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Presynaptic BK channel localization is dependent on the hierarchical organization of alpha-catulin and dystrobrevin and fine-tuned by CaV2 calcium channels

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Large conductance, calcium-activated BK channels regulate many important physiological processes, including smooth muscle excitation, hormone release and synaptic transmission. The biological roles of these channels hinge on their unique ability to respond synergistically to both voltage and cytosolic calcium elevations. Because calcium influx is meticulously regulated both spatially and temporally, the localization of BK channels near calcium channels is critical for their proper function. However, the mechanism underlying BK channel localization near calcium channels is not fully understood.

RESULTS: We show here that in C. elegans the localization of SLO-1/BK channels to presynaptic terminals, where UNC-2/CaV2 calcium channels regulate neurotransmitter release, is controlled by the hierarchical organization of CTN-1/alpha-catulin and DYB-1/dystrobrevin, two proteins that interact with cortical cytoskeletal proteins. CTN-1 organizes a macromolecular SLO-1 channel complex at presynaptic terminals by direct physical interaction. DYB-1 contributes to the maintenance or stabilization of the complex at presynaptic terminals by interacting with CTN-1. We also show that SLO-1 channels are functionally coupled with UNC-2 calcium channels, and that normal localization of SLO-1 to presynaptic terminals requires UNC-2. In the absence of UNC-2, SLO-1 clusters lose the localization specificity, thus accumulating inside and outside of presynaptic terminals. Moreover, CTN-1 is also similarly localized in unc-2 mutants, consistent with the direct interaction between CTN-1 and SLO-1. However, localization of UNC-2 at the presynaptic terminals is not dependent on either CTN-1 or SLO-1. Taken together, our data strongly suggest that the absence of UNC-2 indirectly influences SLO-1 localization via the reorganization of cytoskeletal proteins.

CONCLUSION: CTN-1 and DYB-1, which interact with cortical cytoskeletal proteins, are required for the presynaptic punctate localization of SLO-1 in a hierarchical manner. In addition, UNC-2 calcium channels indirectly control the fidelity of SLO-1 puncta localization at presynaptic terminals. We suggest that the absence of UNC-2 leads to the reorganization of the cytoskeletal structure that includes CTN-1, which in turn influences SLO-1 puncta localization.

A chimeric EBV gp350/220-based VLP replicates the virion B-cell attachment mechanism and elicits long-lasting neutralizing antibodies in mice

Wed, 07/22/2015 - 12:27pm

Epstein-Barr virus (EBV), an oncogenic gammaherpesvirus, causes acute infectious mononucleosis (AIM) and is linked to the development of several human malignancies. There is an urgent need for a vaccine that is safe, prevents infection and/or limits disease. Unique among human herpesviruses, glycoprotein (gp)350/220, which initiates EBV attachment to susceptible host cells, is the major ligand on the EBV envelope and is highly conserved. Interaction between gp350/220 and complement receptor type 2 (CR2)/CD21 and/or (CR1)/CD35 on B-cells is required for infection. Potent antibody responses to gp350/220 occur in animal models and humans. Thus, gp350/220 provides an attractive candidate for prophylactic subunit vaccine development. However, in a recent Phase II clinical trial immunization with soluble recombinant gp350 reduced the incidence of AIM, but did not prevent infection. Despite various attempts to produce an EBV vaccine, no vaccine is licensed. Herein we describe a sub-unit vaccine against EBV based on a novel Newcastle disease virus (NDV)-virus-like particle (VLP) platform consisting of EBVgp350/220 ectodomain fused to NDV-fusion (F) protein. The chimeric protein EBVgp350/220-F is incorporated into the membrane of a VLP composed of the NDV matrix and nucleoprotein. The particles resemble native EBV in diameter and shape and bind CD21 and CD35. Immunization of BALB/c mice with EBVgp350/220-F VLPs elicited strong, long-lasting neutralizing antibody responses when assessed in vitro. This chimeric VLP is predicted to provide a superior safety profile as it is efficiently produced in Chinese hamster ovary (CHO) cells using a platform devoid of human nucleic acid and EBV-transforming genes.

HIV-1 non-macrophage-tropic R5 envelope glycoproteins are not more tropic for entry into primary CD4+ T-cells than envelopes highly adapted for macrophages

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Non-mac-tropic HIV-1 R5 viruses are predominantly transmitted and persist in immune tissue even in AIDS patients who carry highly mac-tropic variants in the brain. Non-mac-tropic R5 envelopes (Envs) require high CD4 levels for infection contrasting with highly mac-tropic Envs, which interact more efficiently with CD4 and mediate infection of macrophages that express low CD4. Non-mac-tropic R5 Envs predominantly target T-cells during transmission and in immune tissue where they must outcompete mac-tropic variants. Here, we investigated whether Env+ pseudoviruses bearing transmitted/founder (T/F), early and late disease non-mac-tropic R5 envelopes mediated more efficient infection of CD4+ T-cells compared to those with highly mac-tropic Envs.

RESULTS: Highly mac-tropic Envs mediated highest infectivity for primary T-cells, Jurkat/CCR5 cells, myeloid dendritic cells, macrophages, and HeLa TZM-bl cells, although this was most dramatic on macrophages. Infection of primary T-cells mediated by all Envs was low. However, infection of T-cells was greatly enhanced by increasing virus attachment with DEAE dextran and spinoculation, which enhanced the three Env+ virus groups to similar extents. Dendritic cell capture of viruses and trans-infection also greatly enhanced infection of primary T-cells. In trans-infection assays, non-mac-tropic R5 Envs were preferentially enhanced and those from late disease mediated levels of T-cell infection that were equivalent to those mediated by mac-tropic Envs.

CONCLUSIONS: Our results demonstrate that T/F, early or late disease non-mac-tropic R5 Envs do not preferentially mediate infection of primary CD4+ T-cells compared to highly mac-tropic Envs from brain tissue. We conclude that non-macrophage-tropism of HIV-1 R5 Envs in vitro is determined predominantly by a reduced capacity to target myeloid cells via low CD4 rather than a specific adaptation for T-cells entry that precludes macrophage infection.

Understanding multifactorial influences on the continuum of maternal weight trajectories in pregnancy and early postpartum: study protocol, and participant baseline characteristics

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Maternal and offspring immediate and long-term health are affected by pregnancy weight gain and maternal weight. This study was designed to determine feasibility of: 1) recruiting a socio-economically and racially/ethnically diverse sample of pregnant women into a longitudinal observational study, including consenting the women for serial biologic specimen evaluations; 2) implementing comprehensive assessments (including biologic, anthropometric, behavioral, cognitive/psychosocial and socio-demographic, and cultural measures) at multiple time points over the study period, including collecting biologic specimens at planned and unplanned pregnancy delivery times; and 3) retaining the sample for one year into the postpartum period. Additionally, the study will provide preliminary data of associations among hypothesized predictors, mediators and moderators of pregnancy and post-partum maternal and infant weight trajectories. The study was conceptualized under a Biopsychosocial Model using a lifespan approach. Study protocol and baseline characteristics are described.

METHODS/DESIGN: We sought to recruit a sample of 100 healthy women age 18-45 years, between 28-34 weeks gestation, with singleton pregnancies, enrolled in care prior to 17 weeks gestation. Women provide written consent for face-to-face (medical history, anthropometrics, biologic specimens), and paper-and-pencil assessments, at five time points: baseline (third trimester), delivery-associated, and 6-weeks, 3-months and 6-months postpartum. Additional telephone-based assessments (diet, physical activity and breastfeeding) administered baseline and three-months postpartum. Infant weights are collected until 1-year of life. We seek to retain 80% of participants at six-months postpartum and 80% of offspring at 12-months. 110 women were recruited. Sample characteristics include: mean age 28.3 years, BMI 25.7 kg/m(2), and gestational age at baseline visit of 32.5 weeks. One-third of cohort was non-white, over a quarter were Latina, and almost a quarter were non-US born. The cohort majority was multigravida, had graduated high school and/or had higher levels of education, and worked outside the home.

DISCUSSION: Documentation of study feasibility and preliminary data for theory-driven hypothesis of maternal and child factors associated with weight trajectories will support future large scale longitudinal studies of risk and protective factors for maternal and child health. This research will also inform intervention targets facilitating healthy maternal and child weight.

Patterns of initiation of second generation antipsychotics for bipolar disorder: a month-by-month analysis of provider behavior

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Several second generation antipsychotics (SGAs) received FDA approval for bipolar disorder in the 2000s. Although efficacious, they have been costly and may cause significant side effects. Little is known about the factors associated with prescribers' decisions to initiate SGA prescriptions for this condition.

METHODS: We gathered administrative data from the Department of Veterans Affairs on 170,713 patients with bipolar disorder between fiscal years 2003-2010. Patients without a prior history of taking SGAs were considered eligible for SGA initiation during the study (n =126,556). Generalized estimating equations identified demographic, clinical, and comorbidity variables associated with initiation of an SGA prescription on a month-by-month basis.

RESULTS: While the number of patients with bipolar disorder using SGAs nearly doubled between 2003 and 2010, analyses controlling for patient characteristics and the rise in the bipolar population revealed a 1.2% annual decline in SGA initiation during this period. Most medical comorbidities were only modestly associated with overall SGA initiation, although significant differences emerged among individual SGAs. Several markers of patient severity predicted SGA initiation, including previous hospitalizations, psychotic features, and a history of other antimanic prescriptions; these severity markers became less firmly linked to SGA initiation over time. Providers in the South were somewhat more likely to initiate SGA treatment.

CONCLUSIONS: The number of veterans with bipolar disorder prescribed SGAs is rising steadily, but this increase appears primarily driven by a corresponding increase in the bipolar population. Month-by-month analyses revealed that higher illness severity predicted SGA initiation, but that this association may be weakening over time.

Antimicrobial susceptibility of Neisseria gonorrhoeae isolates from symptomatic men attending the Nanjing sexually transmitted diseases clinic (2011-2012): genetic characteristics of isolates with reduced sensitivity to ceftriaxone

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Evolving gonococcal antimicrobial resistance (AMR) poses a serious threat to public health. The aim of this study was to: update antimicrobial susceptibility data of Neisseria gonorrhoeae recently isolated in Nanjing, China and identify specific deteminants of antimicrobial resistance and gentoypes of isolates with decreased sensitivity to ceftriaxone.

METHODS: 334 N. gonorrhoeae isolates were collected consecutively from symptomatic men attending the Nanjing STD Clinic between April 2011 and December 2012. The minimum inhibitory concentrations (MICs) for penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone were determined by agar plate dilution for each isolate. Penicillinase-producing N. gonorrhoeae (PPNG) and tetracycline-resistant N. gonorrhoeae (TRNG) were examined and typed for beta-lactamase and tetM encoding plasmids respectively. Isolates that displayed elevated MICs to ceftriaxone (MIC > /=0.125 mg/L) were also tested for mutations in penA, mtrR, porB1b, ponA and pilQ genes and characterized by Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST).

RESULTS: 98.8% (330/334) of N. gonorrhoeae isolates were resistant to ciprofloxacin; 97.9% (327/334) to tetracycline and 67.7% (226/334) to penicillin. All isolates were susceptible to ceftriaxone (MIC < /=0.25 mg/L) and spectinomycin (MIC < /=32 mg/L). Plasmid mediated resistance was exhibited by 175/334 (52%) of isolates: 120/334 (36%) of isolates were PPNG and 104/334 (31%) were TRNG. 90.0% (108/120) of PPNG isolates carried the Asia type beta-lactamase encoding plasmid and 96% (100/104) of TRNG isolates carried the Dutch type tetM containing plasmid. Elevated MICs for ceftriaxone were present in 15 (4.5%) isolates; multiple mutations were found in penA, mtrR, porB1b and ponA genes. The 15 isolates were distributed into diverse NG-MAST sequence types; four different non-mosaic penA alleles were identified, including one new type.

CONCLUSIONS: N. gonorrhoeae isolates in Nanjing generally retained similar antimicrobial resistance patterns to isolates obtained five years ago. Fluctuations in resistance plasmid profiles imply that genetic exchange among gonococcal strains is ongoing and is frequent. Ceftriaxone and spectinomycin remain treatments of choice of gonorrhea in Nanjing, however, decreased susceptibility to ceftriaxone and rising MICs for spectinomycin of N. gonorrhoeae isolates underscore the importance of maintaining surveillance for AMR (both phenotypic and genotypic).

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease.

RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease.

CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target.

A randomized controlled trial of smartphone-based mindfulness training for smoking cessation: a study protocol

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Tobacco use is responsible for the death of about 1 in 10 individuals worldwide. Mindfulness training has shown preliminary efficacy as a behavioral treatment for smoking cessation. Recent advances in mobile health suggest advantages to smartphone-based smoking cessation treatment including smartphone-based mindfulness training. This study evaluates the efficacy of a smartphone app-based mindfulness training program for improving smoking cessation rates at 6-months follow-up.

METHODS/DESIGN: A two-group parallel-randomized clinical trial with allocation concealment will be conducted. Group assignment will be concealed from study researchers through to follow-up. The study will be conducted by smartphone and online. Daily smokers who are interested in quitting smoking and own a smartphone (n = 140) will be recruited through study advertisements posted online. After completion of a baseline survey, participants will be allocated randomly to the control or intervention group. Participants in both groups will receive a 22-day smartphone-based treatment program for smoking. Participants in the intervention group will receive mobile mindfulness training plus experience sampling. Participants in the control group will receive experience sampling-only. The primary outcome measure will be one-week point prevalence abstinence from smoking (at 6-months follow-up) assessed using carbon monoxide breath monitoring, which will be validated through smartphone-based video chat.

DISCUSSION: This is the first intervention study to evaluate smartphone-based delivery of mindfulness training for smoking cessation. Such an intervention may provide treatment in-hand, in real-world contexts, to help individuals quit smoking.

TRIAL REGISTRATION: Clinicaltrials.gov NCT02134509 . Registered 7 May 2014.

Two ways to fold the genome during the cell cycle: insights obtained with chromosome conformation capture

Wed, 07/22/2015 - 12:27pm

Genetic and epigenetic inheritance through mitosis is critical for dividing cells to maintain their state. This process occurs in the context of large-scale re-organization of chromosome conformation during prophase leading to the formation of mitotic chromosomes, and during the reformation of the interphase nucleus during telophase and early G1. This review highlights how recent studies over the last 5 years employing chromosome conformation capture combined with classical models of chromosome organization based on decades of microscopic observations, are providing new insights into the three-dimensional organization of chromatin inside the interphase nucleus and within mitotic chromosomes. One striking observation is that interphase genome organization displays cell type-specific features that are related to cell type-specific gene expression, whereas mitotic chromosome folding appears universal and tissue invariant. This raises the question of whether or not there is a need for an epigenetic memory for genome folding. Herein, the two different folding states of mammalian genomes are reviewed and then models are discussed wherein instructions for cell type-specific genome folding are locally encoded in the linear genome and transmitted through mitosis, e.g., as open chromatin sites with or without continuous binding of transcription factors. In the next cell cycle these instructions are used to re-assemble protein complexes on regulatory elements which then drive three-dimensional folding of the genome from the bottom up through local action and self-assembly into higher order levels of cell type-specific organization. In this model, no explicit epigenetic memory for cell type-specific chromosome folding is required.

Improving patient notification of solid abdominal viscera incidental findings with a standardized protocol

Wed, 07/22/2015 - 12:27pm

BACKGROUND: The increasing use of computed tomography (CT) scans in the evaluation of trauma patients has led to increased detection of incidental radiologic findings. Incidental findings (IFs) of the abdominal viscera are among the most commonly discovered lesions and can carry a risk of malignancy. Despite this, patient notification regarding these findings is often inadequate.

METHODS: We identified patients who underwent abdominopelvic CTs as part of their trauma evaluation during a recent 1-year period (9/2011-8/2012). Patients with IFs of the kidneys, liver, adrenal glands, pancreas and/or ovaries had their charts reviewed for documentation of the lesion in their discharge paperwork or follow-up. A quality improvement project was initiated where patients with abdominal IFs were verbally informed of the finding, it was noted on their discharge summary and/or were referred to specialists for evaluation. Nine months after the implementation of the IF protocol, a second chart review was performed to determine if the rate of patient notification improved.

RESULTS: Of 1,117 trauma patients undergoing abdominopelvic CT scans during the 21 month study period, 239 patients (21.4%) had 292 incidental abdominal findings. Renal lesions were the most common (146 patients, 13% of all patients) followed by hepatic (95/8.4%) and adrenal (38/3.4%) lesions. Pancreatic (10/0.9%) and ovarian lesions (3/0.3%) were uncommon. Post-IF protocol implementation patient notification regarding IFs improved by over 80% (32.4% vs. 17.7% pre-protocol, p = 0.02).

CONCLUSION: IFs of the solid abdominal organs are common in trauma patients undergoing abdominopelvic CT scan. Patient notification regarding these lesions is often inadequate. A systematic approach to the documentation and evaluation of incidental radiologic findings can significantly improve the rate of patient notification.

Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.

RESULTS: Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.

CONCLUSIONS: Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.

A randomized controlled trial of mental health interventions for survivors of systematic violence in Kurdistan, Northern Iraq

Wed, 07/22/2015 - 12:27pm

Background: Experiencing systematic violence and trauma increases the risk of poor mental health outcomes; few interventions for these types of exposures have been evaluated in low resource contexts. The objective of this randomized controlled trial was to assess the effectiveness of two psychotherapeutic interventions, Behavioral Activation Treatment for Depression (BATD) and Cognitive Processing Therapy (CPT), in reducing depression symptoms using a locally adapted and validated version of the Hopkins Symptom Checklist and dysfunction measured with a locally developed scale. Secondary outcomes included posttraumatic stress, anxiety, and traumatic grief symptoms.

Methods: Twenty community mental health workers, working in rural health clinics, were randomly assigned to training in one of the two interventions. The community mental health workers conducted baseline assessments, enrolled survivors of systematic violence based on severity of depression symptoms, and randomly assigned them to treatment or waitlist-control. Blinded community mental health workers conducted post-intervention assessments on average five months later.

Results: Adult survivors of systematic violence were screened (N inverted question mark= inverted question mark732) with 281 enrolled in the trial; 215 randomized to an intervention (114 to BATD; 101 to CPT) and 66 to waitlist-control (33 to BATD; 33 to CPT). Nearly 70% (n = 149) of the intervention participants completed treatment and post-intervention assessments; 53 (80%) waitlist-controls completed post-intervention assessments. Estimated effect sizes for depression and dysfunction were 0.60 and 0.55 respectively, comparing BATD participants to all controls and 0.84 and 0.79 respectively, compared to BATD controls only. Estimated effect sizes for depression and dysfunction were 0.70 and 0.90 respectively comparing CPT participants to all controls and 0.44 and 0.63 respectively compared to CPT controls only. Using a permutation-based hypothesis test that is robust to the model assumptions implicit in regression models, BATD had significant effects on depression (p inverted question mark= inverted question mark.003) and dysfunction (p inverted question mark= inverted question mark.007), while CPT had a significant effect on dysfunction only (p inverted question mark= inverted question mark.004).

Conclusions: Both interventions showed moderate to strong effects on most outcomes. This study demonstrates effectiveness of these interventions in low resource environments by mental health workers with limited prior experience.

Trial Registration: ClinicalTrials.Gov NCT00925262. Registered June 3, 2009.

Sequential dengue virus infections detected in active and passive surveillance programs in Thailand, 1994-2010

Wed, 07/22/2015 - 12:27pm

BACKGROUND: The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease.

METHODS: We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher's exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes.

RESULTS: Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 +/- 3.0 and 11.2 +/- 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95% CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection.

CONCLUSIONS: Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease.

Cognitive testing for dementia is adversely affected by administration in a foreign location

Wed, 07/22/2015 - 12:27pm

BACKGROUND: It is colloquially considered that cognitive tests can be adversely affected by administration in a foreign location. However, a definitive demonstration of this is lacking in the literature. To determine whether or not this is the case, we compared the results of cognitive testing in a familiar versus foreign environment by single test administrator of individuals diagnosed with Alzheimer's disease randomized to placebo in a multi-site clinical study.

FINDINGS: Cognitive tests were administered to 6 long-term residents of an assisted living facility at their residence (the "Familiar" cohort). The identical tests were administered to a newly admitted resident and to 2 community-dwelling individuals who drove to the administrator's office for the first time (the "Foreign" cohort). Secondary testing was administered 3 months later at the same respective locations. Caregivers of participants completed reports of mood, behavior and activities of daily living. The Familiar cohort performed equally well at both visits. The Foreign cohort performed significantly worse than the Familiar cohort at baseline. They improved statistically, and matched Familiar cohort performance, by their second visit. Caregiver reports for both cohorts were unchanged between visits.

CONCLUSIONS: These findings support the notion that a foreign location can adversely affect performance on cognitive tests, and therefore support cognitive testing in a familiar location.

Cognition and mood-related behaviors in l3mbtl1 null mutant mice

Wed, 07/22/2015 - 12:27pm

Alterations in histone lysine methylation and epigenetic regulators of gene expression could play a role in the neurobiology and treatment of patients diagnosed with mood spectrum disorder, including depression and anxiety. Mutations and altered expression of various lysine methyltransferases (KMTs) and demethylases (KDMs) have been linked to changes in motivational and emotional behaviors in preclinical model systems. However, it is not known whether regulators operating downstream of histone lysine methylation could affect mood-related behavior. Malignant Brain Tumor (MBT) domain 'chromatin reader' proteins bind to methylated histone lysine residues and associate with chromatin remodeling complexes to facilitate or repress gene expression. MBT proteins, including the founding member, L3mbtl1, maintain high levels of expression in neurons of the mature brain. Here, we exposed L3mbtl1 null mutant mice to a wide range of tests exploring cognition and mood-relevant behaviors at baseline and in the context of social isolation, as a stressor to elicit depression-related behavior in susceptible mice. L3mbtl1 loss-of-function was associated with significant decreases in depression and and anxiety in some of the behavioral paradigms. This was not associated with a more generalized neurological dysfunction because cognition and memory remained unaltered in comparison to controls. These findings warrant further investigations on the role of MBT chromatin reader proteins in the context of emotional and affective behaviors.

Differential Adjuvant Activities of TLR7 and TLR9 Agonists Inversely Correlate with Nitric Oxide and PGE2 Production

Wed, 07/22/2015 - 12:27pm

Activation of different pattern recognition receptors causes distinct profiles of innate immune responses, which in turn dictate the adaptive immune response. We found that mice had higher CD4+ T cell expansion to an immunogen, ovalbumin, when coadministered with CpG than with CL097 in vivo. To account for this differential adjuvanticity, we assessed the activities of CpG and CL097 on antigen-specific CD4+ T cell expansion in vitro using an OT-II CD4+ T cell/bone marrow-derived dendritic cell (DC) co-culture system. Unexpectedly, ovalbumin-stimulated expansion of OT-II CD4+ T cells in vitro was potently suppressed by both TLR agonists, with CL097 being stronger than CpG. The suppression was synergistically reversed by co-inhibition of cyclooxygenases 1 and 2, and inducible nitric oxide (NO) synthase. In addition, stimulation of OT-II CD4+ T cell/DC cultures with CL097 induced higher levels of CD4+ T cell death than stimulation with CpG, and this CD4+ T cell turnover was reversed by NO and PGE2 inhibition. Consistently, the co-cultures stimulated with CL097 produced higher levels of prostaglandin E2 (PGE2) and NO than stimulation with CpG. CL097 induced higher PGE2 production in DC cultures and higher IFN-gamma in the OT-II CD4+ T cell/DC cultures, accounting for the high levels of PGE2 and NO. This study demonstrates that the adjuvant activities of immunostimulatory molecules may be determined by differential induction of negative regulators, including NO and PGE2 suppressing clonal expansion and promoting cell death of CD4+ T cells.

Prevalence of Human Papillomavirus Genotypes among African Women with Normal Cervical Cytology and Neoplasia: A Systematic Review and Meta-Analysis

Wed, 07/22/2015 - 12:27pm

BACKGROUND: Several meta-analyses confirmed the five most prevalent human papillomavirus (HPV) strains in women with and without cervical neoplastic diseases are HPV16, 18, 31, 52, and 58. HPV16/18 are the predominant oncogenic genotypes, causing approximately 70% of global cervical cancer cases. The vast majority of the women studied in previous analyses were from Europe, North America, Asia, and most recently Latin America and the Caribbean. Despite the high burden of cervical cancer morbidity and mortality in Africa, a robust meta-analysis of HPV genotype prevalence and distribution in African women is lacking.

METHODS AND FINDINGS: We systematically searched 14 major databases from inception to August 2013 without language restriction, following the Meta-Analysis of Observational Studies in Epidemiology and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Seventy-one studies from 23 African countries were identified after screening 1162 citations and data abstracted and study quality appraised from 195 articles. HPV type-specific prevalence and distribution was estimated from 17,273 cases of women with normal cervical cytology; 1019 women with atypical squamous cells of undetermined significance (ASCUS); 1444 women with low-grade squamous intraepithelial lesion (LSIL); 1571 women with high-grade squamous intraepithelial lesion (HSIL); and 4,067 cases of invasive cervical carcinoma (ICC). Overall prevalence of HPV16/18 were 4.4% and 2.8% of women with normal cytology, 12.0% and 4.4% with ASCUS, 14.5% and 10.0% with LSIL, 31.2% and 13.9% with HSIL, and 49.7% and 18.0% with ICC, respectively. Study limitations include the lack of adequate data from Middle and Northern African regions, and variations in the HPV type-specific sensitivity of different genotyping protocols.

CONCLUSIONS: To our knowledge, this study is the most comprehensive assessment of the overall prevalence and distribution of HPV genotypes in African women with and without different cervical neoplasias. We have established that HPV16/18 account for 67.7% of ICC cases among African women. Based on our findings, we highly recommend the administration of existing prophylactic vaccines to younger women not infected with HPV16/18 and an increase in HPV screening efforts for high-risk genotypes to prevent cervical cancer.

REVIEW REGISTRATION: International Prospective Register of Systematic Reviews CRD42013006558.

The p53R172H Mutant Does Not Enhance Hepatocellular Carcinoma Development and Progression

Wed, 07/22/2015 - 12:27pm

Hepatocellular carcinoma is a highly deadly malignancy, accounting for approximately 800,000 deaths worldwide every year. Mutation of the p53 tumor suppressor gene is a common genetic change in HCC, present in 30% of cases. p53R175H (corresponding to p53R172H in mice) is a hotspot for mutation that demonstrates "prometastatic" gain-of-function in other cancer models. Since the frequency of p53 mutation increases with tumor grade in HCC, we hypothesized that p53R172H is a gain-of-function mutation in HCC that contributes to a decrease in tumor-free survival and an increase in metastasis. In an HCC mouse model, we found that p53R172H/flox mice do not have decreased survival, increased tumor incidence, or increased metastasis, relative to p53flox/flox littermates. Analysis of cell lines derived from both genotypes indicated that there are no differences in anchorage-independent growth and cell migration. However, shRNA-mediated knockdown of mutant p53 in p53R172H-expressing HCC cell lines resulted in decreased cell migration and anchorage-independent growth. Thus, although p53 mutant-expressing cells and tumors do not have enhanced properties relative to their p53 null counterparts, p53R172H-expressing HCC cells depend on this mutant for their transformation. p53 mutants have been previously shown to bind and inhibit the p53 family proteins p63 and p73. Interestingly, we find that the levels of p63 and p73 target genes are similar in p53 mutant and p53 null HCC cells. These data suggest that pathways regulated by these p53 family members are similarly impacted by p53R172H in mutant expressing cells, and by alternate mechanisms in p53 null cells, resulting in equivalent phenotypes. Consistent with this, we find that p53 null HCC cell lines display lower levels of the TA isoforms of p63 and p73 and higher levels of DeltaNp63. Taken together these data point to the importance of p63 and p73 in constraining HCC progression.

Genomic and clinical effects associated with a relaxation response mind-body intervention in patients with irritable bowel syndrome and inflammatory bowel disease

Wed, 07/22/2015 - 12:27pm

INTRODUCTION: Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) can profoundly affect quality of life and are influenced by stress and resiliency. The impact of mind-body interventions (MBIs) on IBS and IBD patients has not previously been examined.

METHODS: Nineteen IBS and 29 IBD patients were enrolled in a 9-week relaxation response based mind-body group intervention (RR-MBI), focusing on elicitation of the RR and cognitive skill building. Symptom questionnaires and inflammatory markers were assessed pre- and post-intervention, and at short-term follow-up. Peripheral blood transcriptome analysis was performed to identify genomic correlates of the RR-MBI.

RESULTS: Pain Catastrophizing Scale scores improved significantly post-intervention for IBD and at short-term follow-up for IBS and IBD. Trait Anxiety scores, IBS Quality of Life, IBS Symptom Severity Index, and IBD Questionnaire scores improved significantly post-intervention and at short-term follow-up for IBS and IBD, respectively. RR-MBI altered expression of more genes in IBD (1059 genes) than in IBS (119 genes). In IBD, reduced expression of RR-MBI response genes was most significantly linked to inflammatory response, cell growth, proliferation, and oxidative stress-related pathways. In IBS, cell cycle regulation and DNA damage related gene sets were significantly upregulated after RR-MBI. Interactive network analysis of RR-affected pathways identified TNF, AKT and NF-kappaB as top focus molecules in IBS, while in IBD kinases (e.g. MAPK, P38 MAPK), inflammation (e.g. VEGF-C, NF-kappaB) and cell cycle and proliferation (e.g. UBC, APP) related genes emerged as top focus molecules.

CONCLUSIONS: In this uncontrolled pilot study, participation in an RR-MBI was associated with improvements in disease-specific measures, trait anxiety, and pain catastrophizing in IBS and IBD patients. Moreover, observed gene expression changes suggest that NF-kappaB is a target focus molecule in both IBS and IBD-and that its regulation may contribute to counteracting the harmful effects of stress in both diseases. Larger, controlled studies are needed to confirm this preliminary finding.

TRIAL REGISTRATION: ClinicalTrials.Gov NCT02136745.

OXPHOS-Mediated Induction of NAD+ Promotes Complete Oxidation of Fatty Acids and Interdicts Non-Alcoholic Fatty Liver Disease

Wed, 07/22/2015 - 12:27pm

OXPHOS is believed to play an important role in non-alcoholic fatty liver disease (NAFLD), however, precise mechanisms whereby OXPHOS influences lipid homeostasis are incompletely understood. We previously reported that ectopic expression of LRPPRC, a protein that increases cristae density and OXPHOS, promoted fatty acid oxidation in cultured primary hepatocytes. To determine the biological significance of that observation and define underlying mechanisms, we have ectopically expressed LRPPRC in mouse liver in the setting of NAFLD. Interestingly, ectopic expression of LRPPRC in mouse liver completely interdicted NAFLD, including inflammation. Consistent with mitigation of NAFLD, two markers of hepatic insulin resistance-ROS and PKCepsilon activity-were both modestly reduced. As reported by others, improvement of NAFLD was associated with improved whole-body insulin sensitivity. Regarding hepatic lipid homeostasis, the ratio of NAD+ to NADH was dramatically increased in mouse liver replete with LRPPRC. Pharmacological activators and inhibitors of the cellular respiration respectively increased and decreased the [NAD+]/[NADH] ratio, indicating respiration-mediated control of the [NAD+]/[NADH] ratio. Supporting a prominent role for NAD+, increasing the concentration of NAD+ stimulated complete oxidation of fatty acids. Importantly, NAD+ rescued impaired fatty acid oxidation in hepatocytes deficient for either OXPHOS or SIRT3. These data are consistent with a model whereby augmented hepatic OXPHOS increases NAD+, which in turn promotes complete oxidation of fatty acids and protects against NAFLD.