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Recent documents in eScholarship@UMMS
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On the prospect of identifying adaptive loci in recently bottlenecked populations

Mon, 02/09/2015 - 9:59pm

Identifying adaptively important loci in recently bottlenecked populations - be it natural selection acting on a population following the colonization of novel habitats in the wild, or artificial selection during the domestication of a breed - remains a major challenge. Here we report the results of a simulation study examining the performance of available population-genetic tools for identifying genomic regions under selection. To illustrate our findings, we examined the interplay between selection and demography in two species of Peromyscus mice, for which we have independent evidence of selection acting on phenotype as well as functional evidence identifying the underlying genotype. With this unusual information, we tested whether population-genetic-based approaches could have been utilized to identify the adaptive locus. Contrary to published claims, we conclude that the use of the background site frequency spectrum as a null model is largely ineffective in bottlenecked populations. Results are quantified both for site frequency spectrum and linkage disequilibrium-based predictions, and are found to hold true across a large parameter space that encompasses many species and populations currently under study. These results suggest that the genomic footprint left by selection on both new and standing variation in strongly bottlenecked populations will be difficult, if not impossible, to find using current approaches.

Coincident pre-diabetes is associated with dysregulated cytokine responses in pulmonary tuberculosis

Mon, 02/09/2015 - 9:59pm

BACKGROUND: Cytokines play an important role in the pathogenesis of pulmonary tuberculosis (PTB)--Type 2 diabetes mellitus co-morbidity. However, the cytokine interactions that characterize PTB coincident with pre-diabetes (PDM) are not known.

METHODS: To identify the influence of coincident PDM on cytokine levels in PTB, we examined circulating levels of a panel of cytokines in the plasma of individuals with TB-PDM and compared them with those without PDM (TB-NDM).

RESULTS: TB-PDM is characterized by elevated circulating levels of Type 1 (IFNgamma, TNFalpha and IL-2), Type 17 (IL-17A and IL-17F) and other pro-inflammatory (IL-1beta, IFNbeta and GM-CSF) cytokines. TB-PDM is also characterized by increased systemic levels of Type 2 (IL-5) and regulatory (IL-10 and TGFbeta) cytokines. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory (IFNgamma, TNFalpha and IL-1beta) cytokines as well. However, the cytokines did not exhibit any significant correlation with HbA1C levels or with bacterial burdens.

CONCLUSION: Our data reveal that pre-diabetes in PTB individuals is characterized by heightened cytokine responsiveness, indicating that a balanced pro and anti - inflammatory cytokine milieu is a feature of pre-diabetes--TB co-morbidity.

The machinery at endoplasmic reticulum-plasma membrane contact sites contributes to spatial regulation of multiple Legionella effector proteins

Mon, 02/09/2015 - 9:59pm

The Dot/Icm system of the intracellular pathogen Legionella pneumophila has the capacity to deliver over 270 effector proteins into host cells during infection. Important questions remain as to spatial and temporal mechanisms used to regulate such a large array of virulence determinants after they have been delivered into host cells. Here we investigated several L. pneumophila effector proteins that contain a conserved phosphatidylinositol-4-phosphate (PI4P)-binding domain first described in the effector DrrA (SidM). This PI4P binding domain was essential for the localization of effectors to the early L. pneumophila-containing vacuole (LCV), and DrrA-mediated recruitment of Rab1 to the LCV required PI4P-binding activity. It was found that the host cell machinery that regulates sites of contact between the plasma membrane (PM) and the endoplasmic reticulum (ER) modulates PI4P dynamics on the LCV to control localization of these effectors. Specifically, phosphatidylinositol-4-kinase IIIalpha (PI4KIIIalpha) was important for generating a PI4P signature that enabled L. pneumophila effectors to localize to the PM-derived vacuole, and the ER-associated phosphatase Sac1 was involved in metabolizing the PI4P on the vacuole to promote the dissociation of effectors. A defect in L. pneumophila replication in macrophages deficient in PI4KIIIalpha was observed, highlighting that a PM-derived PI4P signature is critical for biogenesis of a vacuole that supports intracellular multiplication of L. pneumophila. These data indicate that PI4P metabolism by enzymes controlling PM-ER contact sites regulate the association of L. pneumophila effectors to coordinate early stages of vacuole biogenesis.

A system for genome-wide histone variant dynamics in ES cells reveals dynamic MacroH2A2 replacement at promoters

Mon, 02/09/2015 - 9:59pm

Dynamic exchange of a subset of nucleosomes in vivo plays important roles in epigenetic inheritance of chromatin states, chromatin insulator function, chromosome folding, and the maintenance of the pluripotent state of embryonic stem cells. Here, we extend a pulse-chase strategy for carrying out genome-wide measurements of histone dynamics to several histone variants in murine embryonic stem cells and somatic tissues, recapitulating expected characteristics of the well characterized H3.3 histone variant. We extended this system to the less-studied MacroH2A2 variant, commonly described as a "repressive" histone variant whose accumulation in chromatin is thought to fix the epigenetic state of differentiated cells. Unexpectedly, we found that while large intergenic blocks of MacroH2A2 were stably associated with the genome, promoter-associated peaks of MacroH2A2 exhibited relatively rapid exchange dynamics in ES cells, particularly at highly-transcribed genes. Upon differentiation to embryonic fibroblasts, MacroH2A2 was gained primarily in additional long, stably associated blocks across gene-poor regions, while overall turnover at promoters was greatly dampened. Our results reveal unanticipated dynamic behavior of the MacroH2A2 variant in pluripotent cells, and provide a resource for future studies of tissue-specific histone dynamics in vivo.

A conserved dopamine-cholecystokinin signaling pathway shapes context-dependent Caenorhabditis elegans behavior

Mon, 02/09/2015 - 9:59pm

An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output.

A deterministic model predicts the properties of stochastic calcium oscillations in airway smooth muscle cells

Mon, 02/09/2015 - 9:59pm

The inositol trisphosphate receptor ([Formula: see text]) is one of the most important cellular components responsible for oscillations in the cytoplasmic calcium concentration. Over the past decade, two major questions about the [Formula: see text] have arisen. Firstly, how best should the [Formula: see text] be modeled? In other words, what fundamental properties of the [Formula: see text] allow it to perform its function, and what are their quantitative properties? Secondly, although calcium oscillations are caused by the stochastic opening and closing of small numbers of [Formula: see text], is it possible for a deterministic model to be a reliable predictor of calcium behavior? Here, we answer these two questions, using airway smooth muscle cells (ASMC) as a specific example. Firstly, we show that periodic calcium waves in ASMC, as well as the statistics of calcium puffs in other cell types, can be quantitatively reproduced by a two-state model of the [Formula: see text], and thus the behavior of the [Formula: see text] is essentially determined by its modal structure. The structure within each mode is irrelevant for function. Secondly, we show that, although calcium waves in ASMC are generated by a stochastic mechanism, [Formula: see text] stochasticity is not essential for a qualitative prediction of how oscillation frequency depends on model parameters, and thus deterministic [Formula: see text] models demonstrate the same level of predictive capability as do stochastic models. We conclude that, firstly, calcium dynamics can be accurately modeled using simplified [Formula: see text] models, and, secondly, to obtain qualitative predictions of how oscillation frequency depends on parameters it is sufficient to use a deterministic model.

The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria

Mon, 02/09/2015 - 9:59pm

Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-alpha and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection.

Out-of-sequence signal 3 as a mechanism for virus-induced immune suppression of CD8 T cell responses

Mon, 02/09/2015 - 9:59pm

Virus infections are known to induce a transient state of immune suppression often associated with an inhibition of T cell proliferation in response to mitogen or cognate-antigen stimulation. Recently, virus-induced immune suppression has been linked to responses to type 1 interferon (IFN), a signal 3 cytokine that normally can augment the proliferation and differentiation of T cells exposed to antigen (signal 1) and co-stimulation (signal 2). However, pre-exposure of CD8 T cells to IFN-inducers such as viruses or poly(IratioC) prior to antigen signaling is inhibitory, indicating that the timing of IFN exposure is of essence. We show here that CD8 T cells pretreated with poly(IratioC) down-regulated the IFN receptor, up-regulated suppressor of cytokine signaling 1 (SOCS1), and were refractory to IFNbeta-induced signal transducers and activators of transcription (STAT) phosphorylation. When exposed to a viral infection, these CD8 T cells behaved more like 2-signal than 3-signal T cells, showing defects in short lived effector cell differentiation, reduced effector function, delayed cell division, and reduced levels of survival proteins. This suggests that IFN-pretreated CD8 T cells are unable to receive the positive effects that type 1 IFN provides as a signal 3 cytokine when delivered later in the signaling process. This desensitization mechanism may partially explain why vaccines function poorly in virus-infected individuals.

The spatial dynamics of dengue virus in Kamphaeng Phet, Thailand

Mon, 02/09/2015 - 9:59pm

BACKGROUND: Dengue is endemic to the rural province of Kamphaeng Phet, Northern Thailand. A decade of prospective cohort studies has provided important insights into the dengue viruses and their generated disease. However, as elsewhere, spatial dynamics of the pathogen remain poorly understood. In particular, the spatial scale of transmission and the scale of clustering are poorly characterized. This information is critical for effective deployment of spatially targeted interventions and for understanding the mechanisms that drive the dispersal of the virus.

METHODOLOGY/PRINCIPAL FINDINGS: We geocoded the home locations of 4,768 confirmed dengue cases admitted to the main hospital in Kamphaeng Phet province between 1994 and 2008. We used the phi clustering statistic to characterize short-term spatial dependence between cases. Further, to see if clustering of cases led to similar temporal patterns of disease across villages, we calculated the correlation in the long-term epidemic curves between communities. We found that cases were 2.9 times (95% confidence interval 2.7-3.2) more likely to live in the same village and be infected within the same month than expected given the underlying spatial and temporal distribution of cases. This fell to 1.4 times (1.2-1.7) for individuals living in villages 1 km apart. Significant clustering was observed up to 5 km. We found a steadily decreasing trend in the correlation in epidemics curves by distance: communities separated by up to 5 km had a mean correlation of 0.28 falling to 0.16 for communities separated between 20 km and 25 km. A potential explanation for these patterns is a role for human movement in spreading the pathogen between communities. Gravity style models, which attempt to capture population movement, outperformed competing models in describing the observed correlations.

CONCLUSIONS: There exists significant short-term clustering of cases within individual villages. Effective spatially and temporally targeted interventions deployed within villages may target ongoing transmission and reduce infection risk.

Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90

Mon, 02/09/2015 - 9:59pm

Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naive individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naive cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

Allele-specific induction of IL-1beta expression by C/EBPbeta and PU.1 contributes to increased tuberculosis susceptibility

Mon, 02/09/2015 - 9:59pm

Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1beta, have the contrasting potential both to prevent disease by restricting bacterial replication, and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPbeta and PU.1 transcription factors and controls Mtb-induced IL-1beta production. The high-IL-1beta expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1beta expression did not suppress the activity of IFN-gamma-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1beta and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis.

Dengue virus neutralizing antibody levels associated with protection from infection in Thai cluster studies

Mon, 02/09/2015 - 9:59pm

BACKGROUND: Long-term homologous and temporary heterologous protection from dengue virus (DENV) infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs) have not been clearly associated with protection from infection.

METHODOLOGY/PRINCIPAL FINDINGS: Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004-2007), cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009-2012), clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age 6 months and older were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been "susceptible" on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered "non-susceptible." Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT). Seventeen "susceptible" (six DENV-1, five DENV-2, and six DENV-4), and 32 "non-susceptible" (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4) subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC) curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate "susceptible" from "non-susceptible" subjects.

CONCLUSIONS/SIGNIFICANCE: PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant for both dengue epidemiology studies and vaccine development efforts.

PI3K signaling and Stat92E converge to modulate glial responsiveness to axonal injury

Mon, 02/09/2015 - 9:59pm

Glial cells are exquisitely sensitive to neuronal injury but mechanisms by which glia establish competence to respond to injury, continuously gauge neuronal health, and rapidly activate reactive responses remain poorly defined. Here, we show glial PI3K signaling in the uninjured brain regulates baseline levels of Draper, a receptor essential for Drosophila glia to sense and respond to axonal injury. After injury, Draper levels are up-regulated through a Stat92E-modulated, injury-responsive enhancer element within the draper gene. Surprisingly, canonical JAK/STAT signaling does not regulate draper expression. Rather, we find injury-induced draper activation is downstream of the Draper/Src42a/Shark/Rac1 engulfment signaling pathway. Thus, PI3K signaling and Stat92E are critical in vivo regulators of glial responsiveness to axonal injury. We provide evidence for a positive auto-regulatory mechanism whereby signaling through the injury-responsive Draper receptor leads to Stat92E-dependent, transcriptional activation of the draper gene. We propose that Drosophila glia use this auto-regulatory loop as a mechanism to adjust their reactive state following injury.

Improving Selection Criteria for Early Cystectomy in High-Grade T1 Bladder Cancer: A Meta-Analysis of 15,215 Patients

Mon, 02/09/2015 - 3:52pm

PURPOSE: High-grade T1 (HGT1) bladder cancer is the highest risk subtype of non-muscle-invasive bladder cancer, with highly variable prognosis, poorly understood risk factors, and considerable debate about the role of early cystectomy. We aimed to address these questions through a meta-analysis of outcomes and prognostic factors.

METHODS: PubMed, EMBASE, Cochrane Central Register of Controlled Trials, and American Society of Clinical Oncology abstracts were searched for cohort studies in HGT1. We pooled data on recurrence, progression, and cancer-specific survival from 73 studies.

RESULTS: Five-year rates of recurrence, progression, and cancer-specific survival were 42% (95% CI, 39% to 45%), 21% (95% CI, 18% to 23%), and 87% (95% CI, 85% to 89%), respectively (56 studies, n = 15,215). In the prognostic factor meta-analysis (33 studies, n = 8,880), the highest impact risk factor was depth of invasion (T1b/c) into lamina propria (progression: hazard ratio [HR], 3.34; P < .001; cancer-specific survival: HR, 2.02; P = .001). Several other previously proposed factors also predicted progression and cancer-specific survival (lymphovascular invasion, associated carcinoma in situ, nonuse of bacillus Calmette-Guérin, tumor size > 3 cm, and older age; HRs for progression between 1.32 and 2.88, P ≤ .002; HRs for cancer-specific survival between 1.28 and 2.08, P ≤ .02).

CONCLUSION: In this large analysis of outcomes and prognostic factors in HGT1 bladder cancer, deep lamina propria invasion had the largest negative impact, and other previously proposed prognostic factors were also confirmed. These factors should be used for prognostication and patient stratification in future clinical trials, and depth of invasion should be considered for inclusion in TNM staging criteria. This meta-analysis can also help define selection criteria for early cystectomy in HGT1 bladder cancer, particularly for patients with deep lamina propria invasion combined with other risk factors.

Vector sequences are not detected in tumor tissue from research subjects with ornithine transcarbamylase deficiency who previously received adenovirus gene transfer

Mon, 02/09/2015 - 3:02pm

A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.