. RSV is an enveloped, nonsegmented negative-sense RNA virus that is classified in Family Paramyxoviridae and is one of its more complex members. Although the replicative cycle of RSV follows the general pattern of the Paramyxoviridae, it encodes additional proteins. Two of these (NS1 and NS2) inhibit the host type I and type III interferon (IFN) responses, among other functions, and another gene encodes two novel RNA synthesis factors (M2-1 and M2-2). The attachment (G) glycoprotein also exhibits unusual features, such as high sequence variability, extensive glycosylation, cytokine mimicry, and a shed form that helps the virus evade neutralizing antibodies. RSV is notable for being able to efficiently infect early in life, with the peak of hospitalization at 2-3 months of age. It also is notable for the ability to reinfect symptomatically throughout life without need for significant antigenic change, although immunity from prior infection reduces disease. It is widely thought that re-infection is due to an ability of RSV to inhibit or subvert the host immune response. Mechanisms of viral pathogenesis remain controversial. RSV is notable for a historic, tragic pediatric vaccine failure involving a formalin-inactivated virus preparation that was evaluated in the 1960s and that was poorly protective and paradoxically primed for enhanced RSV disease. RSV also is notable for the development of a successful strategy for passive immunoprophylaxis of high-risk infants using RSV-neutralizing antibodies. Vaccines and new antiviral drugs are in pre-clinical and clinical development, but controlling RSV remains a formidable challenge.
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The cDNA-derived investigational human parainfluenza virus type 3 vaccine rcp45 is well tolerated, infectious, and immunogenic in infants and young children.
Pediatr. Infect. Dis. J.
PUBLISHED: 08-11-2011
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Human parainfluenza virus type 3 (HPIV3) is an important yet underappreciated cause of lower respiratory tract illness in children, and a licensed vaccine is not yet available.
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NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature.
Neuro-oncology
PUBLISHED: 07-30-2011
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Glioblastoma multiforme (GBM) is the most common type of primary brain tumor and a highly malignant and heterogeneous cancer. Current conventional therapies fail to eradicate or curb GBM cell growth. Hence, exploring the cellular and molecular basis of GBM cell growth is vital to develop novel therapeutic approaches. Neuroglia (NG)-2 is a transmembrane proteoglycan expressed by NG2+ progenitors and is strongly linked to cell proliferation in the normal brain. By using NG2 as a biomarker we identify a GBM cell population (GBM NG2+ cells) with robust proliferative, clonogenic, and tumorigenic capacity. We show that a significant proportion (mean 83%) of cells proliferating in the tumor mass express NG2 and that over 50% of GBM NG2+ cells are proliferating. Compared with the GBM NG2- cells from the same tumor, the GBM of NG2+ cells overexpress genes associated with aggressive tumorigenicity, including overexpression of Mitosis and Cell Cycling Module genes (e.g., MELK, CDC, MCM, E2F), which have been previously shown to correlate with poor survival in GBM. We also show that the coexpression pattern of NG2 with other glial progenitor markers in GBM does not recapitulate that described in the normal brain. The expression of NG2 by such an aggressive and actively cycling GBM population combined with its location on the cell surface identifies this cell population as a potential therapeutic target in a subset of patients with GBM.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.