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1.  Admixture Analysis of Spontaneous Hepatitis C Virus Clearance in Individuals of African-Descent 
Genes and immunity  2014;15(4):241-246.
Hepatitis C virus (HCV) infects an estimated 3% of the global population with the majority of individuals (75–85%) failing to clear the virus without treatment, leading to chronic liver disease. Individuals of African-descent have lower rates of clearance compared to individuals of European-descent and this is not fully explained by social and environmental factors. This suggests that differences in genetic background may contribute to this difference in clinical outcome following HCV infection. Using 473 individuals and 792,721 SNPs from a genome-wide association study (GWAS), we estimated local African ancestry across the genome. Using admixture mapping and logistic regression we identified two regions of interest associated with spontaneous clearance of HCV (15q24, 20p12). A genome-wide significant variant was identified on chromosome 15 at the imputed SNP, rs55817928 (P=6.18×10−8) between the genes SCAPER and RCN. Each additional copy of the African ancestral C allele is associated with 2.4 times the odds of spontaneous clearance. Conditional analysis using this SNP in the logistic regression model explained one-third of the local ancestry association. Additionally, signals of selection in this area suggest positive selection due to some ancestral pathogen or environmental pressure in African, but not in European populations.
PMCID: PMC4308959  PMID: 24622687
Hepatitis C; Chronic Infection; Admixture; African Ancestry
2.  Hepatitis C Virus Infection Is Not An Independent Risk Factor For Obstructive Lung Disease 
COPD  2013;11(1):10-16.
Several epidemiological studies have suggested that hepatitis C virus (HCV) infection is associated with the presence of obstructive lung disease (OLD). However, there is a strong link between HCV infection and tobacco abuse, a major risk factor for the development of OLD. In this study we analyzed clinical, laboratory and spirometric data from 1068 study participants to assess whether HCV infection, viremia, or HCV-associated end organ damage were associated with OLD. Demographics, risk behavior, serologic status for HCV and HIV, and spirometric measurements were collected from a cross-sectional analysis of the Acquired Immunodeficiency Syndrome (AIDS) Linked to the IntraVenous Experience (ALIVE) study, an observational cohort of IDUs followed in Baltimore, MD since 1988. Of 1,068 participants, 890 (83%) were HCV positive and 174 (16%) met spirometric criteria for OLD. Factors independently associated with OLD were age and BMI. HCV infection, viral load and HCV-associated end organ damage were similar in participants with and without OLD. In summary, there was no independent association between markers of HCV exposure, chronicity, viremia, or HCV-associated end-organ damage with OLD. Our findings support the strong correlation between HCV status, injection drug use, and smoking. These data suggest that HCV may not be a sole contributor to the increased prevalence of OLD described in previous studies of HCV-infected individuals.
PMCID: PMC4302731  PMID: 23862666
chronic viral infections; injection drug users; HIV; Obstructive Lung Disease
3.  Human Immunodeficiency Virus and Liver Disease Forum 2012 
Hepatology (Baltimore, Md.)  2013;59(1):10.1002/hep.26638.
In the U.S. more than 1.1 million individuals are infected with the human immunodeficiency virus (HIV). These patients exhibit a high frequency of coinfections with other hepatotropic viruses and ongoing fibrosis leading to cirrhosis and liver-related mortality. The etiologies of liver disease include viral hepatitis coinfections, drug-related hepatotoxicity, fatty liver disease, and direct and indirect effects from HIV infection including increased bacterial translocation, immune activation, and presence of soluble proteins that modulate the hepatic cytokine environment. New treatments for HCV using direct acting agents appear viable, though issues related to intrinsic toxicities and drug:drug interactions remain. Recent research suggests that acute HCV infection, unrecognized hepatitis D infection, and hepatitis E may all represent emergent areas of concern. Antiretroviral agents, including those used in past years may represent risk factors for hepatic injury and portal hypertension. Key issues in the future include systematic implementation of liver disease management and new treatment in HIV-infected populations with concomitant injection drug use, alcohol use, and low socioeconomic status.
PMCID: PMC3849330  PMID: 23904401
HIV; HCV; HBV; HCC; Injury; Pathogenesis; Treatment; Epidemiology
4.  Electron Paramagnetic Resonance Resolves Effects of Oxidative Stress on Muscle Proteins 
We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to explore the effects of oxidation on muscle function, with particular focus on the actin-myosin interaction. EPR measurements show that aging or oxidative modification cause a decrease in the fraction of myosins in the strong-binding state, which can be traced to the actin-binding cleft of the myosin catalytic domain.
PMCID: PMC3934098  PMID: 24188980
myosin; actin; muscle; Reactive Oxygen Species (ROS); methionine; aging
5.  High Frequency of False-Positive Hepatitis C Virus Enzyme-Linked Immunosorbent Assay in Rakai, Uganda 
The prevalence of hepatitis C virus (HCV) infection in sub-Saharan Africa remains unclear. We tested 1000 individuals from Rakai, Uganda, with the Ortho version 3.0 HCV enzyme-linked immunosorbent assay. All serologically positive samples were tested for HCV RNA. Seventy-six of the 1000 (7.6%) participants were HCV antibody positive; none were confirmed by detection of HCV RNA.
PMCID: PMC3840403  PMID: 24051866
hepatitis C virus; ELISA; Africa
6.  Mycobacterium avium Complex Infection in a Patient with Sickle Cell Disease and Severe Iron Overload 
A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolated Mycobacterium avium complex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die from Mycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.
PMCID: PMC4269307  PMID: 25544913
7.  An Overview of Arsenic Metabolism and Toxicity 
It is likely that at least some of the toxic and carcinogenic effects associated with exposure to inorganic arsenic are, in fact, due to actions of its methylated metabolites. Here, we provide an overview of current models for the biological methylation of arsenicals. This information provides a context for understanding the chemical, biochemical, and genetic approaches to elucidation of the formation and function of metahylated arsenicals which are presented in the following manuscripts.
PMCID: PMC4240622  PMID: 25419261
8.  Alginate Oligosaccharides Inhibit Fungal Cell Growth and Potentiate the Activity of Antifungals against Candida and Aspergillus spp 
PLoS ONE  2014;9(11):e112518.
The oligosaccharide OligoG, an alginate derived from seaweed, has been shown to have anti-bacterial and anti-biofilm properties and potentiates the activity of selected antibiotics against multi-drug resistant bacteria. The ability of OligoG to perturb fungal growth and potentiate conventional antifungal agents was evaluated using a range of pathogenic fungal strains. Candida (n = 11) and Aspergillus (n = 3) spp. were tested using germ tube assays, LIVE/DEAD staining, scanning electron microscopy (SEM), atomic force microscopy (AFM) and high-throughput minimum inhibition concentration assays (MICs). In general, the strains tested showed a significant dose-dependent reduction in cell growth at ≥6% OligoG as measured by optical density (OD600; P<0.05). OligoG (>0.5%) also showed a significant inhibitory effect on hyphal growth in germ tube assays, although strain-dependent variations in efficacy were observed (P<0.05). SEM and AFM both showed that OligoG (≥2%) markedly disrupted fungal biofilm formation, both alone, and in combination with fluconazole. Cell surface roughness was also significantly increased by the combination treatment (P<0.001). High-throughput robotic MIC screening demonstrated the potentiating effects of OligoG (2, 6, 10%) with nystatin, amphotericin B, fluconazole, miconazole, voriconazole or terbinafine with the test strains. Potentiating effects were observed for the Aspergillus strains with all six antifungal agents, with an up to 16-fold (nystatin) reduction in MIC. Similarly, all the Candida spp. showed potentiation with nystatin (up to 16-fold) and fluconazole (up to 8-fold). These findings demonstrate the antifungal properties of OligoG and suggest a potential role in the management of fungal infections and possible reduction of antifungal toxicity.
PMCID: PMC4237368  PMID: 25409186
9.  Inferring Viral Dynamics in Chronically HCV Infected Patients from the Spatial Distribution of Infected Hepatocytes 
PLoS Computational Biology  2014;10(11):e1003934.
Chronic liver infection by hepatitis C virus (HCV) is a major public health concern. Despite partly successful treatment options, several aspects of intrahepatic HCV infection dynamics are still poorly understood, including the preferred mode of viral propagation, as well as the proportion of infected hepatocytes. Answers to these questions have important implications for the development of therapeutic interventions. In this study, we present methods to analyze the spatial distribution of infected hepatocytes obtained by single cell laser capture microdissection from liver biopsy samples of patients chronically infected with HCV. By characterizing the internal structure of clusters of infected cells, we are able to evaluate hypotheses about intrahepatic infection dynamics. We found that individual clusters on biopsy samples range in size from infected cells. In addition, the HCV RNA content in a cluster declines from the cell that presumably founded the cluster to cells at the maximal cluster extension. These observations support the idea that HCV infection in the liver is seeded randomly (e.g. from the blood) and then spreads locally. Assuming that the amount of intracellular HCV RNA is a proxy for how long a cell has been infected, we estimate based on models of intracellular HCV RNA replication and accumulation that cells in clusters have been infected on average for less than a week. Further, we do not find a relationship between the cluster size and the estimated cluster expansion time. Our method represents a novel approach to make inferences about infection dynamics in solid tissues from static spatial data.
Author Summary
Around 170 million people worldwide are chronically infected with the hepatitis C virus (HCV). Although partly successful treatment options are available, several aspects of HCV infection dynamics within the liver are still poorly understood. How many hepatocytes are infected during chronic HCV infection? How does the virus propagate, and how do innate immune responses interfere with the spread of the virus? We developed mathematical and computational methods to study liver biopsy samples of patients chronically infected with HCV that were analyzed by single cell laser capture microdissection, to infer the spatial distribution of infected cells. With these methods, we find that infected cells on biopsy sections tend to occur in clusters comprising 4–50 hepatocytes, and, based on their amount of intracellular viral RNA, that these cells have been infected for less than a week. The observed HCV RNA profile within clusters of infected cells suggests that factors such as local immune responses could have shaped cluster expansion and intracellular viral replication. Our methods can be applied to various types of infections in order to infer infection dynamics from spatial data.
PMCID: PMC4230741  PMID: 25393308
10.  Identification of a transcriptional signature for the wound healing continuum 
Wound Repair and Regeneration  2014;22(3):399-405.
There is a spectrum/continuum of adult human wound healing outcomes ranging from the enhanced (nearly scarless) healing observed in oral mucosa to scarring within skin and the nonhealing of chronic skin wounds. Central to these outcomes is the role of the fibroblast. Global gene expression profiling utilizing microarrays is starting to give insight into the role of such cells during the healing process, but no studies to date have produced a gene signature for this wound healing continuum. Microarray analysis of adult oral mucosal fibroblast (OMF), normal skin fibroblast (NF), and chronic wound fibroblast (CWF) at 0 and 6 hours post-serum stimulation was performed. Genes whose expression increases following serum exposure in the order OMF < NF < CWF are candidates for a negative/impaired healing phenotype (the dysfunctional healing group), whereas genes with the converse pattern are potentially associated with a positive/preferential healing phenotype (the enhanced healing group). Sixty-six genes in the enhanced healing group and 38 genes in the dysfunctional healing group were identified. Overrepresentation analysis revealed pathways directly and indirectly associated with wound healing and aging and additional categories associated with differentiation, development, and morphogenesis. Knowledge of this wound healing continuum gene signature may in turn assist in the therapeutic assessment/treatment of a patient's wounds.
PMCID: PMC4230470  PMID: 24844339
11.  Faecal Microbiota of Forage-Fed Horses in New Zealand and the Population Dynamics of Microbial Communities following Dietary Change 
PLoS ONE  2014;9(11):e112846.
The effects of abrupt dietary transition on the faecal microbiota of forage-fed horses over a 3-week period were investigated. Yearling Thoroughbred fillies reared as a cohort were exclusively fed on either an ensiled conserved forage-grain diet (“Group A”; n = 6) or pasture (“Group B”; n = 6) for three weeks prior to the study. After the Day 0 faecal samples were collected, horses of Group A were abruptly transitioned to pasture. Both groups continued to graze similar pasture for three weeks, with faecal samples collected at 4-day intervals. DNA was isolated from the faeces and microbial 16S and 18S rRNA gene amplicons were generated and analysed by pyrosequencing. The faecal bacterial communities of both groups of horses were highly diverse (Simpson’s index of diversity >0.8), with differences between the two groups on Day 0 (P<0.017 adjusted for multiple comparisons). There were differences between Groups A and B in the relative abundances of four genera, BF311 (family Bacteroidaceae; P = 0.003), CF231 (family Paraprevotellaceae; P = 0.004), and currently unclassified members within the order Clostridiales (P = 0.003) and within the family Lachnospiraceae (P = 0.006). The bacterial community of Group A horses became similar to Group B within four days of feeding on pasture, whereas the structure of the archaeal community remained constant pre- and post-dietary change. The community structure of the faecal microbiota (bacteria, archaea and ciliate protozoa) of pasture-fed horses was also identified. The initial differences observed appeared to be linked to recent dietary history, with the bacterial community of the forage-fed horses responding rapidly to abrupt dietary change.
PMCID: PMC4226576  PMID: 25383707
12.  A Novel SERCA Inhibitor Demonstrates Synergy with Classic SERCA Inhibitors and Targets Multidrug-Resistant AML 
Molecular pharmaceutics  2013;10(11):4358-4366.
Drug resistance exists as a major obstacle in the treatment of cancer and drug molecules that retain effectiveness against resistant cancers are a high clinical priority. Ethyl 2-amino-6-(3,5-dimethoxyphenyl)-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (CXL017) was recently identified as a promising lead for the treatment of multidrug-resistant leukemia, which elicits its cytotoxic effect, in part, through inhibition of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Herein initial experiments with SERCA1a, CXL017 demonstrated no significant effect on calcium affinity, competed with ATP, and induced a dose-dependent decrease in ATPase activity. Among all CXLs tested, (−)-CXL017 exhibited the greatest SERCA inhibition with an IC50 = 13.5 ± 0.5 μM. Inhibitor combination studies were used to assess potential interactions between (−)-CXL017 and well-known SERCA inhibitors: thapsigargin, cyclopiazonic acid, and 2, 5-di-tert-butylhydroquinone. Surprisingly, (−)-CXL017 exhibited marked synergy with each of the known SERCA inhibitors whereas all combinations of the known inhibitors yielded additive effects, indicating that (−)-CXL017 may bind at a unique allosteric site. Treatment of parental (HL60) and multidrug-resistant (HL60/MX2) acute myeloid leukemia cells with the known SERCA inhibitors revealed that all of these inhibitors demonstrate selective cytotoxicity (7.7 to 400 fold) for the resistant cell line. Within the CXL series, a positive correlation exists between SERCA inhibition and cytotoxicity in HL60/MX2 but not HL60. (−)-CXL017 was also shown to enhance the cytotoxicity of thapsigargin in HL60/MX2 cells. Given the elevated SERCA levels and ER calcium content in HL60/MX2, SERCA likely plays a significant role in the collateral sensitivity of this multidrug-resistance cell line to CXL molecules as well as known SERCA inhibitors.
PMCID: PMC3946399  PMID: 24079514
SERCA; inhibitor; synergy; cancer; drug-resistance
13.  A Comparison of Amplitude-based and Phase-based Positron Emission Tomography Gating Algorithms for Segmentation of Internal Target Volumes of Tumors Subject to Respiratory Motion 
To compare quantitatively the accuracy of tumor volume segmentation in amplitude-based and phase-based respiratory gating algorithms in respiratory-correlated positron emission tomography (PET).
Methods and Materials
List-mode fluorodeoxyglucose-PET data was acquired for 10 patients with a total of 12 fluorodeoxyglucose-avid tumors and 9 lymph nodes. Additionally, a phantom experiment was performed in which 4 acrylic spheres with inner diameters ranging from 1 to 4 cm were imaged as they underwent 1-dimensional motion based on 2 measured patient breathing trajectories. PET list-mode data were gated into 8 bins using 2 amplitude-based (equal amplitude bins [A1] and equal counts per bin [A2]) and 2 temporal phase-based gating algorithms. Gated images were segmented using a commercially available gradient-based technique and a fixed 40% threshold of maximum uptake. Internal target volumes (ITVs) were generated by taking the union of all 8 contours per gated image. Segmented phantom ITVs were compared with their respective ground-truth ITVs, defined as the volume subtended by the tumor model positions covering 99% of breathing amplitude. Superior-inferior distances between sphere centroids in the end-inhale and end-exhale phases were also calculated.
Tumor ITVs from amplitude-based methods were significantly larger than those from temporal-based techniques (P= .002). For lymph nodes, A2 resulted in ITVs that were significantly larger than either of the temporal-based techniques (P<.0323). A1 produced the largest and most accurate ITVs for spheres with diameters of ≥2 cm (P = .002). No significant difference was shown between algorithms in the 1-cm sphere data set. For phantom spheres, amplitude-based methods recovered an average of 9.5% more motion displacement than temporal-based methods under regular breathing conditions and an average of 45.7% more in the presence of baseline drift (P<.001).
Target volumes in images generated from amplitude-based gating are larger and more accurate, at levels that are potentially clinically significant, compared with those from temporal phase-based gating.
PMCID: PMC3795431  PMID: 24074930
14.  Genome-Wide Association Study of Hepatitis C Virus- and Cryoglobulin-Related Vasculitis 
Genes and immunity  2014;15(7):500-505.
The host genetic basis of mixed cryoglobulin vasculitis is not well understood and has not been studied in large cohorts. A genome-wide association study was conducted among 356 HCV RNA positive individuals with cryoglobulin-related vasculitis and 447 ethnically-matched, HCV RNA positive controls.
All cases had both serum cryoglobulins as well as a vasculitis syndrome. A total of 899,641 markers from the Illumina HumanOmni1-Quad chip were analyzed using logistic regression adjusted for sex, as well as genetically-determined ancestry. Replication of select single nucleotide polymorphisms (SNPs) was conducted using 91 cases and 180 controls, adjusting for sex and country of origin.
The most significant associations were identified on chromosome 6 near the NOTCH4 and MHC class II genes. A genome-wide significant association was detected on chromosome 6 at SNP rs9461776 (OR= 2.16, p=1.16E-07) between HLA-DRB1 and DQA1: this association was further replicated in additional independent samples (meta-analysis p=7.1×10−9).
A genome-wide significant association with cryoglobulin related vasculitis was identified with SNPs near NOTCH4 and MHC Class II genes. The two regions are correlated and it is difficult to disentangle which gene is responsible for the association with MC vasculitis in this extended MHC region.
PMCID: PMC4208981  PMID: 25030430
Genome-Wide Association Study; Cryoglobulinemia; Human Leukocyte Antigen; SNP
15.  Energy Requirements of Adult Dogs: A Meta-Analysis 
PLoS ONE  2014;9(10):e109681.
A meta-analysis was conducted to determine the maintenance energy requirements of adult dogs. Suitable publications were first identified, and then used to generate relationships amongst energy requirements, husbandry, activity level, methodology, sex, neuter status, dog size, and age in healthy adult dogs. Allometric equations for maintenance energy requirements were determined using log-log linear regression. So that the resulting equations could readily be compared with equations reported by the National Research Council, maintenance energy requirements in the current study were determined in kcal/kg0.75 body weight (BW). Ultimately, the data of 70 treatment groups from 29 publications were used, and mean (± standard deviation) maintenance energy requirements were 142.8±55.3 kcal.kgBW−−1. The corresponding allometric equation was 81.5 kcal.kgBW−−1 (adjusted R2 = 0.64; 70 treatment groups). Type of husbandry had a significant effect on maintenance energy requirements (P<0.001): requirements were greatest in racing dogs, followed by working dogs and hunting dogs, whilst the energy requirements of pet dogs and kennel dogs were least. Maintenance energy requirements were less in neutered compared with sexually intact dogs (P<0.001), but there was no effect of sex. Further, reported activity level tended to effect the maintenance energy requirement of the dog (P = 0.09). This review suggests that estimating maintenance energy requirements based on BW alone may not be accurate, but that predictions that factor in husbandry, neuter status and, possibly, activity level might be superior. Additionally, more information on the nutrient requirements of older dogs, and those at the extremes of body size (i.e. giant and toy breeds) is needed.
PMCID: PMC4196927  PMID: 25313818
16.  Occupational Exposure to Magnetic Fields and Breast Cancer Among Women Textile Workers in Shanghai, China 
American Journal of Epidemiology  2013;178(7):1038-1045.
Exposure to magnetic fields (MFs) is hypothesized to increase the risk of breast cancer by reducing production of melatonin by the pineal gland. A nested case-cohort study was conducted to investigate the association between occupational exposure to MFs and the risk of breast cancer within a cohort of 267,400 female textile workers in Shanghai, China. The study included 1,687 incident breast cancer cases diagnosed from 1989 to 2000 and 4,702 noncases selected from the cohort. Subjects’ complete work histories were linked to a job–exposure matrix developed specifically for the present study to estimate cumulative MF exposure. Hazard ratios and 95% confidence intervals were calculated using Cox proportional hazards modeling that was adapted for the case-cohort design. Hazard ratios were estimated in relation to cumulative exposure during a woman's entire working years. No association was observed between cumulative exposure to MFs and overall risk of breast cancer. The hazard ratio for the highest compared with the lowest quartile of cumulative exposure was 1.03 (95% confidence interval: 0.87, 1.21). Similar null findings were observed when exposures were lagged and stratified by age at breast cancer diagnosis. The findings do not support the hypothesis that MF exposure increases the risk of breast cancer.
PMCID: PMC3783098  PMID: 24043439
breast cancer; electric and magnetic fields; occupation; textile industry
17.  Benefits and costs of ecological restoration: Rapid assessment of changing ecosystem service values at a U.K. wetland 
Ecology and Evolution  2014;4(20):3875-3886.
Restoration of degraded land is recognized by the international community as an important way of enhancing both biodiversity and ecosystem services, but more information is needed about its costs and benefits. In Cambridgeshire, U.K., a long-term initiative to convert drained, intensively farmed arable land to a wetland habitat mosaic is driven by a desire both to prevent biodiversity loss from the nationally important Wicken Fen National Nature Reserve (Wicken Fen NNR) and to increase the provision of ecosystem services. We evaluated the changes in ecosystem service delivery resulting from this land conversion, using a new Toolkit for Ecosystem Service Site-based Assessment (TESSA) to estimate biophysical and monetary values of ecosystem services provided by the restored wetland mosaic compared with the former arable land. Overall results suggest that restoration is associated with a net gain to society as a whole of $199 ha−1y−1, for a one-off investment in restoration of $2320 ha−1. Restoration has led to an estimated loss of arable production of $2040 ha−1y−1, but estimated gains of $671 ha−1y−1 in nature-based recreation, $120 ha−1y−1 from grazing, $48 ha−1y−1 from flood protection, and a reduction in greenhouse gas (GHG) emissions worth an estimated $72 ha−1y−1. Management costs have also declined by an estimated $1325 ha−1y−1. Despite uncertainties associated with all measured values and the conservative assumptions used, we conclude that there was a substantial gain to society as a whole from this land-use conversion. The beneficiaries also changed from local arable farmers under arable production to graziers, countryside users from towns and villages, and the global community, under restoration. We emphasize that the values reported here are not necessarily transferable to other sites.
PMCID: PMC4242572  PMID: 25505517
Arable production; biodiversity conservation; ecosystem services; flood protection; global climate change mitigation; nature-based recreation; wetland restoration
18.  MiR-210 is induced by Oct-2, regulates B-cells and inhibits autoantibody production1 
MicroRNAs are small, non-coding RNAs that regulate gene expression post-transcriptionally. Here, we show that miR-210 is induced by Oct-2, a key transcriptional mediator of B-cell activation. Germline deletion of miR-210 results in the development of autoantibodies from 5 months of age. Overexpression of miR-210 in vivo resulted in cell autonomous expansion of the B1 lineage and impaired fitness of B2 cells. Mice over-expressing miR-210 exhibited impaired class-switched antibody responses, a finding confirmed in wild-type B-cells transfected with a miR-210 mimic. In vitro studies demonstrated a defect in cellular proliferation and cell-cycle entry, which was consistent with the transcriptomic analysis demonstrating down-regulation of genes involved in cellular proliferation and B cell activation. These findings indicate that Oct-2 induction of miR-210 provides a novel inhibitory mechanism for the control of B cells and autoantibody production.
PMCID: PMC4162006  PMID: 23960236
19.  Protection of Islet Grafts Through Transforming Growth Factor-β–Induced Tolerogenic Dendritic Cells 
Diabetes  2013;62(9):3132-3142.
In type 1 diabetes, the insulin-producing β-cells are destroyed by the immune system. One way of restoring glucose control is to transplant β-cells from a donor. Although this procedure may restore endogenous insulin production, immunosuppressive treatment is needed to prevent the recipient from rejecting the donor-derived islets. We investigated the possibilities of transient expression of the immunosuppressive cytokine transforming growth factor (TGF)-β within islets to achieve long-term graft tolerance. We found that brief expression of TGF-β prevented rejection of syngeneic islets, that there was reduction of dendritic cell (DC) activation in the graft, and that there was reduced reactivation of T cells in the graft-draining lymph nodes. In vitro exposure of bone marrow–derived DCs to TGF-β reduced expression of costimulatory molecules CD80 and CD86, as well as production of proinflammatory cytokines such as interleukin-12 p70 in DCs, but did not alter levels of major histocompatibility complex classes I and II. Furthermore, the capacity of TGF-β–treated bone marrow–derived DCs to activate both CD4+ and CD8+ T cells was reduced. Adding TGF-β–conditioned tolerogenic DCs to the grafted islets led to long-term survival of the graft, demonstrating that TGF-β–induced tolerogenic DCs can provide an effective means to restore immune tolerance in an already established autoimmune disease.
PMCID: PMC3749342  PMID: 23715623
20.  Effect of a Family-Centered, Secondhand Smoke Intervention to Reduce Respiratory Illness in Indigenous Infants in Australia and New Zealand: A Randomized Controlled Trial 
Nicotine & Tobacco Research  2014;17(1):48-57.
Secondhand smoke (SHS) is a significant cause of acute respiratory illness (ARI) and 5 times more common in indigenous children. A single-blind randomized trial was undertaken to determine the efficacy of a family centered SHS intervention to reduce ARI in indigenous infants in Australia and New Zealand.
Indigenous mothers/infants from homes with ≥1 smoker were randomized to a SHS intervention involving 3 home visits in the first 3 months of the infants’ lives (plus usual care) or usual care. The primary outcome was number of ARI-related visits to a health provider in the first year of life. Secondary outcomes, assessed at 4 and 12 months of age, included ARI hospitalization rates and mothers’ report of infants’ SHS exposure (validated by urinary cotinine/creatinine ratios [CCRs]), smoking restrictions, and smoking cessation.
Two hundred and ninety-three mother/infant dyads were randomized and followed up. Three quarters of mothers smoked during pregnancy and two thirds were smoking at baseline (as were their partners), with no change for more than 12 months. Reported infant exposure to SHS was low (≥95% had smoke-free homes/cars). Infant CCRs were higher if one or both parents were smokers and if mothers breast fed their infants. There was no effect of the intervention on ARI events [471 intervention vs. 438 usual care (reference); incidence rate ratio = 1.10, 95% confidence intervals (CI) = 0.88–1.37, p = .40].
Despite reporting smoke-free homes/cars, mothers and their partners continue to smoke in the first year of infants’ lives, exposing them to SHS. Emphasis needs to be placed on supporting parents to stop smoking preconception, during pregnancy, and postnatal.
PMCID: PMC4282121  PMID: 25156527
21.  Hepatitis C Virus Vaccines Among People Who Inject Drugs 
Most people who inject drugs (PWID) are infected with hepatitis C virus (HCV), and PWID have the highest risk of HCV infection of any risk group. The incidence of HCV infection is 5%–25% per year, demonstrating continued need for HCV infection prevention in PWID. Existing data in chimpanzees and PWID suggest that protective immunity against persistent HCV infection is achievable. Due to the high incidence of infection, PWID are both the most likely to benefit from a vaccine and a population in which vaccine efficacy could be tested. Challenges to testing a vaccine in PWID are significant. However, the first HCV vaccine trial in at-risk HCV-uninfected PWID was initiated in 2012. The results will likely guide future vaccine development and strategies for vaccination of this and other high-risk populations.
PMCID: PMC3722079  PMID: 23884065
hepatitis C virus; viral hepatitis; injection drug users; vaccine; hepatitis
22.  Resident Satisfaction with Continuity Clinic and Career Choice in General Internal Medicine 
Journal of General Internal Medicine  2013;28(8):1020-1027.
The quality of the continuity clinic experience for internal medicine (IM) residents may influence their choice to enter general internal medicine (GIM), yet few data exist to support this hypothesis.
To assess the relationship between IM residents’ satisfaction with continuity clinic and interest in GIM careers.
Cross-sectional survey assessing satisfaction with elements of continuity clinic and residents' likelihood of career choice in GIM.
IM residents at three urban medical centers.
Main Measures
Bivariate and multivariate associations between satisfaction with 32 elements of outpatient clinic in 6 domains (clinical preceptors, educational environment, ancillary staff, time management, administrative, personal experience) and likelihood of considering a GIM career.
Key Results
Of the 225 (90 %) residents who completed surveys, 48 % planned to enter GIM before beginning their continuity clinic, whereas only 38 % did as a result of continuity clinic. Comparing residents’ likelihood to enter GIM as a result of clinic to likelihood to enter a career in GIM before clinic showed that 59 % of residents had no difference in likelihood, 28 % reported a lower likelihood as a result of clinic, and 11 % reported higher likelihood as a result of clinic. Most residents were very satisfied or satisfied with all clinic elements. Significantly more residents (p ≤ 0.002) were likely vs. unlikely to enter GIM if they were very satisfied with faculty mentorship (76 % vs. 53 %), time for appointments (28 % vs. 11 %), number of patients seen (33 % vs. 15 %), personal reward from work (51 % vs. 23 %), relationship with patients (64 % vs. 42 %), and continuity with patients (57 % vs. 33 %). In the multivariate analysis, being likely to enter GIM before clinic (OR 29.0, 95 % CI 24.0–34.8) and being very satisfied with the continuity of relationships with patients (OR 4.08, 95 % CI 2.50–6.64) were the strongest independent predictors of likelihood to enter GIM as a result of clinic.
Resident satisfaction with most aspects of continuity clinic was high; yet, continuity clinic had an overall negative influence on residents’ attitudes toward GIM careers. Targeting resources toward improving ambulatory patient continuity, workflow efficiency and increasing pre-residency interest in primary care may help build the primary care workforce.
PMCID: PMC3710375  PMID: 23595920
medical education—career choice; medical education—graduate; primary care; ambulatory medicine
23.  Metabolic Changes in the Spinal Cord After Brachial Plexus Root Re-implantation 
Objective. To investigate metabolic changes within the spinal cord using proton magnetic resonance spectroscopy (1H-MRS) and determine their relationship with clinical function in patients with complete brachial plexus avulsion who underwent reimplantation of the ventral roots. Methods. Single-voxel 1H-MRS of the cord between C1 and C3 was performed in 10 patients with normal spinal cord on MRI, who underwent reimplantation of C5 to T1 ventral roots on average 5.5 years earlier, and 19 healthy controls. The ratios of the concentrations of the following main metabolites, with respect to total creatine levels, were obtained: total N-acetyl-aspartate, choline-containing compounds, creatine and phosphocreatine (Cr), and myo-inositol (m-Ins). Patient disability was assessed using upper limb scales. Differences in metabolite concentration ratios and their correlations with disability were investigated. Results. Patients showed increased m-Ins/Cr ratio compared with controls, which was associated with the level of function of the affected arm and time from injury. Conclusions. The finding of increased m-Ins/Cr in patients suggests that reactive gliosis, perhaps in response to the degeneration of avulsed fibers, may occur in the spinal cord above the site of injury and be relevant to motor dysfunction. However, this pathological process appears to diminish with time. These insights underline the need to integrate metabolic imaging with structural and functional magnetic resonance imaging to obtain a complete view of spinal cord plasticity. Last, this study provides the first steps toward identifying markers to serve as outcome measures for trials comparing strategies of plexus repair following avulsion injury.
PMCID: PMC4107801  PMID: 22961264
spinal cord trauma; brachial plexus avulsion; magnetic resonance spectroscopy; brachial plexus reimplantation
24.  Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle 
Biochemistry  2013;52(27):10.1021/bi4004364.
We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1–9 mM) and find that the highest activity occurs at low Mg (0.1–0.3 mM), while there is a 50–60% reduction in activity at high Mg (3–9 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin. ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin. ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.
PMCID: PMC3855573  PMID: 23725637
Myosin; actin; FRET
25.  Curing hepatitis C with pills: a step toward global control 
Lancet  2010;376(9751):1441-1442.
PMCID: PMC4083467  PMID: 20951421

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