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1.  Sequential Myosin Phosphorylation Activates Tarantula Thick Filament via a Disorder-Order Transition 
Molecular bioSystems  2015;11(8):2167-2179.
Phosphorylation of myosin regulatory light chain (RLC) N-terminal extension (NTE) activates myosin in thick filaments. RLC phosphorylation plays a primary regulatory role in smooth muscle and a secondary (modulatory) role in striated muscle, which is regulated by Ca2+ via TnC/TM on the thin filament. Tarantula striated muscle exhibits both regulatory systems: one switches on/off contraction through thin filament regulation, and another through PKC constitutively Ser35 phosphorylated swaying free heads in the thick filaments that produces quick force on twitches regulated from 0 to 50% and modulation is accomplished recruiting additional force-potentiating free and blocked heads via Ca2+4-CaM-MLCK Ser45 phosphorylation. We have used microsecond molecular dynamics (MD) simulations of tarantula RLC NTE to understand the structural basis for phosphorylation-based regulation in tarantula thick filament activation. Trajectories analysis revealed that an inter-domain salt bridges network (R39/E58,E61) facilitates formation of a stable helix-coil-helix (HCH) motif made up by helices P and A in the unphosphorylated NTE of both myosin heads. Phosphorylation of blocked head on Ser45 does not induce any substantial structural change. However, phosphorylation of free head on Ser35 disrupts this salt bridge network and induces a partial extension of helix P along RLC helix A. While not directly participating in the HCH inter-domain folding, phosphorylation of Ser35 unlocks compact structure and allows the NTE to spontaneously undergo coil-helix transitions. The modest structural change induced by subsequent Ser45 diphosphorylation monophosphorylated Ser35 free head, facilitates full helix P extension into a single structurally stable α-helix through a network of intra-domain salt bridges (pS35/R38,R39,R42). We conclude that tarantula thick filament activation is controlled by sequential Ser35-Ser45 phosphorylation via a conserved disorder-to-order transition.
doi:10.1039/c5mb00162e
PMCID: PMC4503533  PMID: 26038232
2.  Purification of Arsenic (+3 Oxidation State) Methyltransferase from Rat Liver Cytosol 
Demonstrating the enzymatic basis of arsenic methylation is critical to further studies of the pathway for the conversion of inorganic arsenic into a variety of methylated metabolites. This protocol describes a procedure for the purification of an arsenic methyltransferase from rat liver cytosol. Purification of this enzyme and subsequent cloning of its gene has permitted studies of enzyme structure and function and has lead to the identification of orthologous genes in genomes of organisms ranging in complexity from sea urchins to humans. These proteins are referred to as arsenic (+3 oxidation state) methyltransferases.
doi:10.1002/0471140856.tx0434s42
PMCID: PMC4500041  PMID: 20949431
arsenic; methylation; methyltransferases; protein purification; chromatofocusing; affinity chromatography
3.  High-Resolution Functional MRI at 3 T: 3D/2D Echo-Planar Imaging with Optimized Physiological Noise Correction 
Magnetic Resonance in Medicine  2012;69(6):1657-1664.
High-resolution functional MRI (fMRI) offers unique possibilities for studying human functional neuroanatomy. Although high-resolution fMRI has proven its potential at 7 T, most fMRI studies are still performed at rather low spatial resolution at 3 T. We optimized and compared single-shot two-dimensional echo-planar imaging (EPI) and multishot three-dimensional EPI high-resolution fMRI protocols. We extended image-based physiological noise correction from two-dimensional EPI to multishot three-dimensional EPI. The functional sensitivity of both acquisition schemes was assessed in a visual fMRI experiment. The physiological noise correction increased the sensitivity significantly, can be easily applied, and requires simple recordings of pulse and respiration only. The combination of three-dimensional EPI with physiological noise correction provides exceptional sensitivity for 1.5 mm high-resolution fMRI at 3 T, increasing the temporal signal-to-noise ratio by more than 25% compared to two-dimensional EPI. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.
doi:10.1002/mrm.24398
PMCID: PMC4495253  PMID: 22821858
high-resolution; functional MRI; echo-planar imaging; three-dimensional; two-dimensional; physiological noise
4.  Time-resolved FRET Reveals the Structural Mechanism of SERCA-PLB Regulation 
We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca2+ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA-PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca2+]. In all cases, two structural states of the SERCA-PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA-PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca2+ completely relieved inhibition but did not induce a detectable change in SERCA-PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca2+ and PLB phosphorylation relieve SERCA-PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA-PLB complex, not by dissociation of that complex.
doi:10.1016/j.bbrc.2014.04.166
PMCID: PMC4054823  PMID: 24813991
phospholamban; SERCA; phosphorylation; FRET
7.  Clearance of Hepatitis C infection is associated with early appearance of broad neutralizing antibody responses 
Hepatology (Baltimore, Md.)  2014;59(6):2140-2151.
Background & Aims
The contribution of humoral immune responses to spontaneous control of Hepatitis C virus (HCV) infection remains unclear. We assessed nAb responses during acute HCV infection to determine whether infection outcome is associated with the neutralizing antibody (nAb) response, specifically its timing or breadth (neutralization of multiple genotype-matched variants).
Methods
A representative genotype 1 HCV pseudoparticle (HCVpp) library, consisting of 19 genetically-distinct genotype 1 HCVpp that comprise the natural variability of genotype 1 E1E2 sequences, was used to assess anti-genotype 1 nAb responses during acute infection in at-risk persons followed prospectively. Neutralization of individual library HCVpp by the last viremic plasma sample obtained before clearance was compared to either one-year post-initial viremia or clearance time-matched specimens obtained from subjects developing persistent infection.
Results
In persistently infected persons nAb responses were delayed then progressively broadened whereas in persons who controlled viremia broader responses were detected early and contracted after clearance of viremia. Surprisingly, the breadth of anti-genotype 1 nAb responses was not dependent upon subjects’ infection genotype. Also, individual library HCVpp neutralization sensitivity was not associated with any known E2 sequence determinants. Interestingly, two single nucleotide polymorphisms in the HLA-DQ locus were associated with nAb breadth.
Conclusions
Taken together, these data demonstrate that control of HCV infection is associated with more rapid development of a broad nAb response, independent of the infection viral genotype, providing further evidence for the role of nAb in controlling HCV infection and the potential benefit of generating broad anti-HCV nAb responses by vaccination.
doi:10.1002/hep.27013
PMCID: PMC4043926  PMID: 24425349
neutralizing antibody; HCV infection outcome; acute infection
8.  3D Bioprinting of Carboxymethylated-Periodate Oxidized Nanocellulose Constructs for Wound Dressing Applications 
BioMed Research International  2015;2015:925757.
Nanocellulose has a variety of advantages, which make the material most suitable for use in biomedical devices such as wound dressings. The material is strong, allows for production of transparent films, provides a moist wound healing environment, and can form elastic gels with bioresponsive characteristics. In this study, we explore the application of nanocellulose as a bioink for modifying film surfaces by a bioprinting process. Two different nanocelluloses were used, prepared with TEMPO mediated oxidation and a combination of carboxymethylation and periodate oxidation. The combination of carboxymethylation and periodate oxidation produced a homogeneous material with short nanofibrils, having widths <20 nm and lengths <200 nm. The small dimensions of the nanofibrils reduced the viscosity of the nanocellulose, thus yielding a material with good rheological properties for use as a bioink. The nanocellulose bioink was thus used for printing 3D porous structures, which is exemplified in this study. We also demonstrated that both nanocelluloses did not support bacterial growth, which is an interesting property of these novel materials.
doi:10.1155/2015/925757
PMCID: PMC4452270  PMID: 26090461
9.  Direct Acting Antiviral Agents and the Path to Interferon Independence 
Summary
Chronic infection with hepatitis C virus (HCV) is a major global health problem—there are approximately 120–130 million chronic infections worldwide. Since discovery of HCV 24 y ago, there has been a relentless effort to develop successful antiviral therapies. Studies of interferon- □(IFN)-based therapies have helped define treatment parameters, and these treatment strategies have cured a substantial percentage of patients. However, IFN must be injected, and there are problems with tolerability, adherence, and incomplete response in a large percentage of patients. New drug candidates designed to target the virus or the host have recently been introduced at an unprecedented pace. In phase I–III studies, these agents have exceeded expectations and achieved rates of response previously not thought possible. We are therefore entering a new era of therapy for HCV infection and interferon independence.
doi:10.1016/j.cgh.2013.06.024
PMCID: PMC4049632  PMID: 23872239
DAA; NS3/4A protease inhibitor; nucleoside/nucleotide; non-nucleoside
10.  A Novel Fast Helical 4D-CT Acquisition Technique to Generate Low-Noise Sorting Artifact–Free Images at User-Selected Breathing Phases 
Purpose
To develop a novel 4-dimensional computed tomography (4D-CT) technique that exploits standard fast helical acquisition, a simultaneous breathing surrogate measurement, deformable image registration, and a breathing motion model to remove sorting artifacts.
Methods and Materials
Ten patients were imaged under free-breathing conditions 25 successive times in alternating directions with a 64-slice CT scanner using a low-dose fast helical protocol. An abdominal bellows was used as a breathing surrogate. Deformable registration was used to register the first image (defined as the reference image) to the subsequent 24 segmented images. Voxel-specific motion model parameters were determined using a breathing motion model. The tissue locations predicted by the motion model in the 25 images were compared against the deformably registered tissue locations, allowing a model prediction error to be evaluated. A low-noise image was created by averaging the 25 images deformed to the first image geometry, reducing statistical image noise by a factor of 5. The motion model was used to deform the low-noise reference image to any user-selected breathing phase. A voxel-specific correction was applied to correct the Hounsfield units for lung parenchyma density as a function of lung air filling.
Results
Images produced using the model at user-selected breathing phases did not suffer from sorting artifacts common to conventional 4D-CT protocols. The mean prediction error across all patients between the breathing motion model predictions and the measured lung tissue positions was determined to be 1.19 ± 0.37 mm.
Conclusions
The proposed technique can be used as a clinical 4D-CT technique. It is robust in the presence of irregular breathing and allows the entire imaging dose to contribute to the resulting image quality, providing sorting artifact–free images at a patient dose similar to or less than current 4D-CT techniques.
doi:10.1016/j.ijrobp.2014.01.016
PMCID: PMC4097042  PMID: 24613815
11.  Electron Paramagnetic Resonance: A High-Resolution Tool for Muscle Physiology 
Skeletal muscle function can be altered by changes in protein structure and motion. Electron paramagnetic resonance (EPR) paired with site-directed spin labeling has been used to study the relationships between (a) muscle force and myosin structure and (b) muscle relaxation and Ca-ATPase motion and structure.
PMCID: PMC4404760  PMID: 11210444
skeletal muscle; aging; myosin; Ca-ATPase; protein structure; aggregation
12.  Functional, structural, and chemical changes in myosin associated with hydrogen peroxide treatment of skeletal muscle fibers 
To understand the molecular mechanism of oxidation-induced inhibition of muscle contractility, we have studied the effects of hydrogen peroxide on permeabilized rabbit psoas muscle fibers, focusing on changes in myosin purified from these fibers. Oxidation by 5 mM peroxide decreased fiber contractility (isometric force and shortening velocity) without significant changes in the enzymatic activity of myofibrils and isolated myosin. The inhibitory effects were reversed by treating fibers with dithiothreitol. Oxidation by 50 mM peroxide had a more pronounced and irreversible inhibitory effect on fiber contractility and also affected enzymatic activity of myofibrils, myosin, and actomyosin. Peroxide treatment also affected regulation of contractility, resulting in fiber activation in the absence of calcium. Electron paramagnetic resonance of spinlabeled myosin in muscle fibers showed that oxidation increased the fraction of myosin heads in the strong-binding structural state under relaxing conditions (low calcium) but had no effect under activating conditions (high calcium). This change in the distribution of structural states of myosin provides a plausible explanation for the observed changes in both contractile and regulatory functions. Mass spectroscopy analysis showed that 50 mM but not 5 mM peroxide induced oxidative modifications in both isoforms of the essential light chains and in the heavy chain of myosin subfragment 1 by targeting multiple methionine residues. We conclude that 1) inhibition of muscle fiber contractility via oxidation of myosin occurs at high but not low concentrations of peroxide and 2) the inhibitory effects of oxidation suggest a critical and previously unknown role of methionines in myosin function.
doi:10.1152/ajpcell.00232.2007
PMCID: PMC4397896  PMID: 18003749
oxidation; proteomics
13.  Behavioral and neurochemical effects of repeated MDMA administration during late adolescence in the rat 
Adolescents and young adults disproportionately abuse 3,4-methylenedioxymethamphetamine (MDMA; ‘Ecstasy’); however, since most MDMA research has concentrated on adults, the effects of MDMA on the developing brain remain obscure. Therefore, we evaluated place conditioning to MDMA (or saline) during late adolescence and assessed anxiety-like behavior and monoamine levels during abstinence. Rats were conditioned to associate 5 or 10 mg/kg MDMA or saline with contextual cues over 4 twice-daily sessions. Five days after conditioning, anxiety-like behavior was examined with the open field test and brain tissue was collected to assess serotonin (5-hydroxytryptamine, 5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the dorsal raphe, amygdala, and hippocampus by high-pressure liquid chromatography (HPLC). In a separate group of rats, anxiety-like and avoidant behaviors were measured using the light–dark box test under similar experimental conditions. MDMA conditioning caused a place aversion at 10, but not at 5, mg/kg, as well as increased anxiety-like behavior in the open field and avoidant behavior in light–dark box test at the same dose. Additionally, 10 mg/kg MDMA decreased 5-HT in the dorsal raphe, increased 5-HT and 5-HIAA in the amygdala, and did not alter levels in the hippocampus. Overall, we show that repeated high (10 mg/kg), but not low (5 mg/kg), dose MDMA during late adolescence in rats increases anxiety-like and avoidant behaviors, accompanied by region-specific alterations in 5-HT levels during abstinence. These results suggest that MDMA causes a region-specific dysregulation of the serotonin system during adolescence that may contribute to maladaptive behavior.
doi:10.1016/j.pnpbp.2013.09.021
PMCID: PMC4348097  PMID: 24121061
Amygdala; Anxiety; Conditioned place preference; Dorsal raphe; MDMA; Serotonin
14.  Fibrosis progression in human immunodeficiency virus/hepatitis C virus coinfected adults: Prospective analysis of 435 liver biopsy pairs 
Hepatology (Baltimore, Md.)  2014;59(3):767-775.
Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection is associated with progressive liver disease. However, the rate of progression is variable and the ability to differentiate patients with stable versus progressive HCV disease is limited. The objective of this study was to assess the incidence of and risk factors for fibrosis progression in a prospective cohort of coinfected patients. Overall, 435 liver biopsy pairs from 282 non-cirrhotic patients were analyzed. Biopsies were scored according to the METAVIR system by a single pathologist blind to biopsy sequence. Fibrosis progression was defined as an increase of at least one METAVIR fibrosis stage between paired biopsies. The majority of patients were African American (84.8%), male (67.7%) and infected with HCV genotype 1 (93.4%). On initial biopsy, no or minimal fibrosis was identified in 243 patients (86%). The median interval between biopsies was 2.5 years. Fibrosis progression was observed in 97 of 282 (34%) patients and 149 of 435 (34%) biopsy pairs. After adjustment, greater body mass index (adjusted odds ratio [aOR]: 1.04 per 1 unit increase), diabetes (aOR: 1.56) and hepatic steatosis (aOR: 1.78) at time of initial biopsy were associated with subsequent fibrosis progression. Between biopsies, elevated serum aspartate and alanine aminotransferase (AST, ALT) (aOR AST: 3.34, ALT: 2.18 for >25% values >100 U/l vs. < 25% values >100 U/l) were strongly associated with fibrosis progression.
Conclusion
Fibrosis progression is common among HIV/HCV coinfected patients; these data suggest that progression can be rapid. Persistent elevations in serum transaminase levels may serve as important non-invasive markers to identify subsets of patients who are more likely to progress and thus warrant closer monitoring and consideration of HCV treatment.
doi:10.1002/hep.26741
PMCID: PMC3943751  PMID: 24436062
cirrhosis; antiretroviral therapy; hepatic steatosis; AIDS; hepatitis C virus treatment
15.  Anthropometric and Hormonal Risk Factors for Male Breast Cancer: Male Breast Cancer Pooling Project Results 
Background
The etiology of male breast cancer is poorly understood, partly because of its relative rarity. Although genetic factors are involved, less is known regarding the role of anthropometric and hormonally related risk factors.
Methods
In the Male Breast Cancer Pooling Project, a consortium of 11 case–control and 10 cohort investigations involving 2405 case patients (n = 1190 from case–control and n = 1215 from cohort studies) and 52013 control subjects, individual participant data were harmonized and pooled. Unconditional logistic regression generated study design–specific (case–control/cohort) odds ratios (ORs) and 95% confidence intervals (CIs), with exposure estimates combined using fixed effects meta-analysis. All statistical tests were two-sided.
Results
Risk was statistically significantly associated with weight (highest/lowest tertile: OR = 1.36; 95% CI = 1.18 to 1.57), height (OR = 1.18; 95% CI = 1.01 to 1.38), and body mass index (BMI; OR = 1.30; 95% CI = 1.12 to 1.51), with evidence that recent rather than distant BMI was the strongest predictor. Klinefelter syndrome (OR = 24.7; 95% CI = 8.94 to 68.4) and gynecomastia (OR = 9.78; 95% CI = 7.52 to 12.7) were also statistically significantly associated with risk, relations that were independent of BMI. Diabetes also emerged as an independent risk factor (OR = 1.19; 95% CI = 1.04 to 1.37). There were also suggestive relations with cryptorchidism (OR = 2.18; 95% CI = 0.96 to 4.94) and orchitis (OR = 1.43; 95% CI = 1.02 to 1.99). Although age at onset of puberty and histories of infertility were unrelated to risk, never having had children was statistically significantly related (OR = 1.29; 95% CI = 1.01 to 1.66). Among individuals diagnosed at older ages, a history of fractures was statistically significantly related (OR = 1.41; 95% CI = 1.07 to 1.86).
Conclusions
Consistent findings across case–control and cohort investigations, complemented by pooled analyses, indicated important roles for anthropometric and hormonal risk factors in the etiology of male breast cancer. Further investigation should focus on potential roles of endogenous hormones.
doi:10.1093/jnci/djt465
PMCID: PMC3975166  PMID: 24552677
16.  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.
doi:10.1038/gene.2014.11
PMCID: PMC4308959  PMID: 24622687
Hepatitis C; Chronic Infection; Admixture; African Ancestry
17.  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.
doi:10.3109/15412555.2013.800854
PMCID: PMC4302731  PMID: 23862666
chronic viral infections; injection drug users; HIV; Obstructive Lung Disease
18.  Fructan synthesis, accumulation, and polymer traits. I. Festulolium chromosome substitution lines 
The fructans found as storage carbohydrates in temperate forage grasses have a physiological role in regrowth and stress tolerance. They are also important for the nutritional value of fresh and preserved livestock feeds, and are potentially useful as feedstocks for biorefining. Seasonal variation in fructan content and the capacity for de novo fructan synthesis have been examined in a Festulolium monosomic substitution line family to investigate variation in the polymers produced by grasses in the ryegrass-fescue complex. There were significant differences between ryegrass and fescue. Fescue had low polymeric fructan content and a high oligomer/polymer ratio; synthesis of polymers longer than degree of polymerization 6 (DP6) from oligomers was slow. However, extension of polymer length from DP10/DP20 upward appeared to occur relatively freely, and, unlike ryegrass, fescue had a relatively even spread of polymer chain lengths above DP20. This included the presence of some very large polymers. Additionally fescue retained high concentrations of fructan, both polymeric and oligomeric, during conditions of low source/high sink demand. There were indications that major genes involved in the control of some of these traits might be located on fescue chromosome 3 opening the possibility to develop grasses optimized for specific applications.
doi:10.3389/fpls.2015.00486
PMCID: PMC4495318  PMID: 26217346
fescue (Festuca); genetic control; genetic variation; polymer chain length; ryegrass (Lolium); water-soluble carbohydrate
19.  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.
doi:10.1002/hep.26638
PMCID: PMC3849330  PMID: 23904401
HIV; HCV; HBV; HCC; Injury; Pathogenesis; Treatment; Epidemiology
20.  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.
doi:10.1249/JES.0000000000000004
PMCID: PMC3934098  PMID: 24188980
myosin; actin; muscle; Reactive Oxygen Species (ROS); methionine; aging
21.  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.
doi:10.1093/cid/cit602
PMCID: PMC3840403  PMID: 24051866
hepatitis C virus; ELISA; Africa
22.  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.
doi:10.1155/2014/405323
PMCID: PMC4269307  PMID: 25544913
23.  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.
doi:10.1002/0471140856.tx0431s42
PMCID: PMC4240622  PMID: 25419261
24.  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.
doi:10.1371/journal.pone.0112518
PMCID: PMC4237368  PMID: 25409186
25.  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.
doi:10.1371/journal.pcbi.1003934
PMCID: PMC4230741  PMID: 25393308

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