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1.  Anti-α4 Antibody Treatment Blocks Virus Traffic to the Brain and Gut Early, and Stabilizes CNS Injury Late in Infection 
PLoS Pathogens  2014;10(12):e1004533.
Four SIV-infected monkeys with high plasma virus and CNS injury were treated with an anti-α4 blocking antibody (natalizumab) once a week for three weeks beginning on 28 days post-infection (late). Infection in the brain and gut were quantified, and neuronal injury in the CNS was assessed by MR spectroscopy, and compared to controls with AIDS and SIV encephalitis. Treatment resulted in stabilization of ongoing neuronal injury (NAA/Cr by 1H MRS), and decreased numbers of monocytes/macrophages and productive infection (SIV p28+, RNA+) in brain and gut. Antibody treatment of six SIV infected monkeys at the time of infection (early) for 3 weeks blocked monocyte/macrophage traffic and infection in the CNS, and significantly decreased leukocyte traffic and infection in the gut. SIV – RNA and p28 was absent in the CNS and the gut. SIV DNA was undetectable in brains of five of six early treated macaques, but proviral DNA in guts of treated and control animals was equivalent. Early treated animals had low-to-no plasma LPS and sCD163. These results support the notion that monocyte/macrophage traffic late in infection drives neuronal injury and maintains CNS viral reservoirs and lesions. Leukocyte traffic early in infection seeds the CNS with virus and contributes to productive infection in the gut. Leukocyte traffic early contributes to gut pathology, bacterial translocation, and activation of innate immunity.
Author Summary
To determine whether ongoing cell traffic is required for SIV-associated tissue damage, we blocked monocyte and T lymphocyte traffic to the brain and gut during a) ongoing infection or, b) at the time of infection. When animals were treated at four weeks post infection (late), once significant neuronal injury and accumulation of infected macrophages had already occurred, neuronal injury was stabilized, and CNS infection and the number of CNS lesions decreased. In the gut, there were significantly fewer productively infected cells and decreased inflammatory macrophages post treatment. Treatment at the time of infection (early) blocked infection of the CNS (SIV –DNA, RNA, or protein) and macrophage accumulation. In the gut, treatment at the time of infection blocked productive infection (SIV –RNA and protein) but not SIV –DNA. Interestingly, with treatment at the time of infection, there was no evidence of microbial translocation or elevated sCD163 in plasma, demonstrating that leukocyte traffic early plays a role in damage to gut tissues. Overall, these data point to the role of monocyte traffic and possibly lymphocytes to the CNS and leukocyte traffic to the gut to establish and maintain viral reservoirs. They underscore the role of monocyte/macrophage traffic and accumulation in the CNS for neuronal injury and maintenance of CNS lesions.
PMCID: PMC4263764  PMID: 25502752
2.  MoeH5: a natural glycorandomizer from the moenomycin biosynthetic pathway 
Molecular microbiology  2013;90(6):1324-1338.
The biosynthesis of the phosphoglycolipid antibiotic moenomycin A attracts the attention of researchers hoping to develop new moenomycin-based antibiotics against multidrug resistant Gram-positive infections. There is detailed understanding of most steps of this biosynthetic pathway in Streptomyces ghanaensis (ATCC14672), except for the ultimate stage, where a single pentasaccharide intermediate is converted into a set of unusually modified final products. Here we report that only one gene, moeH5, encoding a homologue of the glutamine amidotransferase (GAT) enzyme superfamily, is responsible for the observed diversity of terminally decorated moenomycins. Genetic and biochemical evidence support the idea that MoeH5 is a novel member of the GAT superfamily, whose homologues are involved in the synthesis of various secondary metabolites as well as K and O antigens of bacterial lipopolysaccharide. Our results provide insights into the mechanism of MoeH5 and its counterparts, and give us a new tool for the diversification of phosphoglycolipid antibiotics.
PMCID: PMC3961715  PMID: 24164498
3.  Effective interventions to improve medication adherence in Type 2 diabetes: a systematic review 
Medication adherence is associated with improved outcomes in diabetes. Interventions have been established to help improve medication adherence; however, the most effective interventions in patients with Type 2 diabetes remain unclear. The goal of this study was to distinguish whether interventions were effective and identify areas for future research.
Medline was searched for articles published between January 2000 and May 2013, and a reproducible strategy was used. Study eligibility criteria included interventions measuring medication adherence in adults with Type 2 diabetes.
Twenty seven studies met the inclusion criteria and 13 showed a statistically significant change in medication adherence.
Heterogeneity of the study designs and measures of adherence made it difficult to identify effective interventions that improved medication adherence. Additionally, medication adherence may not be solely responsible for achieving glycemic control. Researchers must emphasize tailored interventions that optimize management and improve outcomes, and examine the need for clear indicators of medication adherence.
PMCID: PMC4157681  PMID: 25214893
4.  Beyond Crossing Fibers: Bootstrap Probabilistic Tractography Using Complex Subvoxel Fiber Geometries 
Diffusion magnetic resonance imaging fiber tractography is a powerful tool for investigating human white matter connectivity in vivo. However, it is prone to false positive and false negative results, making interpretation of the tractography result difficult. Optimal tractography must begin with an accurate description of the subvoxel white matter fiber structure, includes quantification of the uncertainty in the fiber directions obtained, and quantifies the confidence in each reconstructed fiber tract. This paper presents a novel and comprehensive pipeline for fiber tractography that meets the above requirements. The subvoxel fiber geometry is described in detail using a technique that allows not only for straight crossing fibers but for fibers that curve and splay. This technique is repeatedly performed within a residual bootstrap statistical process in order to efficiently quantify the uncertainty in the subvoxel geometries obtained. A robust connectivity index is defined to quantify the confidence in the reconstructed connections. The tractography pipeline is demonstrated in the human brain.
PMCID: PMC4211389  PMID: 25389414
diffusion MRI; fiber orientation distribution function; high angular resolution diffusion imaging; fiber dispersion; curve inference
5.  Cost-effectiveness of point-of-care viral load monitoring of ART in resource-limited settings: Mathematical modelling study 
AIDS (London, England)  2013;27(9):10.1097/QAD.0b013e328360a4e5.
Monitoring of HIV viral load in patients on combination antiretroviral therapy (ART) is not generally available in resource-limited settings. We examined the cost-effectiveness of qualitative point-of-care viral load tests (POC-VL) in sub-Saharan Africa.
Mathematical model based on longitudinal data from the Gugulethu and Khayelitsha township ART programmes in Cape Town, South Africa.
Cohorts of patients on ART monitored by POC-VL, CD4 cell count or clinically were simulated. Scenario A considered the more accurate detection of treatment failure with POC-VL only, Scenario B also considered the effect on HIV transmission. Scenario C further assumed that the risk of virologic failure is halved with POC-VL due to improved adherence. We estimated the change in costs per quality-adjusted life-year gained (incremental cost-effectiveness ratios, ICER) of POC-VL compared to CD4 and clinical monitoring.
POC-VL tests with detection limits <1000 copies/ml increased costs due to unnecessary switches to second-line ART, without improving survival. Assuming POC-VL unit costs between US$5–US$20 and detection limits between 1000 and 10000 copies/ml, the ICER of POC-VL was US$4010–US$9230 compared to clinical and US$5960–US$25540 compared to CD4 monitoring. In Scenario B the corresponding ICERs were US$2450–US$5830 and US$2230–US$10380. In Scenario C the ICER ranged between US$960–US$2500 compared to clinical monitoring and between cost-saving and US$2460 compared to CD4 monitoring.
The cost-effectiveness of POC-VL for monitoring ART is improved by a higher detection limit, by taking the reduction in new HIV infections into account and when assuming that failure of first-line ART is reduced due to targeted adherence counselling.
PMCID: PMC3813830  PMID: 23462219
Antiretroviral therapy; cost-effectiveness; mathematical model; sub-Saharan Africa; viral load monitoring
6.  Stress-Induced Antibiotic Susceptibility Testing on a Chip 
We have developed a rapid microfluidic method for antibiotic susceptibility testing in a stress-based environment. Fluid is passed at high speeds over bacteria immobilized on the bottom of a microfluidic channel. In the presence of stress and antibiotic, susceptible strains of bacteria die rapidly. However, resistant bacteria survive these stressful conditions. The hypothesis behind this method is new: stress activation of biochemical pathways, which are targets of antibiotics, can accelerate antibiotic susceptibility testing. As compared to standard antibiotic susceptibility testing methods, the rate-limiting step – bacterial growth – is omitted during antibiotic application. The technical implementation of the method is in a combination of standard techniques and innovative approaches. The standard parts of the method include bacteria culture protocols, defining microfluidic channels in poly(dimethylsiloxane), PDMS, cell viability monitoring with fluorescence, and batch image processing for bacteria counting. Innovative parts of the method are in the use of culture media flow for mechanical stress application, use of enzymes to damage but not kill the bacteria, and use of microarray substrates for bacterial attachment. The developed platform can be used in antibiotic and non-antibiotic related drug development and testing. As compared to the standard bacterial suspension experiments, the effect of the drug can be turned on and off repeatedly over controlled time periods. Repetitive observation of the same bacterial population is possible over the course of the same experiment.
PMCID: PMC4009506  PMID: 24430495
antibiotic; susceptibility; resistance; microfluidics; microscopy; rapid; testing; stress; bacteria; fluorescence
7.  Glucose control in diabetes: the impact of racial differences on monitoring and outcomes 
Endocrine  2012;42(3):471-482.
Type 2 diabetes is the seventh leading cause of death in the US and is projected to increase in prevalence globally. Minorities are disproportionately affected by diabetes and data suggest that clinical outcomes consistently fall below American Diabetes Association recommendations. The purpose of this systematic review was to examine ethnic differences in self-monitoring and outcomes in adults with type 2 diabetes.
Medline was searched for articles published between January 1990 through January 2012 using a reproducible strategy. Inclusion criteria included: (1) published in English, (2) targeted African Americans, Hispanic or Asian adults, ages 18+ years with type 2 diabetes, (3) cross-sectional, cohort, or intervention study, and (4) measured change in glycemic control, BP, lipids, or quality of life by race.
Twenty-two papers met the inclusion criteria and were reviewed. Overall, significant racial differences and barriers were found in published studies in diabetes management as it pertains to self-monitoring and outcomes. African Americans tend to consistently exhibit worse outcomes and control when compared to other minority populations and non-Hispanic Whites.
Significant racial differences and barriers exist in diabetes management as it pertains to self-monitoring and outcomes when compared to non-Hispanic Whites. Explanatory and intervention studies are needed to determine the mechanisms and mediators of these differences and strategies to reduce these disparities. In addition, more research is needed to investigate the impact of racial differences in self-monitoring and outcomes on quality of life.
PMCID: PMC3779599  PMID: 22815042
Diabetes; Racial differences; Self-monitoring; Glucose control; Lipids; Blood pressure
8.  A cross-sectional study of public attitudes towards safer drug use practices in British Columbia, Canada 
Harm reduction programs are often vulnerable to political and vocal opposition despite documented evidence of their effectiveness and economic benefit. It is not well understood if opponents to harm reduction represent the general public’s attitudes.
To understand the attitudes of the people of British Columbia (BC) towards various harm reduction strategies and services, and factors associated with support for harm reduction.
A random-digit dialing telephone survey assessing attitudes towards various harm reduction strategies was administered to British Columbians in August 2011 (n = 2000). We compared the level of support for general harm reduction by sex, age, education level, and area of residence (Health Authority region) (χ2). Multivariate logistic regression was used to assess odds of support for harm reduction.
Overall support for general harm reduction among participants was 76%; needle distribution 72%; needle distribution in one’s local community 65%; and safer inhalation equipment distribution 52%. In the multivariate analysis, those with significantly lower odds of supporting harm reduction were male, older, had equal or less than high school education or completed a certificate/diploma program, and resided in the Fraser Health Authority region. The Health Authority region with a municipality that has introduced a bylaw prohibiting the implementation of harm reduction services was found to have 69% support for harm reduction. Another Health Authority region with a municipality that closed a long-standing needle distribution site was found to have over 78% support.
In contrast to some local policies, our results show the British Columbians surveyed in our study support harm reduction. It is unclear whether policy makers are swayed by a vocal minority or block harm reduction activities for other reasons. Tailoring messages towards segments of the public less likely to support harm reduction, as well civic policy-makers and the media, may help to reduce stigma and gain support for harm reduction services designed to protect and improve the health of the individual and the public.
PMCID: PMC4221987  PMID: 24289280
Harm reduction; Safer drug use; Public attitude
9.  Increased Coronary Atherosclerosis and Immune Activation in HIV-1 Elite Controllers 
AIDS (London, England)  2012;26(18):2409-2412.
HIV-1 elite controllers (EC) spontaneously maintain suppressed levels of viremia, but exhibit significant immune activation. We investigated coronary atherosclerosis by coronary CT angiography (CTA) in: 1) EC, 2) non EC, chronically HIV-1 infected, ART-treated patients with undetectable viral load (“chronic HIV”), and 3) HIV-negative controls. Prevalence of atherosclerosis (78% vs. 42%, P<0.05) and markers of immune activation were increased in EC compared to HIV-negative controls. sCD163, a monocyte activation marker, was increased in EC compared to chronic HIV-1 (P<0.05) and compared to HIV-negative controls (P< 0.05). These data suggest a significant degree of coronary atherosclerosis and monocyte activation among EC.
PMCID: PMC3660105  PMID: 23032411
10.  The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data 
Nucleic Acids Research  2013;42(Database issue):D966-D974.
The Human Phenotype Ontology (HPO) project, available at, provides a structured, comprehensive and well-defined set of 10,088 classes (terms) describing human phenotypic abnormalities and 13,326 subclass relations between the HPO classes. In addition we have developed logical definitions for 46% of all HPO classes using terms from ontologies for anatomy, cell types, function, embryology, pathology and other domains. This allows interoperability with several resources, especially those containing phenotype information on model organisms such as mouse and zebrafish. Here we describe the updated HPO database, which provides annotations of 7,278 human hereditary syndromes listed in OMIM, Orphanet and DECIPHER to classes of the HPO. Various meta-attributes such as frequency, references and negations are associated with each annotation. Several large-scale projects worldwide utilize the HPO for describing phenotype information in their datasets. We have therefore generated equivalence mappings to other phenotype vocabularies such as LDDB, Orphanet, MedDRA, UMLS and phenoDB, allowing integration of existing datasets and interoperability with multiple biomedical resources. We have created various ways to access the HPO database content using flat files, a MySQL database, and Web-based tools. All data and documentation on the HPO project can be found online.
PMCID: PMC3965098  PMID: 24217912
11.  A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus aureus 
Lab on a chip  2012;12(21):4523-4532.
The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies the need for fast phenotype-based clinical tests for determining antibiotic susceptibility. Standard susceptibility testing relies on the passive observation of bacterial growth inhibition in the presence of antibiotics. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing basedon stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a β-lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. Bacteria, covalently-bound to the bottom of the microfluidic channel, were subjected to mechanical shear stress created by flowing culture media through the microfluidic channel and to enzymatic stress with sub-inhibitory concentrations of the bactericidal agent lysostaphin. Bacterial cell death was monitored via fluorescence using the Sytox Green dead cell stain, and rates of killing were measured for the bacterial samples in the presence and absence of oxacillin. Using model susceptible (Sanger 476) and resistant (MW2) S. aureus strains, a metric was established to separate susceptible and resistant staphylococci based on normalized fluorescence values after 60 minutes of exposure to stress and antibiotic. Because this groundbreaking approach is not based on standard methodology, it circumvents the need for minimum inhibitory concentration (MIC) measurements and long wait times. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional clinically relevant S. aureus strains in a blinded study. In addition to future clinical utility, this method has great potential for studying the effects of various stresses on bacteria and their antibiotic susceptibility.
PMCID: PMC3489182  PMID: 22968495
12.  Effect of Diabetes Fatalism on Medication Adherence and Self-Care Behaviors in Adults with Diabetes 
General hospital psychiatry  2012;34(6):598-603.
Diabetes fatalism is defined as “a complex psychological cycle characterized by perceptions of despair, hopelessness, and powerlessness” and associated with poor glycemic control. This study examined the association between diabetes fatalism and medication adherence and self-care behaviors in adults with diabetes.
Data on 378 subjects with type 2 diabetes recruited from two primary care clinics in the Southeastern United States were examined. Previously validated scales were used to measure diabetes fatalism, medication adherence, diabetes knowledge, and diabetes self-care behaviors (diet, physical activity, blood sugar testing and foot care). Multiple linear regression was used to assess the independent effect of diabetes fatalism on medication adherence and self-care behaviors controlling for relevant covariates.
Fatalism correlated significantly with medication adherence (r = 0.24, p<0.001), diet (r = −0.26, p<0.001), exercise (r = −0.20, p<0.001) and blood sugar testing (r = −0.19, p<0.001). In the linear regression model, diabetes fatalism was significantly associated with medication adherence (β= 0.029, 95% CI 0.016, 0.043); diabetes knowledge (β= −0.042, 95% CI −0.001, −0.084); diet (β= −0.063, 95% CI −0.039, −0.087), exercise (β= −0.055, 95% CI −0.028, −0.083), and blood sugar testing (β= −0.055, 95% CI −0.023, −0.087). There was no significant association between diabetes fatalism and foot care (β= −0.018, 95% CI −0.047, 0.011). The association between diabetes fatalism and medication adherence, diabetes knowledge and diabetes self-care behaviors did not change significantly when depression was added to the models, suggesting that the associations are independent of depression.
Diabetes fatalism is associated with poor medication adherence and self-care and may be an important target for education and skills interventions in diabetes care. In addition, the effect of diabetes fatalism is independent of depression, suggesting that interventions that target depression may not be sufficient to deal with diabetes fatalism.
PMCID: PMC3479321  PMID: 22898447
fatalism; medication adherence; self-care; diabetes
13.  Wall teichoic acid protects Staphylococcus aureus from inhibition by Congo red and other dyes 
Polyanionic polymers, including lipoteichoic acid and wall teichoic acid, are important determinants of the charged character of the staphylococcal cell wall. This study was designed to investigate the extent to which teichoic acid contributes to protection from anionic azo dyes and to identify barriers to drug penetration for development of new antibiotics for multidrug-resistant Staphylococcus aureus infection.
We studied antimicrobial activity of azo dyes against S. aureus strains with or without inhibition of teichoic acid in vitro and in vivo.
We observed that inhibition of wall teichoic acid expression resulted in an ∼1000-fold increase in susceptibility to azo dyes such as Congo red, reducing its MIC from >1024 to <4 mg/L. Sensitization occurred when the first step in the wall teichoic acid pathway, catalysed by TarO, was inhibited either by mutation or by chemical inhibition. In contrast, genetic blockade of lipoteichoic acid biosynthesis did not confer Congo red susceptibility. Based on this finding, combination therapy was tested using the highly synergistic combination of Congo red plus tunicamycin at sub-MIC concentrations (to inhibit wall teichoic acid biosynthesis). The combination rescued Caenorhabditis elegans from a lethal challenge of S. aureus.
Our studies show that wall teichoic acid confers protection to S. aureus from anionic azo dyes and related compounds, and its inhibition raises the prospect of development of new combination therapies based on this inhibition.
PMCID: PMC3584970  PMID: 22615298
bacteria; antibiotics; S. aureus
14.  Associations between Coping, Diabetes Knowledge, Medication Adherence, and Self-Care Behaviors in Adults with Type 2 Diabetes 
General Hospital Psychiatry  2012;34(4):385-389.
Few studies have examined the emotional approach to coping on diabetes outcomes. This study examined the relationship between emotional coping and diabetes knowledge, medication adherence, and self-care behaviors in adults with type 2 diabetes.
Data on 378 subjects with type 2 diabetes recruited from two primary care clinics in the Southeastern United States were examined. Previously validated scales were used to measure coping, medication adherence, diabetes knowledge, and diabetes self-care behaviors (including diet, physical activity, blood sugar testing and foot care). Multiple linear regression was used to assess the independent effect of coping through emotional approach on medication adherence and self-care behaviors while controlling for relevant covariates.
Significant correlations were observed between emotional coping (as measured by emotional expression (EE) and emotional processing (EP)) and self-care behaviors. In the linear regression model, EP was significantly associated with medication adherence (β −0.17, 95% CI −0.32 to −0.015), diabetes knowledge (β 0.76, 95% CI 0.29 to 1.24), diet (β 0.52, 95% CI 0.24 to 0.81), exercise (β 0.51, 95% CI 0.19 to 0.82), blood sugar testing (β 0.54, 95% CI 0.16 to 0.91), and foot care (β 0.32, 95% CI −0.02 to 0.67). On the other hand, EE was associated with diet (β 0.38, 95% CI 0.13 to 0.64), exercise (β 0.54, 95% CI 0.27 to 0.82), blood sugar testing (β 0.42, 95% CI 0.09 to 0.76) and foot care (β 0.36, 95% CI 0.06 to 0.66), but it was not associated with diabetes knowledge.
These findings indicate that coping through an emotional approach is significantly associated with behaviors that lead to positive diabetes outcomes.
PMCID: PMC3383912  PMID: 22554428
Coping; medication adherence; self-care; diabetes
15.  Diabetes Empowerment, Medication Adherence and Self-Care Behaviors in Adults with Type 2 Diabetes 
Evidence suggests that empowerment is an important factor to address everyday aspects of dealing with a chronic disease. This study evaluated the effect of diabetes empowerment on medication adherence and self-care behaviors in adults with type 2 diabetes.
Subjects and Methods
Data on 378 subjects with type 2 diabetes recruited from two primary care clinics in the southeastern United States were examined. Previously validated scales were used to measure diabetes empowerment, medication adherence, diabetes knowledge, and diabetes self-care behaviors (including diet, physical activity, blood sugar testing, and foot care). Multiple linear regression was used to assess the independent effect of diabetes empowerment on medication adherence and self-care behaviors controlling for relevant covariates.
Eighty-three percent were non-Hispanic blacks, 69% were women, 22% were 65 years or older, 68% were not married, 26% had less than high school education, 60% were unemployed, 39% were uninsured, and 47% had a yearly income <$10,000. Empowerment had significant correlations with medication adherence (r=0.17, P<0.003), diabetes knowledge (r=0.16, P=0.007), diet (r=0.24, P<0.001), exercise (r=0.25, P<0.001), blood sugar testing (r=0.12, P=0.043), and foot care (r=0.18, P=0.002). In the regression model, diabetes empowerment was significantly associated with medication adherence (β=−0.04, P=0.001), diabetes knowledge (β=0.09, P=0.012), diet (β=0.09, P<0.001), exercise (β=0.10, P<0.001), blood sugar testing (β=0.07, P=0.016), and foot care (β=0.08, P=0.001).
In this sample, diabetes empowerment was related to better diabetes knowledge, medication adherence and improved self-care behaviors. Emphasis on empowerment and self-efficacy is relevant to improve outcomes in the management of diabetes.
PMCID: PMC3389377  PMID: 22524548
16.  Lack of GNAQ and GNA11 Germ-Line Mutations in Familial Melanoma Pedigrees with Uveal Melanoma or Blue Nevi 
Frontiers in Oncology  2013;3:160.
Approximately 10% of melanoma cases are familial, but only 25–40% of familial melanoma cases can be attributed to germ-line mutations in the CDKN2A – the most significant high-risk melanoma susceptibility locus identified to date. The pathogenic mutation(s) in most of the remaining familial melanoma pedigrees have not yet been identified. The most common mutations in nevi and sporadic melanoma are found in BRAF and NRAS, both of which result in constitutive activation of the MAPK pathway. However, these mutations are not found in uveal melanomas or the intradermal melanocytic proliferations known as blue nevi. Rather, multiple studies report a strong association between these lesions and somatic mutations in Guanine nucleotide-binding protein G(q) subunit alpha (GNAQ), Guanine nucleotide-binding protein G(q) subunit alpha-11 (GNA11), and BRCA1-associated protein-1 (BAP1). Recently, germ-line mutations in BAP1, the gene encoding a tumor suppressing deubiquitinating enzyme, have been associated with predisposition to a variety of cancers including uveal melanoma, but no studies have examined the association of germ-line mutations in GNAQ and GNA11 with uveal melanoma and blue nevi. We have now done so by sequencing exon 5 of both of these genes in 13 unique familial melanoma pedigrees, members of which have had either uveal or cutaneous melanoma and/or blue nevi. Germ-line DNA from a total of 22 individuals was used for sequencing; however no deleterious mutations were detected. Nevertheless, such candidate gene studies and the discovery of novel germ-line mutations associated with an increased MM susceptibility can lead to a better understanding of the pathways involved in melanocyte transformation, formulation of risk assessment, and the development of specific drug therapies.
PMCID: PMC3695489  PMID: 23825798
GNAQ; GNA11; familial melanoma; germ-line; blue nevi; uveal melanoma
17.  Regional and temporal variation in the treatment of rheumatoid arthritis across the UK: a descriptive register-based cohort study 
BMJ Open  2012;2(6):e001603.
To describe current disease-modifying antirheumatic drugs (DMARDs) prescription in rheumatoid arthritis (RA) with reference to best practice and to identify temporal and regional trends in the UK.
Descriptive, register-based cohort study.
Permanently registered patients aged ≥18 years with a recorded diagnosis of RA between 1 January 1995 and 31 March 2010 and matched controls. Participants with RA were identified through screening of all patients in the General Practice Research Database (GPRD) with a clinical or referral record for RA and at least 1 day of follow-up.
639 general practices in the UK supplying data to the GPRD.
Main outcome measures
Medication prescribing between 3 and 12 months of RA diagnosis by region and time period (1995–1999, 2000–2005 and 2006–April 2010).
Of the 35 911 patients in the full RA cohort, 15 259 patients (42%) had incident RA. Analysis of prescribing in incident RA patients demonstrated that between 1995 (baseline) and 2010 there was a substantial increase in DMARD, and specifically methotrexate, prescribing across all regions with a less marked increase in combination DMARD prescribing. Taking 12-month prescribing as a snapshot: DMARD prescribing was 19–49% at baseline increasing to 45–74% by 2006–April 2010; methotrexate prescribing was 4–16% at baseline increasing to 32–60%; combination DMARD prescribing was 0–8% at baseline increasing to 3–17%. However, there was marked regional variation in the proportion of RA patients receiving DMARD regardless of time period.
There has been a substantial increase in prescribing of DMARDs for RA since 1995; however, regional variation persists across the UK with relative undertreatment, according to established best practice. Improved implementation of evidence-based best clinical practice to facilitate removal of treatment variation is warranted. This may occur as a result of the implementation of published national guidance.
PMCID: PMC3533005  PMID: 23144258
Primary Care; Rheumatology
18.  High-throughput assessment of bacterial growth inhibition by optical density measurements 
The increasing incidence of antibiotic-resistant bacterial infections both in hospitals and in the community intensifies the need for new antibacterial strategies and targets. Although high-throughput screening against live bacteria allows rapid discovery of compounds with growth-inhibitory activities, these efforts have failed to fill the pipeline with the anticipated antibacterial compounds because target identification is often onerous. Recently, a strategy was reported that employs a bacteria growth inhibition assay readout using optical density measurements on paired strains – both a wildtype strain and a pathway-null mutant – to find inhibitors of wildtype bacterial growth that specifically target conditionally essential enzymes in the pathway of interest. Protocols are provided here for determining the robustness of an assay, screening in a high-throughput format and setting up dose-response curves in paired Staphylococcus aureus strains. However, the protocols can be used to screen for growth-inhibitory compounds in any bacterial strain of interest.
PMCID: PMC3182142  PMID: 21966637
high-throughput screening; Staphylococcus aureus; optical density; cell-based assay; pathway specific screen; bacterial growth
19.  An Antibiotic That Inhibits a Late Step in Wall Teichoic Acid Biosynthesis Induces the Cell Wall Stress Stimulon in Staphylococcus aureus 
Wall teichoic acids (WTAs) are phosphate-rich, sugar-based polymers attached to the cell walls of most Gram-positive bacteria. In Staphylococcus aureus, these anionic polymers regulate cell division, protect cells from osmotic stress, mediate host colonization, and mask enzymatically susceptible peptidoglycan bonds. Although WTAs are not required for survival in vitro, blocking the pathway at a late stage of synthesis is lethal. We recently discovered a novel antibiotic, targocil, that inhibits a late acting step in the WTA pathway. Its target is TarG, the transmembrane component of the ABC transporter (TarGH) that exports WTAs to the cell surface. We examined here the effects of targocil on S. aureus using transmission electron microscopy and gene expression profiling. We report that targocil treatment leads to multicellular clusters containing swollen cells displaying evidence of osmotic stress, strongly induces the cell wall stress stimulon, and reduces the expression of key virulence genes, including dltABCD and capsule genes. We conclude that WTA inhibitors that act at a late stage of the biosynthetic pathway may be useful as antibiotics, and we present evidence that they could be particularly useful in combination with beta-lactams.
PMCID: PMC3318382  PMID: 22290958
20.  The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays 
Owing to its ease of collection, saliva is potentially the sample of choice in diagnosis. Salivary biomolecules have provided a porthole in surveying a person’s health and well-being. Our study aims were (1) to demonstrate the effects of pre-analytical steps, collection and pre-processing techniques on salivary protein detection and (2) to establish an indication of salivary reference intervals for 3 biomolecules of clinical interest.
Saliva samples were collected from participants (n = 25, ages 20–35 years) using the following methods: no stimulation (resting/unstimulated), mechanical, and acid stimulation. The saliva was prepared for analysis by: unprocessed, post standard centrifugation in a container without any additives, and centrifugation using Centrifugal Filter Unit (Amicon® Ultra-0.5). AlphaLisa® assays were used to measure the levels of C-Reactive Protein (CRP), Immunoglobin (IgE) and myoglobin in saliva samples.
Saliva flow rates were lowest with the resting/drooling collection method. The lowest total protein concentration was with acid stimulation. Unstimulated and mechanically stimulated collections produced no effect on the CRP and IgE levels while myoglobin levels were highest with the unstimulated collection. Acid stimulation had a negative impact on the measured concentrations of IgE and myoglobin (except for CRP levels).
Mechanical stimulation was the most viable option for collecting saliva without affecting the levels of CRP and myoglobin. The processing methods had an adverse effect on the concentration of total protein as well as on CRP and IgE concentrations.
PMCID: PMC3560976  PMID: 23369566
Human saliva; Homogeneous bead-based assay; Salivary stimulations; Salivary pre-processing techniques; Non-invasive
21.  Brain Creatine Elevation and NAA Reduction Indicates Neuronal Dysfunction in the Setting of Enhanced Glial Energy Metabolism in a Macaque Model of neuroAIDS 
Proton magnetic resonance spectroscopy (1H MRS) has emerged as one of the most informative neuroimaging modalities for studying the effect of HIV infection in the brain, providing surrogate markers by which to assess disease progression and monitor treatment. Reductions in the level of N-Acetylaspartate (NAA) and NAA/creatine (NAA/Cr) are established markers of neuronal injury or loss. However, the biochemical basis of altered creatine levels in neuroAIDS is not well understood. This study used a rapid progression macaque model of neuroAIDS to elucidate the changes in creatine. As the disease progressed 1H MRS revealed a decrease in NAA, indicative of neuronal injury, and an increase in creatine yet to be elucidated. Subsequently, immunohistochemistry and stereology measures of decreased synaptophysin, microtubule-associated protein 2, and neuronal density confirmed neuronal injury. Furthermore, increases in ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein indicated microglial and astroglial activation, respectively. Given these data, elevated creatine may reflect enhanced high-energy phosphate turnover in highly metabolizing activated astrocytes and microglia.
PMCID: PMC3139817  PMID: 21381104
HIV; SIV; Macaques; MR Spectroscopy; Creatine; Gliosis
22.  Detection of Favorable Oral Cephalosporin-Clavulanate Interactions by In Vitro Disk Approximation Susceptibility Testing of Extended-Spectrum-Beta-Lactamase-Producing Members of the Enterobacteriaceae 
Journal of Clinical Microbiology  2012;50(3):1023-1026.
Extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacteriaceae are often resistant to multiple drug classes, making therapy of urinary infections with oral antibiotics difficult. Previously it was shown that amoxicillin-clavulanate can provide clavulanate inhibition of ESBLs and protect an oral cephalosporin present in combination when tested by broth microdilution. This study has shown that disk approximation testing could detect favorable cephalosporin-clavulanate interactions among a group of 101 previously characterized members of the Enterobacteriaceae with CTX-M, SHV, or TEM ESBLs.
PMCID: PMC3295132  PMID: 22170910
23.  Predictors and effects of alcohol use on liver function among young HCV-infected injection drug users in a behavioral intervention 
Journal of hepatology  2010;55(1):45-52.
Background & Aims
Hepatitis C virus (HCV) screening can provide opportunities to reduce disease progression through counseling against alcohol use, but empirical data on this issue are sparse. We determined the efficacy of a behavioral intervention in reducing alcohol use among young, HCV-infected injection drug users (IDUs) (n=355) and assessed whether changes in liver enzymes were associated with changes in alcohol consumption.
Both the intervention and attention-control groups were counseled to avoid alcohol use, but the intervention group received enhanced counseling. Logistic regression, ANOVA, and continuous time Markov models were used to identify factors associated with alcohol use, changes in mean ALT and AST levels and change in alcohol use post-intervention.
Six months post-intervention, alcohol abstinence increased 22.7% in both groups, with no difference by intervention arm. Transition from alcohol use to abstinence was associated with a decrease in liver enzymes, with a marginally greater decrease in the intervention group (p=0.05 for ALT; p=0.06 for AST). In multivariate Markov models, those who used marijuana transitioned from alcohol abstinence to consumption more rapidly than non-users (RR=3.11); those who were homeless transitioned more slowly to alcohol abstinence (RR=0.47); and those who had ever received a clinical diagnosis of liver disease transitioned more rapidly to abstinence (RR=1.88).
Although, behavioral counseling to reduce alcohol consumption among HCV-infected IDUs had a modest effect, reductions in alcohol consumption were associated with marked improvements in liver function. Interventions to reduce alcohol use among HCV-infected IDUs may benefit from being integrated into clinical care and monitoring of HCV infection.
PMCID: PMC3094600  PMID: 21145862
24.  Synthetic lethal compound combinations reveal a fundamental connection between wall teichoic acid and peptidoglycan biosyntheses in Staphylococcus aureus 
ACS chemical biology  2010;6(1):106-116.
Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase (TP) with reduced susceptibility to beta-lactam antibiotics. PBP2A crosslinks nascent peptidoglycan when the native TPs are inhibited by beta-lactams. Although mecA expression is essential for beta-lactam resistance, it is not sufficient. Here we show that blocking the expression of wall teichoic acids (WTAs) by inhibiting the first enzyme in the pathway, TarO, sensitizes MRSA strains to beta-lactams even though the beta-lactam-resistant transpeptidase, PBP2A, is still expressed. The dramatic synergy between TarO inhibitors and beta-lactams is noteworthy not simply because strategies to overcome methicillin-resistant S. aureus (MRSA) are desperately needed, but because neither TarO nor the activities of the native TPs are essential in MRSA strains. The “synthetic lethality” of inhibiting TarO and the native TPs suggests a functional connection between ongoing WTA expression and peptidoglycan assembly in S. aureus. Indeed, transmission electron microscopy shows that S. aureus cells blocked in WTA synthesis have extensive defects in septation and cell separation, indicating dysregulated cell wall assembly and degradation. Our studies imply that WTAs play a fundamental role in S. aureus cell division and raise the possibility that synthetic lethal compound combinations may have therapeutic utility for overcoming antibiotic resistant bacterial infections.
PMCID: PMC3025082  PMID: 20961110
25.  Role of Wall Teichoic Acids in Staphylococcus aureus Endophthalmitis 
This study shows that wall teichoic acids, which are major polyanionic polymer components of the cell wall of Staphylococcus aureus, are necessary for growth and survival of S. aureus in the eye.
Wall teichoic acids (WTAs) are major polyanionic polymer components of the cell wall of Staphylococcus aureus. However, little is known about their role at the host–pathogen interface, especially in endophthalmitis. This study was designed to investigate the extent to which WTAs contribute to the pathogenicity of S. aureus in models of endophthalmitis and to determine whether there would be value in targeting their biosynthesis as a new therapeutic approach.
S. aureus RN6390 and its isogenic WTA-null mutant (RN6390ΔtarO) were used to evaluate the role of WTAs in endophthalmitis. RN6390 and RN6390ΔtarO were cultured in bovine vitreous humor (VH) in vitro or inoculated into the vitreous chamber of C57B6 mice. Changes in the number of bacteria, organ function as determined by electroretinography (ERG), and histopathologic changes were assessed throughout the course of infection. In addition, the efficacy of WTA biosynthesis inhibitors in VH in vitro was examined.
It was observed that a component of VH synergized with WTA biosynthesis inhibitors in vitro and killed the S. aureus. This effect was also seen when mutants incapable of expressing WTA were exposed to VH. The killing activity of VH was lost on treatment with a protease inhibitor. RN6390ΔtarO could not survive in mouse eyes and did not affect organ function, nor was it able to establish endophthalmitis.
WTAs are essential cellular constituents for the manifestation of virulence by S. aureus in endophthalmitis, and appears to be a viable target for treating the endophthalmitis caused by S. aureus strains.
PMCID: PMC3109022  PMID: 21345983

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