Second site escape of a T20-dependent HIV-1 variant by a single amino acid change in the CD4 binding region of the envelope glycoprotein

BACKGROUND: We previously described the selection of a T20-dependent human immunodeficiency virus type-1 (HIV-1) variant in a patient on T20 therapy. The fusion inhibitor T20 targets the viral envelope (Env) protein by blocking a conformational switch that is critical for viral entry into the host cell. T20-dependent viral entry is the result of 2 mutations in Env (GIA-SKY), creating a protein that undergoes a premature conformational switch, and the presence of T20 prevents this premature switch and rescues viral entry. In the present study, we performed 6 independent evolution experiments with the T20-dependent HIV-1 variant in the absence of T20, with the aim to identify second site compensatory changes, which may provide new mechanistic insights into Env function and the T20-dependence mechanism. RESULTS: Escape variants with improved replication capacity appeared within 42 days in 5 evolution cultures. Strikingly, 3 cultures revealed the same single amino acid change in the CD4 binding region of Env (glycine at position 431 substituted for arginine: G431R). This mutation was sufficient to abolish the T20-dependence phenotype and restore viral replication in the absence of T20. The GIA-SKY-G431R escape variant produces an Env protein that exhibits reduced syncytia formation and reduced cell-cell fusion activity. The escape variant was more sensitive to an antibody acting on an early gp41 intermediate, suggesting that the G431R mutation helps preserve a pre-fusion Env conformation, similar to T20 action. The escape variant was also less sensitive to soluble CD4, suggesting a reduced CD4 receptor affinity. CONCLUSION: The forced evolution experiments indicate that the premature conformational switch of the T20-dependent HIV-1 Env variant (GIA-SKY) can be corrected by a second site mutation in Env (GIA-SKY-G431R) that affects the interaction with the CD4 receptor

The alpha 7 nicotinic receptor agonist PHA-543613 hydrochloride inhibits <i>Porphyromonas gingivalis</i>-induced expression of interleukin-8 by oral keratinocytes

Objective: The alpha 7 nicotinic receptor (α7nAChR) is expressed by oral keratinocytes. α7nAChR activation mediates anti-inflammatory responses. The objective of this study was to determine if α7nAChR activation inhibited pathogen-induced interleukin-8 (IL-8) expression by oral keratinocytes.&lt;p&gt;&lt;/p&gt; Materials and methods: Periodontal tissue expression of α7nAChR was determined by real-time PCR. OKF6/TERT-2 oral keratinocytes were exposed to &lt;i&gt;Porphyromonas gingivalis&lt;/i&gt; in the presence and absence of a α7nAChR agonist (PHA-543613 hydrochloride) alone or after pre-exposure to a specific α7nAChR antagonist (α-bungarotoxin). Interleukin-8 (IL-8) expression was measured by ELISA and real-time PCR. Phosphorylation of the NF-κB p65 subunit was determined using an NF-κB p65 profiler assay and STAT-3 activation by STAT-3 in-cell ELISA. The release of ACh from oral keratinocytes in response to &lt;i&gt;P. gingivalis&lt;/i&gt; lipopolysaccharide was determined using a GeneBLAzer M3 CHO-K1-blacell reporter assay.&lt;p&gt;&lt;/p&gt; Results: Expression of α7nAChR mRNA was elevated in diseased periodontal tissue. PHA-543613 hydrochloride inhibited &lt;i&gt;P. Gingivalis&lt;/i&gt;-induced expression of IL-8 at the transcriptional level. This effect was abolished when cells were pre-exposed to a specific α7nAChR antagonist, α-bungarotoxin. PHA-543613 hydrochloride downregulated NF-κB signalling through reduced phosphorylation of the NF-κB p65-subunit. In addition, PHA-543613 hydrochloride promoted STAT-3 signalling by maintenance of phosphorylation. Furthermore, oral keratinocytes upregulated ACh release in response to &lt;i&gt;P. Gingivalis&lt;/i&gt; lipopolysaccharide.&lt;p&gt;&lt;/p&gt; Conclusion: These data suggest that α7nAChR plays a role in regulating the innate immune responses of oral keratinocytes.&lt;p&gt;&lt;/p&gt

A spatio-temporal mining approach towards summarizing and analyzing protein folding trajectories

Understanding the protein folding mechanism remains a grand challenge in structural biology. In the past several years, computational theories in molecular dynamics have been employed to shed light on the folding process. Coupled with high computing power and large scale storage, researchers now can computationally simulate the protein folding process in atomistic details at femtosecond temporal resolution. Such simulation often produces a large number of folding trajectories, each consisting of a series of 3D conformations of the protein under study. As a result, effectively managing and analyzing such trajectories is becoming increasingly important. In this article, we present a spatio-temporal mining approach to analyze protein folding trajectories. It exploits the simplicity of contact maps, while also integrating 3D structural information in the analysis. It characterizes the dynamic folding process by first identifying spatio-temporal association patterns in contact maps, then studying how such patterns evolve along a folding trajectory. We demonstrate that such patterns can be leveraged to summarize folding trajectories, and to facilitate the detection and ordering of important folding events along a folding path. We also show that such patterns can be used to identify a consensus partial folding pathway across multiple folding trajectories. Furthermore, we argue that such patterns can capture both local and global structural topology in a 3D protein conformation, thereby facilitating effective structural comparison amongst conformations. We apply this approach to analyze the folding trajectories of two small synthetic proteins-BBA5 and GSGS (or Beta3S). We show that this approach is promising towards addressing the above issues, namely, folding trajectory summarization, folding events detection and ordering, and consensus partial folding pathway identification across trajectories

Monitoring HSVtk suicide gene therapy: the role of [18F]FHPG membrane transport

Favourable pharmacokinetics of the prodrug are essential for successful HSVtk/ganciclovir (GCV) suicide gene therapy. [F-18] FHPG PET might be a suitable technique to assess the pharmacokinetics of the prodrug GCV noninvasively, provided that [F-18] FHPG mimics the behaviour of GCV. Since membrane transport is an important aspect of the pharmacokinetics of the prodrug, we investigated the cellular uptake mechanism of [F-18] FHPG in an HSVtk expressing C6 rat glioma cell line and in tumour- bearing rats. The nucleoside transport inhibitors dipyridamol, NBMPR and 2- chloroadenosine did not significantly affect the [F-18] FHPG uptake in vitro. Thymidine and uridine significantly decreased [F-18] FHPG uptake by 84 and 58%, respectively, but an enzyme assay revealed that this decline was due to inhibition of the HSVtk enzyme rather than membrane transport. Nucleobase transport inhibitors, thymine and adenine, caused a 58 and 55% decline in tracer uptake, respectively. In vivo, the ratio of [F-18] FHPG uptake in C6tk and C6 tumours decreased from 3.070.5 to 1.070.2 after infusion of adenine. Thus, in our tumour model, [F-18] FHPG transport exclusively occurred via purine nucleobase transport. In this respect, FHPG does not resemble GCV, which is predominantly taken up via the nucleoside transporter, but rather acyclovir, which is also taken up via the purine nucleobase carrier

Police Strategies and Suspect Responses in Real-Life Serious Crime Interviews

This research focuses exclusively on real-life taped interviews with serious crime suspects and examines the strategies used and types of questions asked by police, and suspects’ responses to these. The information source was audio-tape-recorded interviews with 56 suspects. These recordings were obtained from 11 police services across England and Wales and were analysed using a specially designed coding frame. It was found that interviewers employed a range of strategies with presentation of evidence and challenge the most frequently observed. Closed questions were by far the most frequently used, and open questions, although less frequent, were found to occur more during the opening phases of the interviews. The frequency of ineffective question types (e.g. negative, repetitive, multiple) was low. A number of significant associations were observed between interviewer strategies and suspect responses. Rapport/empathy and open-type questions were associated with an increased likelihood of suspects admitting the offence whilst describing trauma, and negative questions were associated with a decreased likelihood

TEV GAMMA-RAYS FROM PROTON BLAZARS

Proton acceleration in nearby blazars can be diagnosed measuring their intense TeV $\gamma$-ray emission. Flux predictions for 1101+384 (Mrk421) and 1219+285 (ON231), both strong EGRET sources (0.1-10 GeV), are obtained from model spectra of unsaturated synchrotron pair cascades fitted to publicly available multifrequency data. An experimental effort to confirm the predicted emission in the range 1-10 TeV would be of great importance for the problems of the origin of cosmic rays, the era of galaxy formation and the cosmological distance scale.Comment: 10 pages of latex using Kluwer spacekap.sty, to appear in Space Science Review

Enhanced effects of cigarette smoke extract on inflammatory cytokine expression in IL-1β-activated human mast cells were inhibited by Baicalein via regulation of the NF-κB pathway

Background: Human mast cells are capable of a wide variety of inflammatory responses and play a vital role in the pathogenesis of inflammatory diseases such as allergy, asthma, and atherosclerosis. We have reported that cigarette smoke extract (CSE) significantly increased IL-6 and IL-8 production in IL-1β-activated human mast cell line (HMC-1). Baicalein (BAI) has anti-inflammatory properties and inhibits IL-1β- and TNF-α-induced inflammatory cytokine production from HMC-1. The goal of the present study was to examine the effect of BAI on IL-6 and IL-8 production from CSE-treated and IL-1β-activated HMC-1.Methods: Main-stream (Ms) and Side-stream (Ss) cigarette smoke were collected onto fiber filters and extracted in RPMI-1640 medium. Two ml of HMC-1 at 1 × 10 6 cells/mL were cultured with CSE in the presence or absence of IL-1β (10 ng/mL) for 24 hrs. A group of HMC-1 cells stimulated with both IL-1β (10 ng/ml) and CSE was also treated with BAI. The expression of IL-6 and IL-8 was assessed by ELISA and RT-PCR. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA) and IκBα degradation by Western blot.Results: Both Ms and Ss CSE significantly increased IL-6 and IL-8 production (p \u3c 0.001) in IL-1β-activated HMC-1. CSE increased NF-κB activation and decreased cytoplasmic IκBα proteins in IL-1β-activated HMC-1. BAI (1.8 to 30 μM) significantly inhibited production of IL-6 and IL-8 in a dose-dependent manner in IL-1β-activated HMC-1 with the optimal inhibition concentration at 30 μM, which also significantly inhibited the enhancing effect of CSE on IL-6 and IL-8 production in IL-1β-activated HMC-1. BAI inhibited NF-κB activation and increased cytoplasmic IκBα proteins in CSE-treated and IL-1β-activated HMC-1.Conclusions: Our results showed that CSE significantly increased inflammatory cytokines IL-6 and IL-8 production in IL-1β-activated HMC-1. It may partially explain why cigarette smoke contributes to lung and cardiovascular diseases. BAI inhibited the production of inflammatory cytokines through inhibition of NF-κB activation and IκBα phosphorylation and degradation. This inhibitory effect of BAI on the expression of inflammatory cytokines induced by CSE suggests its usefulness in the development of novel anti-inflammatory therapies

Informing evaluation of a smartphone application for people with acquired brain injury: a stakeholder engagement study

Background Brain in Hand is a smartphone application (app) that allows users to create structured diaries with problems and solutions, attach reminders, record task completion and has a symptom monitoring system. Brain in Hand was designed to support people with psychological problems, and encourage behaviour monitoring and change. The aim of this paper is to describe the process of exploring the barriers and enablers for the uptake and use of Brain in Hand in clinical practice, identify potential adaptations of the app for use with people with acquired brain injury (ABI), and determine whether the behaviour change wheel can be used as a model for engagement. Methods We identified stakeholders: ABI survivors and carers, National Health Service and private healthcare professionals, and engaged with them via focus groups, conference presentations, small group discussions, and through questionnaires. The results were evaluated using the behaviour change wheel and descriptive statistics of questionnaire responses. Results We engaged with 20 ABI survivors, 5 carers, 25 professionals, 41 questionnaires were completed by stakeholders. Comments made during group discussions were supported by questionnaire results. Enablers included smartphone competency (capability), personalisation of app (opportunity), and identifying perceived need (motivation). Barriers included a physical and cognitive inability to use smartphone (capability), potential cost and reliability of technology (opportunity), and no desire to use technology or change from existing strategies (motivation). The stakeholders identified potential uses and changes to the app, which were not easily mapped onto the behaviour change wheel, e.g. monitoring fatigue levels, method of logging task completion, and editing the diary on their smartphone. Conclusions The study identified that both ABI survivors and therapists could see a use for Brain in Hand, but wanted users to be able to personalise it themselves to address individual user needs, e.g. monitoring activity levels. The behaviour change wheel is a useful tool when designing and evaluating engagement activities as it addresses most aspects of implementation, however additional categories may be needed to explore the specific features of assistive technology interventions, e.g. technical functions

Complementary and alternative medicine use among US Navy and Marine Corps personnel

<p>Abstract</p> <p>Background</p> <p>Recently, numerous studies have revealed an increase in complementary and alternative medicine (CAM) use in US civilian populations. In contrast, few studies have examined CAM use within military populations, which have ready access to conventional medicine. Currently, the prevalence and impact of CAM use in US military populations remains unknown.</p> <p>Methods</p> <p>To investigate CAM use in US Navy and Marine Corps personnel, the authors surveyed a stratified random sample of 5,000 active duty and Reserve/National Guard members between December 2000 and July 2002. Chi-square tests and multivariable logistic regression were used to assess univariate associations and adjusted odds of CAM use in this population.</p> <p>Results and discussion</p> <p>Of 3,683 service members contacted, 1,446 (39.3%) returned a questionnaire and 1,305 gave complete demographic and survey data suitable for study. Among respondents, more than 37% reported using at least one CAM therapy during the past year. Herbal therapies were among the most commonly reported (15.9%). Most respondents (69.8%) reported their health as being very good or excellent. Modeling revealed that CAM use was most common among personnel who were women, white, and officers. Higher levels of recent physical pain and lower levels of satisfaction with conventional medical care were significantly associated with increased odds of reporting CAM use.</p> <p>Conclusion</p> <p>These data suggest that CAM use is prevalent in the US military and consistent with patterns in other US civilian populations. Because there is much to be learned about CAM use along with allopathic therapy, US military medical professionals should record CAM therapies when collecting medical history data.</p

A Multitrait–Multimethod Analysis of the Construct Validity of Child Anxiety Disorders in a Clinical Sample

The present study examines the construct validity of separation anxiety disorder (SAD), social phobia (SoP), panic disorder (PD), and generalized anxiety disorder (GAD) in a clinical sample of children. Participants were 174 children, 6 to 17 years old (94 boys) who had undergone a diagnostic evaluation at a university hospital based clinic. Parent and child ratings of symptom severity were assessed using the Multidimensional Anxiety Scale for Children (MASC). Diagnostician ratings were obtained from the Anxiety Disorders Interview Schedule for Children and Parents (ADIS: C/P). Discriminant and convergent validity were assessed using confirmatory factor analytic techniques to test a multitrait–multimethod model. Confirmatory factor analyses supported the current classification of these child anxiety disorders. The disorders demonstrated statistical independence from each other (discriminant validity of traits), the model fit better when the anxiety syndromes were specified than when no specific syndromes were specified (convergent validity), and the methods of assessment yielded distinguishable, unique types of information about child anxiety (discriminant validity of methods). Using a multi-informant approach, these findings support the distinctions between childhood anxiety disorders as delineated in the current classification system, suggesting that disagreement between informants in psychometric studies of child anxiety measures is not due to poor construct validity of these anxiety syndromes