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1.  Mephedrone Does not Damage Dopamine Nerve Endings of the Striatum but Enhances the Neurotoxicity of Methamphetamine, Amphetamine and MDMA 
Journal of neurochemistry  2013;125(1):102-110.
Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine. One of the most powerful actions associated with mephedrone is the ability to stimulate dopamine (DA) release and block its reuptake through its interaction with the dopamine transporter (DAT). Although mephedrone does not cause toxicity to DA nerve endings, its ability to serve as a DAT blocker could provide protection against methamphetamine-induced neurotoxicity like other DAT inhibitors. To test this possibility, mice were treated with mephedrone (10, 20 or 40 mg/kg) prior to each injection of a neurotoxic regimen of methamphetamine (4 injections of 2.5 or 5.0 mg/kg at 2 hr intervals). The integrity of DA nerve endings of the striatum was assessed through measures of DA, DAT and tyrosine hydroxylase levels. The moderate to severe DA toxicity associated with the different doses of methamphetamine was not prevented by any dose of mephedrone but was, in fact, significantly enhanced. The hyperthermia caused by combined treatment with mephedrone and methamphetamine was the same as seen after either drug alone. Mephedrone also enhanced the neurotoxic effects of amphetamine and MDMA on DA nerve endings. In contrast, nomifensine protected against methamphetamine-induced neurotoxicity. Because mephedrone increases methamphetamine neurotoxicity, the present results suggest that it interacts with the DAT in a manner unlike that of other typical DAT inhibitors. The relatively innocuous effects of mephedrone alone on DA nerve endings mask a potentially dangerous interaction with drugs that are often co-abused with it, leading to heightened neurotoxicity.
PMCID: PMC3604033  PMID: 23205838
mephedrone; methamphetamine; MDMA; amphetamine; dopamine; neurotoxicity
2.  Genome-wide Association Study Identifies Two Susceptibility Loci for Osteosarcoma 
Nature genetics  2013;45(7):799-803.
Osteosarcoma is the most common primary bone malignancy of adolescents and young adults. In order to better understand the genetic etiology of osteosarcoma, we performed a multi-stage genome-wide association study (GWAS) consisting of 941 cases and 3,291 cancer-free adult controls of European ancestry. Two loci achieved genome-wide significance: rs1906953 at 6p21.3, in the glutamate receptor metabotropic 4 [GRM4] gene (P = 8.1 ×10-9), and rs7591996 and rs10208273 in a gene desert on 2p25.2 (P = 1.0 ×10-8 and 2.9 ×10-7). These two susceptibility loci warrant further exploration to uncover the biological mechanisms underlying susceptibility to osteosarcoma.
PMCID: PMC3910497  PMID: 23727862
3.  Measuring biexponential transverse relaxation of the ASL signal at 9.4 T to estimate arterial oxygen saturation and the time of exchange of labeled blood water into cortical brain tissue 
The transverse decay of the arterial spin labeling (ASL) signal was measured at four inflow times in the rat brain cortex at 9.4 T. Biexponential T2 decay was observed that appears to derive from different T2 values associated with labeled water in the intravasculature (IV) and extravascular (EV) compartments. A two compartment biexponential model was used to assess the relative contribution of the IV and EV compartments to the ASL signal, without assuming a value for T2 of labeled blood water in the vessels. This novel methodology was applied to estimate the exchange time of blood water into EV tissue space and the oxygen saturation of blood on the arterial side of the vasculature. The mean exchange time of labeled blood water was estimated to be 370±40 ms. The oxygen saturation of the arterial side of the vasculature was significantly less than 100% (∼85%), which may have implications for quantitative functional magnetic resonance imaging studies where the arterial oxygen saturation is frequently assumed to be 100%.
PMCID: PMC3564190  PMID: 23168531
arterial spin labeling; ASL; cerebral hemodynamics; exchange; functional MRI (fMRI); MRI
4.  Identification of small molecules for human hepatocyte expansion and iPS differentiation 
Nature chemical biology  2013;9(8):514-520.
Cell-based therapies hold the potential to alleviate the growing burden of liver diseases. Such therapies require human hepatocytes, which, within the stromal context of the liver, are capable of many rounds of replication. However, this ability is lost ex vivo and human hepatocyte sourcing has been limiting many fields of research for decades. Here, we developed a high-throughput screening platform for primary human hepatocytes to identify small molecules in two different classes that can be used to generate renewable sources of functional human hepatocytes. One class induced functional proliferation of primary human hepatocytes in vitro. The second class enhanced hepatocyte functions and promoted differentiation of iPS-derived hepatocytes, toward a phenotype more mature than what was previously obtainable. The identification of these small molecules can help to address a major challenge impacting many facets of liver research and may lead to the development of novel therapeutics for liver diseases.
PMCID: PMC3720805  PMID: 23728495
5.  Bacterial community dynamics and activity in relation to dissolved organic matter availability during sea-ice formation in a mesocosm experiment 
MicrobiologyOpen  2014;3(1):139-156.
The structure of sea-ice bacterial communities is frequently different from that in seawater. Bacterial entrainment in sea ice has been studied with traditional microbiological, bacterial abundance, and bacterial production methods. However, the dynamics of the changes in bacterial communities during the transition from open water to frozen sea ice is largely unknown. Given previous evidence that the nutritional status of the parent water may affect bacterial communities during ice formation, bacterial succession was studied in under ice water and sea ice in two series of mesocosms: the first containing seawater from the North Sea and the second containing seawater enriched with algal-derived dissolved organic matter (DOM). The composition and dynamics of bacterial communities were investigated with terminal restriction fragment length polymorphism (T-RFLP), and cloning alongside bacterial production (thymidine and leucine uptake) and abundance measurements (measured by flow cytometry). Enriched and active sea-ice bacterial communities developed in ice formed in both unenriched and DOM-enriched seawater (0–6 days). γ-Proteobacteria dominated in the DOM-enriched samples, indicative of their capability for opportunistic growth in sea ice. The bacterial communities in the unenriched waters and ice consisted of the classes Flavobacteria, α-and γ-Proteobacteria, which are frequently found in natural sea ice in polar regions. Furthermore, the results indicate that seawater bacterial communities are able to adapt rapidly to sudden environmental changes when facing considerable physicochemical stress such as the changes in temperature, salinity, nutrient status, and organic matter supply during ice formation.
PMCID: PMC3937737  PMID: 24443388
16S rDNA; bacteria; DOM; flow cytometry; mesocosm; sea ice; T-RFLP
6.  A microscale human liver platform that supports the hepatic stages of Plasmodium falciparum and vivax 
Cell host & microbe  2013;14(1):104-115.
The Plasmodium liver stage is an attractive target for the development of anti-malarial drugs and vaccines, as it provides an opportunity to interrupt the life cycle of the parasite at a critical early stage. However, targeting the liver stage has been difficult. Undoubtedly, a major barrier has been the lack of robust, reliable and reproducible in vitro liver stage cultures. Here, we establish the liver stages for both Plasmodium falciparum and Plasmodium vivax in a microscale human liver platform composed of cryopreserved, micropatterned human primary hepatocytes surrounded by supportive stromal cells. Using this system, we have successfully recapitulated the full liver stage of P. falciparum including the release of infected merozoites and infection of overlaid erythrocytes, and also the establishment of small forms in late liver stages of P. vivax. Finally, we validate the potential of this platform as a tool for medium-throughput anti-malarial drug screening and vaccine development.
PMCID: PMC3780791  PMID: 23870318
7.  Anterior Glenohumeral Laxity and Stiffness After a Shoulder-Strengthening Program in Collegiate Cheerleaders 
Journal of Athletic Training  2013;48(1):25-30.
Approximately 62% of all cheerleaders sustain some type of orthopaedic injury during their cheerleading careers. Furthermore, the occurrence of such injuries has led to inquiry regarding optimal prevention techniques. One possible cause of these injuries may be related to inadequate conditioning in cheerleaders.
To determine whether a strength and conditioning program produces quantifiable improvements in anterior glenohumeral (GH) laxity and stiffness.
Descriptive laboratory study.
University laboratory.
Patients or Other Participants
A sample of 41 collegiate cheerleaders (24 experimental and 17 control participants) volunteered. No participants had a recent history (in the past 6 months) of upper extremity injury or any history of upper extremity surgery.
The experimental group completed a 6-week strength and conditioning program between the pretest and posttest measurements; the control group did not perform any strength training between tests.
Main Outcome Measure(s)
We measured anterior GH laxity and stiffness with an instrumented arthrometer. We conducted a group × time analysis of variance with repeated measures on time (P < .05) to determine differences between groups.
A significant interaction was demonstrated, with the control group having more anterior GH laxity at the posttest session than the strengthening group (P = .03, partial η2 = 0.11). However, no main effect for time (P = .92) or group (P = .97) was observed. In another significant interaction, the control group had less anterior GH stiffness at the posttest session than the strengthening group (P = .03, partial η2 = 0.12). Main effects for time (P = .02) and group (P = .004) were also significant.
Cheerleaders who participate in a shoulder-strengthening program developed less anterior GH laxity and more stiffness than cheerleaders in the control group.
PMCID: PMC3554029  PMID: 23672322
instability; conditioning; injury prevention
8.  High-Throughput FRET Assay Yields Allosteric SERCA Activators 
Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.
PMCID: PMC3721969  PMID: 22923787
calcium pump; calcium transport; phospholamban; reconstituted membrane; fluorescence lifetime
9.  Diabetic nephropathy: Is it time yet for routine kidney biopsy? 
World Journal of Diabetes  2013;4(6):245-255.
Diabetic nephropathy (DN) is one of the most important long-term complications of diabetes. Patients with diabetes and chronic kidney disease have an increased risk of all-cause mortality, cardiovascular mortality, and kidney failure. The clinical diagnosis of DN depends on the detection of microalbuminuria. This usually occurs after the first five years from the onset of diabetes, and predictors of DN development and progression are being studied but are not yet implemented into clinical practice. Diagnostic tests are useful tools to recognize onset, progression and response to therapeutic interventions. Microalbuminuria is an indicator of DN, and it is considered the only noninvasive marker of early onset. However, up to now there is no diagnostic tool that can predict which patients will develop DN before any damage is present. Pathological renal injury is hard to predict only with clinical and laboratory findings. An accurate estimate of damage in DN can only be achieved by the histological analysis of tissue samples. At the present time, renal biopsy is indicated on patients with diabetes under the suspicion of the presence of nephropathies other than DN. Results from renal biopsies in patients with diabetes had made possible the classification of renal biopsies in three major groups associated with different prognostic features: diabetic nephropathy, non-diabetic renal disease (NDRD), and a superimposed non-diabetic condition on underlying diabetic nephropathy. In patients with type 2 diabetes with a higher degree of suspicion for NDRD, it is granted the need of a renal biopsy. It is important to identify and differentiate these pathologies at an early stage in order to prevent progression and potential complications. Therefore, a more extensive use of biopsy is advisable.
PMCID: PMC3874483  PMID: 24379914
Diabetic nephropathy; Kidney biopsy; Non-diabetic renal disease
10.  Intra-patient dose escalation in Ewing’s sarcoma treated with vincristine, doxorubicin, cyclophosphamide alternating with ifosfamide and etoposide: a retrospective review 
Data suggests that males experience less toxicity and poorer survival than females treated for Ewing’s sarcoma. We instituted an intra-patient dose escalation (DE) policy with Vincristine/Doxorubicin/Cyclophosphamide (VDC) alternating with Ifosfamide/Etoposide (IE) based on hematological nadirs and report its feasibility and safety.
A retrospective review of adherence to DE guidelines and toxicities was conducted for patients who received DE with VDC/IE over 3 years at a single cancer center. Absolute neutrophil counts (ANC) was collected on days 8, 12 and 15 for cycles 1–6. DE of 10%/cycle was applied if ANC > 1.5×109/L and platelet > 100×109/L on all blood results. The primary endpoint was the proportion of patients who received appropriate DE. The secondary endpoint was to assess morbidity, changes in hematologic nadirs between gender and age and a comparison with a prior cohort of ESFT patients who did not receive DE. Gender comparisons were assessed via independent 2-sample t-tests assuming unequal variances. Within cycle changes in hematologic nadirs were assessed using repeated measures ANOVA. Relapse free survival and overall survival (OS) curves were estimated using the Kaplan-Meier method.
23 patients were identified (mean age: 27; range 17–54). 91 decisions for DE were made (1 decision excluded because of progressive disease) with 90% concordance with guidelines. No adverse outcomes occurred as a result of the inappropriate escalation. Grade 3/4 febrile neutropenia (FN) during VDC and IE was 26.1% (6/23 patients) and 17.4% respectively with no difference for those who were DE. Males were less neutropenic after C1 and C3 of VDC compared to females (P-value C1 = 0.003; C3 = 0.005). VDC was associated with greater neutropenia on day 8 whereas IE had greater neutropenia on day 12 (P-value <0.001). During VDC, a non statistical difference in neutropenia was seen for individuals aged 15–25 (n = 13) compared with older individuals (P-value = 0.09). OS comparison for those with localized disease with a prior cohort who were not DE showed similar outcomes (P-value = 0.37).
DE is deliverable without increased adverse outcomes. Males have less myelosuppression during VDC, and should be especially considered for DE.
PMCID: PMC3866566  PMID: 24321600
Ewing’s sarcoma; Dose escalation; Toxicity; Neutropenia; Chemotherapy
11.  Structural dynamics of muscle protein phosphorylation 
We have used site-directed spectroscopic probes to detect structural changes, motions, and interactions due to phosphorylation of proteins involved in the regulation of muscle contraction and relaxation. Protein crystal structures provide static snapshots that provide clues to the conformations that are sampled dynamically by proteins in the cellular environment. Our site-directed spectroscopic experiments, combined with computational simulations, extend these studies into functional assemblies in solution, and reveal details of protein regions that are too dynamic or disordered for crystallographic approaches. Here, we discuss phosphorylation-mediated structural transitions in the smooth muscle myosin regulatory light chain (RLC), the striated muscle accessory protein myosin binding protein-C (MyBP-C), and the cardiac membrane Ca2+ pump modulator phospholamban (PLB). In each of these systems, phosphorylation near the N terminus of the regulatory protein relieves an inhibitory interaction between the phosphoprotein and its regulatory target. Several additional unifying themes emerge from our studies: (a) The effect of phosphorylation is not to change the affinity of the phosphoprotein for its regulated binding partner, but to change the structure of the bound complex without dissociation. (b) Phosphorylation induces transitions between order and dynamic disorder. (c) Structural states are only loosely coupled to phosphorylation; i.e., complete phosphorylation induces dramatic functional effects with only a partial shift in the equilibrium between ordered and disordered structural states. These studies, which offer atomic-resolution insight into the structural and functional dynamics of these phosphoproteins, were inspired in part by the ground-breaking work in this field by Michael and Kate Barany.
PMCID: PMC3652605  PMID: 22930331
Muscle Regulation; Spectroscopy; Structural Dynamics; Phospholamban; Regulatory Light Chain; Myosin Binding Protein-C
12.  Post-Weaning Diet Affects Faecal Microbial Composition but Not Selected Adipose Gene Expression in the Cat (Felis catus) 
PLoS ONE  2013;8(11):e80992.
The effects of pre- (i.e., gestation and during lactation) and post-weaning diet on the composition of faecal bacterial communities and adipose expression of key genes in the glucose and insulin pathways were investigated in the cat. Queens were maintained on a moderate protein:fat:carbohydrate kibbled (“Diet A”; 35:20:28% DM; n  =  4) or high protein:fat:carbohydrate canned (“Diet B”; 45:37:2% DM; n = 3) diet throughout pregnancy and lactation. Offspring were weaned onto these diets in a nested design (n  =  5 per treatment). Faecal samples were collected at wk 8 and 17 of age. DNA was isolated from faeces and bacterial 16S rRNA gene amplicons were analysed by pyrosequencing. RNA was extracted from blood (wk 18) and adipose tissue and ovarian/testicular tissues (wk 24) and gene expression levels determined using RT-qPCR. Differences (P<0.05) in composition of faecal bacteria were observed between pregnant queens fed Diet A or B. However, pre-weaning diet had little effect on faecal bacterial composition in weaned kittens. In contrast, post-weaning diet altered bacterial population profiles in the kittens. Increased (P<0.05) abundance of Firmicutes (77% vs 52% of total reads) and Actinobacteria (0.8% vs 0.2% of total reads), and decreased (P<0.05) abundance of Fusobacteria (1.6% vs 18.4% of total reads) were observed for kittens fed the Diet A compared to those fed Diet B post-weaning. Feeding Diet B pre-weaning increased (P<0.05) the expression levels of INRS, LEPT, PAI-1 and tended to increase GLUT1, while the expression levels of IRS-1 in blood increased in kittens fed Diet A pre-weaning. Post-weaning diet had no effect on expression levels of target genes. Correlations between the expression levels of genes involved in glucose and insulin pathways and faecal Bacteriodetes and Firmicutes phyla were identified. The reasons for why post-weaning diet affects microbial populations and not gene expression levels are of interest.
PMCID: PMC3842929  PMID: 24312255
13.  Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation 
The Journal of Clinical Investigation  2013;123(12):5351-5360.
Ionizing radiation (IR) and germline mutations in the retinoblastoma tumor suppressor gene (RB1) are the strongest risk factors for developing osteosarcoma. Recapitulating the human predisposition, we found that Rb1+/– mice exhibited accelerated development of IR-induced osteosarcoma, with a latency of 39 weeks. Initial exposure of osteoblasts to carcinogenic doses of IR in vitro and in vivo induced RB1-dependent senescence and the expression of a panel of proteins known as senescence-associated secretory phenotype (SASP), dominated by IL-6. RB1 expression closely correlated with that of the SASP cassette in human osteosarcomas, and low expression of both RB1 and the SASP genes was associated with poor prognosis. In vivo, IL-6 was required for IR-induced senescence, which elicited NKT cell infiltration and a host inflammatory response. Mice lacking IL-6 or NKT cells had accelerated development of IR-induced osteosarcomas. These data elucidate an important link between senescence, which is a cell-autonomous tumor suppressor response, and the activation of host-dependent cancer immunosurveillance. Our findings indicate that overcoming the immune response to senescence is a rate-limiting step in the formation of IR-induced osteosarcoma.
PMCID: PMC3859382  PMID: 24231354
14.  Occupational exposures and mortality from cardiovascular disease among women textile workers in Shanghai, China 
Exposure to textile fiber dusts, like particulate air pollution, may be associated with cardiovascular disease (CVD) mortality. Bacterial endotoxin, a potent inflammagen found in cotton dust, may be a specific risk factor.
Female textile workers (N=267,400) in Shanghai, China were followed for CVD mortality (1989-2000). Factory exposures were approximated by sector classifications based on materials and processes. Quantitative endotoxin and cotton dust measures were available for a subcohort (n=3,188). Cox proportional hazards modeling was used to estimate hazard ratios (HR) and 95% confidence interval (CI).
Slightly elevated mortality risk for the cotton sector was seen for ischemic stroke (HR=1.12, 95%CI 0.97-1.31) and hemorrhagic stroke (HR=1.12, 95%CI 1.02-1.23). Similar hemorrhagic stroke mortality risk was observed in high dust sectors (HR=1.12, 95%CI 1.02-1.24). No association was observed for ischemic heart disease.
Exposures in textile factories may have contributed to CVD mortality among this cohort. The specific components of these exposures that may be harmful are not clear and should be further investigated.
PMCID: PMC3515077  PMID: 22968969
Cardiovascular diseases; stroke; mortality; endotoxin; China
15.  Relationship of Liver Disease Stage and Antiviral Therapy With Liver-Related Events and Death in Adults Coinfected With HIV/HCV 
JAMA : the journal of the American Medical Association  2012;308(4):10.1001/jama.2012.7844.
Human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV) disease progression; however, the effect of liver disease stage and antiviral therapy on the risk of clinical outcomes is incompletely understood.
To determine the incidence of end-stage liver disease (ESLD), hepatocellular carcinoma (HCC), or death according to baseline hepatic fibrosis and antiviral treatment for HIV/HCV coinfected individuals.
Design, Setting, and Participants
Prospective cohort of 638 coinfected adults (80% black, 66% men) receiving care at the Johns Hopkins HIV clinic and receiving a liver biopsy and who were prospectively monitored for clinical events between July 1993 and August 2011 (median follow-up, 5.82 years; interquartile range, 3.42–8.85 years). Histological specimens were scored for hepatic fibrosis stage according to the METAVIR scoring system.
Main Outcome Measure
Incidence of composite outcome of ESLD, HCC, or death.
Patients experienced a graded increased risk in incidence of clinical outcomes based on baseline hepatic fibrosis stage (classification range, F0-F4): F0, 23.63 (95% CI, 16.80–33.24); F1, 36.33 (95% CI, 28.03–47.10); F2, 53.40 (95% CI, 33.65–84.76); F3, 56.14 (95% CI, 31.09–101.38); and F4, 79.43 (95% CI, 55.86–112.95) per 1000 person-years (P<.001). In multivariable negative binomial regression, fibrosis stages F2 through F4 and antiretroviral therapy were independently associated with composite ESLD, HCC, or all-cause mortality after adjustment for demographic characteristics, injection drug use, and CD4 cell count. Compared with F0, the incidence rate ratio (RR) for F2 was 2.31 (95% CI, 1.23–4.34; P=.009); F3, 3.18 (95% CI, 1.47–6.88; P=.003); and F4, 3.57 (95% CI, 2.06–6.19; P<.001). Human immunodeficiency virus treatment was associated with fewer clinical events (incidence RR, 0.27; 95% CI, 0.19–0.38; P<.001). For the 226 patients who underwent HCV treatment, the incidence of clinical events did not significantly differ between treatment nonresponders and untreated patients (incidence RR, 1.27; 95% CI, 0.86–1.86; P=.23). In contrast, no events were observed in the 51 patients with sustained virologic response (n= 36) and relapse (n= 15), including 19 with significant fibrosis.
In this cohort of patients with HIV/HCV coinfection, hepatic fibrosis stage was independently associated with a composite outcome of ESLD, HCC, or death.
PMCID: PMC3807214  PMID: 22820790
16.  Interleukin-28b: A Key Piece of the Hepatitis C Virus Recovery Puzzle 
Gastroenterology  2010;138(4):1240-1243.
PMCID: PMC3805379  PMID: 20184973
17.  Aqueous sample in an EPR cavity: sensitivity considerations 
The radial mode matching (RMM) method has been used to calculate accurately the microwave field distribution of the TE0 1 1 mode in a spherical EPR cavity containing a linear aqueous sample, in order to understand in detail the factors affecting sensitivity in EPR measurements at X band. Specific details of the experiment were included in the calculations, such as the cavity geometry, the presence of a quartz dewar, the size of the aqueous sample, and the sample’s dielectric properties. From the field distribution, several key physical parameters were calculated, including cavity Q, filling factor, mean microwave magnetic field at the sample, and cavity efficiency parameter Λ. The dependence of EPR signal intensity on sample diameter for a cylindrical aqueous sample was calculated and measured experimentally for non-saturated and half-saturated samples. The optimal aqueous sample diameter was determined for both cases. The impact of sample temperature, conductivity, and cavity Q on sensitivity of EPR is discussed.
PMCID: PMC3804294  PMID: 14987608
EPR; Water; Sensitivity; Cavity; Field distribution
18.  Limited Uptake of Hepatitis C Treatment among Injection Drug Users 
Journal of community health  2008;33(3):126-133.
We characterized HCV treatment knowledge, experience and barriers in a cohort of community-based injection drug users (IDUs) in Baltimore, MD. In 2005, a questionnaire on HCV treatment knowledge, experience and barriers was administered to HCV-infected IDUs. Self-reported treatment was confirmed from medical records. Of 597 participants, 71% were male, 95% African-American, 31% HIV co-infected and 94% were infected with HCV genotype 1; 70% were aware that treatment was available, but only 22% understood that HCV could be cured. Of 418 who had heard of treatment, 86 (21%) reported an evaluation by a provider that included a discussion of treatment of whom 30 refused treatment, 20 deferred and 36 reported initiating treatment (6% overall). The most common reasons for refusal were related to treatment-related perceptions and a low perceived need of treatment. Compared to those who had discussed treatment with their provider, those who had not were more likely to be injecting drugs, less likely to have health insurance, and less knowledgeable about treatment. Low HCV treatment effectiveness was observed in this IDU population. Comprehensive integrated care strategies that incorporate education, case-management and peer support are needed to improve care and treatment of HCV-infected IDUs.
PMCID: PMC3800027  PMID: 18165889
Hepatitis C virus; Injection drug use; Antiviral therapy; Health care access
19.  Hepatic steatosis associated with increased central body fat by dual-energy X-ray absorptiometry and uncontrolled HIV in HIV/hepatitis C co-infected persons 
AIDS (London, England)  2010;24(6):811-817.
To evaluate the relationship between regional body composition and liver disease (fibrosis or steatosis) in HIV/HCV co-infected individuals.
Whole body dual-energy X-ray absorptiometry (DXA) was performed in 173 HIV/HCV co-infected persons within 12 months of a liver biopsy. Significant fibrosis was defined as a METAVIR stage greater than 1. Steatosis was graded as: 0, none; 1, steatosis involving less than 5% of hepatocytes; 2, 5–29%; 3, 30–60%; 4 greater than 60%, and was defined as more than 0. Poisson regression with robust variance was used to estimate prevalence ratios of the outcome measures.
The population was 62% male and 84% black with a median body mass index of 25.2 kg/m2 (interquartile range 22.5, 29.3 kg/m2). No differences in regional body fat or fat distribution were observed in 42 patients with significant fibrosis compared to others with less fibrosis. However, the 77 individuals (45%) with steatosis had greater central fat than those without steatosis [prevalence ratio 1.04 per kg trunk fat; 95% confidence interval (CI) 1.04, 1.11], after adjusting for hepatic fibrosis (prevalence ratio 1.77; 95% CI 1.29, 2.42), uncontrolled HIV replication (viral load >400 copies/ml) (prevalence ratio 1.57; 95% CI 1.12, 2.22), age, sex, race and diabetes mellitus.
In HIV/HCV co-infected individuals, measures of regional body fat or fat distribution were not associated with hepatic fibrosis. In contrast, increased central adiposity by DXA, as well as concomitant fibrosis and uncontrolled HIV, were associated with hepatic steatosis. The extent to which weight loss and effective antiretroviral therapy can reduce the risk of steatosis deserves further investigation.
PMCID: PMC3800050  PMID: 20186036
body composition; hepatic fibrosis; hepatitis C; HIV; steatosis
20.  Development of a high-resolution fat and CSF-suppressed optic nerve DTI protocol at 3T: application in multiple sclerosis 
Functional Neurology  2013;28(2):93-100.
Clinical trials of neuroprotective interventions in multiple sclerosis require outcome measures that reflect the disease pathology. Measures of neuroaxonal integrity in the anterior visual pathways are of particular interest in this context, however imaging of the optic nerve is technically challenging. We therefore developed a 3T optic nerve diffusion tensor imaging protocol incorporating fat and cerebrospinal fluid suppression and without parallel imaging. The sequence used a scheme with six diffusion-weighted directions, b=600smm−2 plus one b≈0 (b0) and 40 repetitions, averaged offline, giving an overall scan time of 30 minutes. A coronal oblique orientation was used with voxel size 1.17mm×1.17mm×4mm, We validated the sequence in 10 MS patients with a history of optic neuritis and 11 healthy controls: mean fractional anisotropy was reduced in the patients: 0.346(±0.159) versus 0.528(±0.123), p<0.001; radial diffusivity was increased: 0.940(±0.370)×10−6mm2s−1 compared to 0.670(± 0.221)×10−6mm2s−1 (p<0.01). No significant differences were seen for mean diffusivity or mean axial diffusivity.
PMCID: PMC3812733  PMID: 24125558
3T; diffusion; multiple sclerosis; optic nerve; optic neuritis
21.  Human Immunodeficiency Virus and Liver Disease Forum 2010: Conference Proceedings 
Hepatology (Baltimore, Md.)  2011;54(6):2245-2253.
Liver disease continues to represent a critical mediator of morbidity and mortality in those with human immunodeficiency virus (HIV) infection. The frequent presence and overlap of concomitant injurious processes, including hepatitis C virus and hepatitis B virus infections, hepatoxicity associated with antiretroviral therapeutic agents, alcohol, and other toxins, in the setting of immunosuppression lead to rapid fibrotic progression and early development of end-stage liver disease. This conference summary describes the proceedings of a state-of-the-art gathering of international experts designed to highlight the status of current research in epidemiology, natural history, pathogenesis, and treatment of HIV and liver disease.
PMCID: PMC3795389  PMID: 21898501
22.  A variant upstream of IFNL3 (IL28B) creating a novel interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus 
Nature genetics  2013;45(2):164-171.
Chronic infection with hepatitis C virus (HCV) is a common cause of liver cirrhosis and cancer. We performed RNA-sequencing in primary human hepatocytes activated with synthetic dsRNA to mimic HCV infection. Upstream of IFNL3 (IL28B) on chromosome 19q13.13, we discovered a novel, transiently induced region that harbors dinucleotide variant ss469415590 (TT/ΔG), which is in high linkage disequilibrium with rs12979860, a genetic marker strongly associated with HCV clearance. ss469415590-ΔG is a frame-shift variant that creates a novel primate-specific gene, designated interferon lambda 4 (IFNL4), which encodes a protein of moderate similarity with IFNL3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, whereas it provides comparable information in Europeans and Asians. Transient over-expression of IFNL4 in a hepatoma cell line induced STAT1/STAT2 phosphorylation and expression of interferon-stimulated genes. Our findings provide new insights into the genetic regulation of HCV clearance and its clinical management.
PMCID: PMC3793390  PMID: 23291588
23.  Vitamin D deficiency and its relation to bone mineral density and liver fibrosis in HIV–HCV coinfection 
Antiviral therapy  2012;18(2):237-242.
Fractures and cirrhosis are major causes of morbidity and mortality among HIV–HCV-coinfected individuals. It is not known whether vitamin D deficiency is associated with these outcomes.
Between 2005 and 2007, 116 HIV–HCV- coinfected individuals underwent dual-energy X-ray absorptiometry within 1 year of a liver biopsy. 25-Hydroxy-vitamin D (25OHD) and parathyroid hormone were measured from archived samples. Low bone mineral density (BMD) was defined as BMD≥2 standard deviations lower than age-, sex- and race-matched controls (Z-score ≤−2.0) at the total hip, femoral neck or lumbar spine. Histological fibrosis staging was assessed according to the METAVIR system (0 [no fibrosis] to 4 [cirrhosis]).
The cohort was 87% African-American and 63% male. The median age (IQR) was 49.9 years (46.5–53.3). A total of 89% had a CD4+ T-cell count >200 cells/mm3 and 64% were receiving HAART. The median 25OHD was 19 ng/ ml (IQR 11.0–26.0). Hypovitaminosis D (25OHD≤15 ng/ml) was present in 41% and secondary hyperparathyroidism, defined by parathyroid hormone >65 pg/ml, was present in 24%. In total, 27% had low BMD (Z-score ≤−2) at the spine, femoral neck or total hip, and 39% had significant hepatic fibrosis (METAVIR≥2). In multivariate analysis, vitamin D deficiency was not associated with significant fibrosis or with BMD at any site.
Vitamin D deficiency was highly prevalent in this mostly African-American HIV–HCV-coinfected population, but was not related to BMD or liver disease severity. These data suggest that efforts to increase vitamin D levels in this population may not improve bone or liver outcomes.
PMCID: PMC3790468  PMID: 22910231
24.  Methylation of Arsenic by Recombinant Human Wild-Type Arsenic (+3 Oxidation State) Methyltransferase and its Methionine 287 Threonine (M287T) Polymorph: Role of Glutathione 
Toxicology and applied pharmacology  2012;264(1):121-130.
Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency of about 10% among populations worldwide. Here, we compared catalytic properties of recombinant human wild-type (wt) AS3MT and AS3MT/M287T in reaction mixtures containing S-adenosylmethionine, arsenite (iAsIII) or methylarsonous acid (MAsIII) as substrates and endogenous or synthetic reductants, including glutathione (GSH), a thioredoxin reductase (TR)/thioredoxin (Trx)/NADPH reducing system, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP). With either TR/Trx/NADPH or TCEP, wtAS3MT or AS3MT/M287T catalyzed conversion of iAsIII to MAsIII, methylarsonic acid (MAsV), dimethylarsinous acid (DMAsIII), and dimethylarsinic acid (DMAsV); MAsIII was converted to DMAsIII and DMAsV. Although neither enzyme required GSH to support methylation of iAsIII or MAsIII, addition of 1 mM GSH decreased Km and increased Vmax estimates for either substrate in reaction mixtures containing TR/Trx/NADPH. Without GSH, Vmax and Km values were significantly lower for AS3MT/M287T than for wtAS3MT. In the presence of 1 mM GSH, significantly more DMAsIII was produced from iAsIII in reactions catalyzed by the M287T variant than in wtAS3MT-catalyzed reactions. Thus, 1 mM GSH modulates AS3MT activity, increasing both methylation rates and yield of DMAsIII. AS3MT genotype exemplified by differences in regulation of wtAS3MT and AS3MT/M287T-catalyzed reactions by GSH may contribute to differences in the phenotype for arsenic methylation and, ultimately, to differences in the disease susceptibility in individuals chronically exposed to inorganic arsenic.
PMCID: PMC3439589  PMID: 22868225
Arsenic methylation; AS3MT polymorphism; thioredoxin reductase; glutathione
25.  Allosteric Communication in Dictyostelium Myosin II 
Myosin’s affinities for nucleotides and actin are reciprocal. Actin-binding substantially reduces the affinity of ATP for myosin, but the effect of actin on myosin’s ADP affinity is quite variable among myosin isoforms, serving as the principal mechanism for tuning the actomyosin system to specific physiological purposes. To understand the structural basis of this variable relationship between actin and ADP binding, we studied several constructs of the catalytic domain of Dictyostelium myosin II, varying their length (from the N-terminal origin) and cysteine content. The constructs varied considerably in their actin-activated ATPase activity and in the effect of actin on ADP affinity. Actin had no significant effect on ADP affinity for a single-cysteine catalytic domain construct, a double-cysteine construct partially restored the actin-dependence of ADP binding, and restoration of all native Cys restored it further, but full restoration of function (similar to that of skeletal muscle myosin II) was obtained only by adding all native Cys and an artificial lever arm extension. Pyrene-actin fluorescence confirmed these effects on ADP binding to actomyosin. We conclude that myosin’s Cys content and lever arm both allosterically modulate the reciprocal affinities of myosin for ADP and actin, a key determinant of the biological functions of myosin isoforms.
PMCID: PMC3600821  PMID: 22752265
actin; cysteine; lever arm; ADP; fluorescence

Results 1-25 (328)