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Recent documents in eScholarship@UMMS
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The PsychENCODE project

Thu, 01/14/2016 - 5:31pm

Recent research on disparate psychiatric disorders has implicated rare variants in genes involved in global gene regulation and chromatin modification, as well as many common variants located primarily in regulatory regions of the genome. Understanding precisely how these variants contribute to disease will require a deeper appreciation for the mechanisms of gene regulation in the developing and adult human brain. The PsychENCODE project aims to produce a public resource of multidimensional genomic data using tissue- and cell type–specific samples from approximately 1,000 phenotypically well-characterized, high-quality healthy and disease-affected human post-mortem brains, as well as functionally characterize disease-associated regulatory elements and variants in model systems. We are beginning with a focus on autism spectrum disorder, bipolar disorder and schizophrenia, and expect that this knowledge will apply to a wide variety of psychiatric disorders. This paper outlines the motivation and design of PsychENCODE.

Bunched and Madm Function Downstream of Tuberous Sclerosis Complex to Regulate the Growth of Intestinal Stem Cells in Drosophila

Thu, 01/14/2016 - 5:31pm

The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. We show here that the leucine zipper protein Bunched and the adaptor protein Madm are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, our data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells.

Utilizing CMP-Sialic Acid Analogs to Unravel Neisseria gonorrhoeae Lipooligosaccharide-Mediated Complement Resistance and Design Novel Therapeutics

Thu, 01/14/2016 - 5:30pm

Neisseria gonorrhoeae deploys a novel immune evasion strategy wherein the lacto-N-neotetraose (LNnT) structure of lipooligosaccharide (LOS) is capped by the bacterial sialyltransferase, using host cytidine-5'-monophosphate (CMP)-activated forms of the nine-carbon nonulosonate (NulO) sugar N-acetyl-neuraminic acid (Neu5Ac), a sialic acid (Sia) abundant in humans. This allows evasion of complement-mediated killing by recruiting factor H (FH), an inhibitor of the alternative complement pathway, and by limiting classical pathway activation ("serum-resistance"). We utilized CMP salts of six additional natural or synthetic NulOs, Neu5Gc, Neu5Gc8Me, Neu5Ac9Ac, Neu5Ac9Az, legionaminic acid (Leg5Ac7Ac) and pseudaminic acid (Pse5Ac7Ac), to define structural requirements of Sia-mediated serum-resistance. While all NulOs except Pse5Ac7Ac were incorporated into the LNnT-LOS, only Neu5Gc incorporation yielded high-level serum-resistance and FH binding that was comparable to Neu5Ac, whereas Neu5Ac9Az and Leg5Ac7Ac incorporation left bacteria fully serum-sensitive and did not enhance FH binding. Neu5Ac9Ac and Neu5Gc8Me rendered bacteria resistant only to low serum concentrations. While serum-resistance mediated by Neu5Ac was associated with classical pathway inhibition (decreased IgG binding and C4 deposition), Leg5Ac7Ac and Neu5Ac9Az incorporation did not inhibit the classical pathway. Remarkably, CMP-Neu5Ac9Az and CMP-Leg5Ac7Ac each prevented serum-resistance despite a 100-fold molar excess of CMP-Neu5Ac in growth media. The concomitant presence of Leg5Ac7Ac and Neu5Ac on LOS resulted in uninhibited classical pathway activation. Surprisingly, despite near-maximal FH binding in this instance, the alternative pathway was not regulated and factor Bb remained associated with bacteria. Intravaginal administration of CMP-Leg5Ac7Ac to BALB/c mice infected with gonorrhea (including a multidrug-resistant isolate) reduced clearance times and infection burden. Bacteria recovered from CMP-Leg5Ac7Ac-treated mice were sensitive to human complement ex vivo, simulating in vitro findings. These data reveal critical roles for the Sia exocyclic side-chain in gonococcal serum-resistance. Such CMP-NulO analogs may provide a novel therapeutic strategy against the global threat of multidrug-resistant gonorrhea.

The Pattern of Adipose Tissue Accumulation during Early Infancy Provides an Environment for the Development of Dengue Hemorrhagic Fever

Thu, 01/14/2016 - 5:30pm

BACKGROUND: Dengue is the most prevalent arthropod-borne viral illness in humans with half of the world's population at risk. During early infancy, severe dengue can develop after a primary dengue virus infection. There has been a clinical observation that severe dengue during the first year of life is seen only in chubby infants.

METHODOLOGY/PRINCIPAL FINDINGS: We examined the associations between the development of severe dengue and adipose tissue accumulation patterns during the first year of life in a prospective observational clinical study of infants and dengue virus infections. We found that adipose tissue contains two potential targets for dengue virus infection and production- adipocytes and adipose tissue macrophages. During the first year of life, total body adiposity and visceral adipose tissue stores were at their highest levels in early infancy. Early infancy was also characterized by a relative decrease in alternatively activated (anti-inflammatory) macrophages, and a relative increase in circulating pro-inflammatory cytokines.

CONCLUSIONS/SIGNIFICANCE: The data has been used to propose a model where the adipose tissue accumulation pattern and pro-inflammatory environment during early infancy provide the conditions for the potential development of severe dengue in immune-susceptible infants.

Epidemiology of Infant Dengue Cases Illuminates Serotype-Specificity in the Interaction between Immunity and Disease, and Changes in Transmission Dynamics

Thu, 01/14/2016 - 5:30pm

BACKGROUND: Infants born to dengue immune mothers acquire maternal antibodies to dengue. These antibodies, though initially protective, decline during the first year of life to levels thought to be disease enhancing, before reaching undetectable levels. Infants have long been studied to understand the interaction between infection and disease on an individual level.
METHODS/FINDINGS: Considering infants (cases < 1 year old) as a unique group, we analyzed serotype specific dengue case data from patients admitted to a pediatric hospital in Bangkok, Thailand. We show differences in the propensity of serotypes to cause disease in individuals with dengue antibodies (infants and post-primary cases) and in individuals without dengue antibodies (primary cases). The mean age of infant cases differed among serotypes, consistent with previously observed differential waning of maternal antibody titers by serotype. We show that trends over time in epidemiology of infant cases are consistent with those observed in the whole population, and therefore with trends in the force of infection.

CONCLUSIONS/SIGNIFICANCE: Infants with dengue are informative about the interaction between antibody and the dengue serotypes, confirming that in this population DENV-2 and DENV-4 almost exclusively cause disease in the presence of dengue antibody despite infections occurring in others. We also observe differences between the serotypes in the mean age in infant cases, informative about the interaction between waning immunity and disease for the different serotypes in infants. In addition, we show that the mean age of infant cases over time is informative about transmission in the whole population. Therefore, ongoing surveillance for dengue in infants could provide useful insights into dengue epidemiology, particularly after the introduction of a dengue vaccine targeting adults and older children.

Preliminary Evaluation of a New German Translated Tobacco Quality of Life Impact Tool to Discriminate Between Healthy Current and Former Smokers and to Explore the Effect of Switching Smokers to a Reduced Toxicant Prototype Cigarette

Thu, 01/14/2016 - 5:30pm

BACKGROUND: Assessment of health-related quality of life (HRQoL) is well established in clinical research, but ceiling effects in validated tools might prevent detection of changes in well respondents. Tobacco Quality of Life Impact Tool (TQOLITv1) uses conceptual and psychometric advances to enhance detection of HRQoL changes.

METHODS: In a 6-month, forced-switch study, the German TQOLITv1 was assessed in healthy adult (age 23-55 years) current and matched former-smokers. At baseline, smokers were switched to reduced toxicant prototype (RTP) or conventional cigarette for 6 months. TQOLITv1 responses were collected at baseline, 3 and 6 months from current smokers whilst former smokers completed it at the latter two time points. TQOLITv1 includes SF-36v2 and new smoking-specific, physical and general-health measures.

RESULTS: Reliability at baseline was good (Cronbach's coefficient alpha > 0.70) for all measures. The baseline percentage with the best possible score (ceiling effect) for former and current smokers was substantially better for the new physical function than SF-36 physical function measure (35% vs. 59% at ceiling, respectively). New smoking-specific measures discriminated current from former smokers better than general health measures. Smoking-specific symptoms (r = 0.73) were more stable from baseline to 6 months than other measures (r = 0.38-0.54) particularly more than the SF-36 mental component score (r = 0.24). Although both product smoking groups worsened in most HRQoL measures, changes in general and smoking-specific HRQoL impact measures favored RTP smokers.

CONCLUSIONS: The German TQOLITv1 is sufficiently reliable and valid to assess HRQoL and may be more useful than SF-36v2 in evaluation of interventions in well smoking populations including those consuming RTPs.

Feasibility of an Assessment Tool for Children's Competence to Consent to Predictive Genetic Testing: a Pilot Study

Thu, 01/14/2016 - 5:30pm

Knowledge on children's capacities to consent to medical treatment is limited. Also, age limits for asking children's consent vary considerably between countries. Decision-making on predictive genetic testing (PGT) is especially complicated, considering the ongoing ethical debate. In order to examine just age limits for alleged competence to consent in children, we evaluated feasibility of a standardized assessment tool, and investigated cutoff ages for children's competence to consent to PGT. We performed a pilot study, including 17 pediatric outpatients between 6 and 18 years at risk for an autosomal dominantly inherited cardiac disease, eligible for predictive genetic testing. The reference standard for competence was established by experts trained in the relevant criteria for competent decision-making. The MacArthur Competence Assessment Tool for Treatment (MacCAT-T) served as index test. Data analysis included raw agreement between competence classifications, difference in mean ages between children judged competent and judged incompetent, and estimation of cutoff ages for judgments of competence. Twelve (71 %) children were considered competent by the reference standard, and 16 (94 %) by the MacCAT-T, with an overall agreement of 76 %. The expert judgments disagreed in most cases, while the MacCAT-T judgments agreed in 65 %. Mean age of children judged incompetent was 9.3 years and of children judged competent 12.1 years (p = .035). With 90 % sensitivity, children younger than 10.0 years were judged incompetent, with 90 % specificity children older than 11.8 years were judged competent. Feasibility of the MacCAT-T in children is confirmed. Initial findings on age cutoffs are indicative for children between the age of 12 and 18 to be judged competent for involvement in the informed consent process. Future research on appropriate age-limits for children's alleged competence to consent is needed.

HiC-Pro: an optimized and flexible pipeline for Hi-C data processing

Thu, 01/14/2016 - 5:30pm

HiC-Pro is an optimized and flexible pipeline for processing Hi-C data from raw reads to normalized contact maps. HiC-Pro maps reads, detects valid ligation products, performs quality controls and generates intra- and inter-chromosomal contact maps. It includes a fast implementation of the iterative correction method and is based on a memory-efficient data format for Hi-C contact maps. In addition, HiC-Pro can use phased genotype data to build allele-specific contact maps. We applied HiC-Pro to different Hi-C datasets, demonstrating its ability to easily process large data in a reasonable time. Source code and documentation are available at .

The Conformation of Yeast Chromosome III Is Mating Type Dependent and Controlled by the Recombination Enhancer

Thu, 01/14/2016 - 5:30pm

Mating-type switching in yeast occurs through gene conversion between the MAT locus and one of two silent loci (HML or HMR) on opposite ends of the chromosome. MATa cells choose HML as template, whereas MATalpha cells use HMR. The recombination enhancer (RE) located on the left arm regulates this process. One long-standing hypothesis is that switching is guided by mating-type-specific and possibly RE-dependent chromosome folding. Here, we use Hi-C, 5C, and live-cell imaging to characterize the conformation of chromosome III in both mating types. We discovered a mating-type-specific conformational difference in the left arm. Deletion of a 1-kb subregion within the RE, which is not necessary during switching, abolished mating-type-dependent chromosome folding. The RE is therefore a composite element with one subregion essential for donor selection during switching and a separate region involved in modulating chromosome conformation.

Discriminating Active Tuberculosis from Latent Tuberculosis Infection by flow cytometric measurement of CD161-expressing T cells

Thu, 01/14/2016 - 5:30pm

Interferon-gamma Release Assays (IGRAs) significantly increases the possibility for early diagnosis of tuberculosis, but IGRAs alone cannot discriminate active TB from LTBI. Therefore, fast and reliable discrimination of active tuberculosis, especially bacteriology negative tuberculosis, from LTBI is a great necessity. Here we established an assay based on flow cytometric multiparameter assay assessing expression of CD161 along with CD3, CD4, and CD8, whereby a set of indices formulated by the percentages of CD3(+)CD161(+), CD3(+)CD4(+)CD161(+) and CD3(+)CD8(+)CD161(+) T cells multiplied with lymphocyte/monocyte ratio were established. Application of the CD3(+)CD8(+)CD161(+) index to compare a cohort of active tuberculosis with a cohort of LTBI or health control yielded 0.7662 (95% confidence interval [CI] 0.6559-0.8552) or 0.7922 (95% CI 0.6846-0.8763) for sensitivity and 0.9048 (95% CI 0.8209-0.9580) or 0.8939 (95% CI 0.8392-0.9349) for specificity when the TB cohort was AFB(+); the corresponding results were 0.7481 (95% CI 0.6648-0.8198) or 0.7557 (95% CI 0.6730-0.8265) for sensitivity and 0.8571 (95% CI 0.7637-0.9239) or 0.8603 (95% CI 0.8008-0.9075) for specificity when the TB cohort was AFB(-). Our results reveal that in combination with IGRAs, CD161-based indices provide a novel, fast diagnostic solution addressing the limitation of current tuberculosis diagnostics.

Near-infrared photoactivatable control of Ca signaling and optogenetic immunomodulation

Thu, 01/14/2016 - 5:30pm

The application of current channelrhodopsin-based optogenetic tools is limited by the lack of strict ion selectivity and the inability to extend the spectra sensitivity into the near-infrared (NIR) tissue transmissible range. Here we present an NIR-stimulable optogenetic platform (termed "Opto-CRAC") that selectively and remotely controls Ca2+ oscillations and Ca2+-responsive gene expression to regulate the function of non-excitable cells, including T lymphocytes, macrophages and dendritic cells. When coupled to upconversion nanoparticles, the optogenetic operation window is shifted from the visible range to NIR wavelengths to enable wireless photoactivation of Ca2+-dependent signaling and optogenetic modulation of immunoinflammatory responses. In a mouse model of melanoma by using ovalbumin as surrogate tumor antigen, Opto-CRAC has been shown to act as a genetically-encoded "photoactivatable adjuvant" to improve antigen-specific immune responses to specifically destruct tumor cells. Our study represents a solid step forward towards the goal of achieving remote control of Ca2+-modulated activities with tailored function.

GGGGCC microsatellite RNA is neuritically localized, induces branching defects, and perturbs transport granule function

Thu, 01/14/2016 - 5:30pm

Microsatellite expansions are the leading cause of numerous neurodegenerative disorders. Here we demonstrate that GGGGCC and CAG microsatellite repeat RNAs associated with C9orf72 in ALS/FTD and with polyglutamine diseases, respectively, localize to neuritic granules that undergo active transport into distal neuritic segments. In cultured mammalian spinal cord neurons, the presence of neuritic GGGGCC repeat RNA correlates with neuronal branching defects and the repeat RNA localizes to granules that label with FMRP, a transport granule component. Using a Drosophila GGGGCC expansion disease model, we characterize dendritic branching defects that are modulated by FMRP and Orb2. The human orthologues of these modifiers are misregulated in induced pluripotent stem cell-differentiated neurons from GGGGCC expansion carriers. These data suggest that expanded repeat RNAs interact with the mRNA transport and translation machinery, causing transport granule dysfunction. This could be a novel mechanism contributing to the neuronal defects associated with C9orf72 and other microsatellite expansion diseases.

MMB-GUI: a fast morphing method demonstrates a possible ribosomal tRNA translocation trajectory

Thu, 01/14/2016 - 5:30pm

Easy-to-use macromolecular viewers, such as UCSF Chimera, are a standard tool in structural biology. They allow rendering and performing geometric operations on large complexes, such as viruses and ribosomes. Dynamical simulation codes enable modeling of conformational changes, but may require considerable time and many CPUs. There is an unmet demand from structural and molecular biologists for software in the middle ground, which would allow visualization combined with quick and interactive modeling of conformational changes, even of large complexes. This motivates MMB-GUI. MMB uses an internal-coordinate, multiscale approach, yielding as much as a 2000-fold speedup over conventional simulation methods. We use Chimera as an interactive graphical interface to control MMB. We show how this can be used for morphing of macromolecules that can be heterogeneous in biopolymer type, sequence, and chain count, accurately recapitulating structural intermediates. We use MMB-GUI to create a possible trajectory of EF-G mediated gate-passing translocation in the ribosome, with all-atom structures. This shows that the GUI makes modeling of large macromolecules accessible to a wide audience. The morph highlights similarities in tRNA conformational changes as tRNA translocates from A to P and from P to E sites and suggests that tRNA flexibility is critical for translocation completion.