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1.  Epidemiological, clinical, outcome and antibiotic susceptibility differences between PVL positive and PVL negative Staphylococcus aureus infections in Western Australia: a case control study 
Panton Valentine Leukocidin (PVL) has been associated with invasive Staphylococcus aureus soft tissue and pneumonic infections.
From September 2007 to January 2009 at Royal Perth Hospital we tested for the PVL gene in S. aureus isolates from an invasive site, a suspected PVL-related soft tissue infection and all MRSA isolates. We could access medical records for 141 PVL positive (PVL + ve) infections and compared these to a control group comprised of 148 PVL negative (PVL-ve) infections.
In the PVL + ve group 62 isolates were MRSA (48 were ST93-MRSA-IV) and 79 isolates were methicillin-sensitive S. aureus, and in the PVL-ve group 56 were MRSA (50 were WA-MRSA strains) and 92 were methicillin-sensitive S. aureus. We found the presence of PVL to be significantly associated with younger age, aboriginality, intravenous drug use, community acquisition, shorter length of hospital stay and lower mortality at 1 year. Overall PVL + ve infections more often required surgical intervention (73.0% versus 44.6%, p < 0.001) and were less often polymicrobial (8.5% versus 41.2%, p < 0.001). PVL + ve isolates were more often susceptible to clindamycin (87.9% versus 73.0%, p = 0.002).
This study demonstrates that PVL + ve infections are associated with a distinct clinical picture, predominantly pyogenic skin and soft tissue infections often requiring surgery, disproportionately affecting patients who are younger, indigenous or with fewer health-care risk factors.
PMCID: PMC4297412  PMID: 25572896
Staphylococcus aureus; MRSA; Panton Valentine Leucocidin; PVL
2.  Specific sequences commonly found in the V3 domain of HIV-1 subtype C isolates affect the overall conformation of native Env and induce a neutralization-resistant phenotype independent of V1/V2 masking 
Virology  2013;448:363-374.
Primary HIV-1 isolates are relatively resistant to neutralization by antibodies commonly induced after infection or vaccination. This is generally attributed to masking of sensitive epitopes by the V1/V2 domain and/or glycans situated at various positions in Env. Here we identified a novel masking effect mediated by subtype C-specific V3 sequences that contributes to the V1/V2-independent and glycan-independent neutralization resistance of chimeric and primary Envs to antibodies directed against multiple neutralization domains. Positions at several conserved charged and hydrophobic sites in the V3 crown and stem were also shown to affect neutralization phenotype. These results indicated that substitutions typically present in subtype C and related V3 sequences influence the overall conformation of native Env in a way that occludes multiple neutralization targets located both within and outside of the V3 domain, and may reflect an alternative mechanism for neutralization resistance that is particularly active in subtype C and related isolates.
PMCID: PMC3913561  PMID: 24314667
HIV-1; Subtype C; V3 domain; Conformational effect; Epitope masking
3.  CD4-Mimetic Small Molecules Sensitize Human Immunodeficiency Virus to Vaccine-Elicited Antibodies 
Journal of Virology  2014;88(12):6542-6555.
Approaches to prevent human immunodeficiency virus (HIV-1) transmission are urgently needed. Difficulties in eliciting antibodies that bind conserved epitopes exposed on the unliganded conformation of the HIV-1 envelope glycoprotein (Env) trimer represent barriers to vaccine development. During HIV-1 entry, binding of the gp120 Env to the initial receptor, CD4, triggers conformational changes in Env that result in the formation and exposure of the highly conserved gp120 site for interaction with the coreceptors, CCR5 and CXCR4. The DMJ compounds (+)-DMJ-I-228 and (+)-DMJ-II-121 bind gp120 within the conserved Phe 43 cavity near the CD4-binding site, block CD4 binding, and inhibit HIV-1 infection. Here we show that the DMJ compounds sensitize primary HIV-1, including transmitted/founder viruses, to neutralization by monoclonal antibodies directed against CD4-induced (CD4i) epitopes and the V3 region, two gp120 elements involved in coreceptor binding. Importantly, the DMJ compounds rendered primary HIV-1 sensitive to neutralization by antisera elicited by immunization of rabbits with HIV-1 gp120 cores engineered to assume the CD4-bound state. Thus, small molecules like the DMJ compounds may be useful as microbicides to inhibit HIV-1 infection directly and to sensitize primary HIV-1 to neutralization by readily elicited antibodies.
IMPORTANCE Preventing HIV-1 transmission is a priority for global health. Eliciting antibodies that can neutralize many different strains of HIV-1 is difficult, creating problems for the development of a vaccine. We found that certain small-molecule compounds can sensitize HIV-1 to particular antibodies. These antibodies can be elicited in rabbits. These results suggest an approach to prevent HIV-1 sexual transmission in which a virus-sensitizing microbicide is combined with a vaccine.
PMCID: PMC4054336  PMID: 24696475
4.  Development of a High-Throughput Screening–Compatible Cell-Based Functional Assay to Identify Small Molecule Probes of the Galanin 3 Receptor (GalR3) 
The galanin 3 receptor (GalR3) belongs to the large G protein–coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening–compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.
PMCID: PMC3804082  PMID: 24116939
6.  Interaction with Cellular CD4 Exposes HIV-1 Envelope Epitopes Targeted by Antibody-Dependent Cell-Mediated Cytotoxicity 
Journal of Virology  2014;88(5):2633-2644.
Anti-HIV-1 envelope glycoprotein (Env) antibodies without broadly neutralizing activity correlated with protection in the RV144 clinical trial, stimulating interest in other protective mechanisms involving antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC). Env epitopes targeted by many antibodies effective at mediating ADCC are poorly exposed on the unliganded Env trimer. Here we investigated the mechanism of exposure of ADCC epitopes on Env and showed that binding of Env and CD4 within the same HIV-1-infected cell effectively exposes these epitopes. Env capacity to transit to the CD4-bound conformation is required for ADCC epitope exposure. Importantly, cell surface CD4 downregulation by Nef and Vpu accessory proteins and Vpu-mediated BST-2 antagonism modulate exposure of ADCC-mediating epitopes and reduce the susceptibility of infected cells to this effector function in vitro. Significantly, Env conformational changes induced by cell surface CD4 are conserved among Env from HIV-1 and HIV-2/SIVmac lineages. Altogether, our observations describe a highly conserved mechanism required to expose ADCC epitopes that might help explain the evolutionary advantage of downregulation of cell surface CD4 by the HIV-1 Vpu and Nef proteins.
IMPORTANCE HIV-1 envelope epitopes targeted by many antibodies effective at mediating antibody-dependent cell-mediated cytotoxicity (ADCC) are poorly exposed on the unliganded envelope trimer. Here we investigated the mechanism of exposure of these epitopes and found that envelope interaction with the HIV-1 CD4 receptor is required to expose some of these epitopes. Moreover, our results suggest that HIV-1 CD4 downregulation might help avoid the killing of HIV-1-infected cells by this immune mechanism.
PMCID: PMC3958102  PMID: 24352444
7.  A novel high-temperature furnace for combined in situ synchrotron X-ray diffraction and infrared thermal imaging to investigate the effects of thermal gradients upon the structure of ceramic materials 
Journal of Synchrotron Radiation  2014;21(Pt 5):1134-1139.
A combined X-ray diffraction and thermal imaging technique is described to investigate the effect of thermal gradients on high-temperature composite materials.
A new technique combining in situ X-ray diffraction using synchrotron radiation and infrared thermal imaging is reported. The technique enables the application, generation and measurement of significant thermal gradients, and furthermore allows the direct spatial correlation of thermal and crystallographic measurements. The design and implementation of a novel furnace enabling the simultaneous thermal and X-ray measurements is described. The technique is expected to have wide applicability in material science and engineering; here it has been applied to the study of solid oxide fuel cells at high temperature.
PMCID: PMC4161039  PMID: 25178003
X-ray diffraction; solid oxide fuel cell; infrared imaging; thermal imaging; stress analysis; composite materials
8.  Genotype List String: a grammar for describing HLA and KIR genotyping results in a text string 
Tissue antigens  2013;82(2):106-112.
Knowledge of an individual’s HLA genotype is essential for modern medical genetics, and is crucial for hematopoietic stem cell and solid-organ transplantation. However, the high levels of polymorphism known for the HLA genes make it difficult to generate an HLA genotype that unambiguously identifies the alleles that are present at a given HLA locus in an individual. For the last twenty years, the histocompatibility and immunogenetics community has recorded this HLA genotyping ambiguity using allele codes developed by the National Marrow Donor Program (NMDP). While these allele codes may have been effective for recording an HLA genotyping result when initially developed, their use today results in increased ambiguity in an HLA genotype, and they are no longer suitable in the era of rapid allele discovery and ultra-high allele polymorphism. Here, we present a text string format capable of fully representing HLA genotyping results. This Genotype List (GL) String format is an extension of a proposed standard for reporting KIR genotype data that can be applied to any genetic data that employs a standard nomenclature for identifying variants. The GL String format employs a hierarchical set of operators to describe the relationships between alleles, lists of possible alleles, phased alleles, genotypes, lists of possible genotypes, and multilocus unphased genotypes, without losing typing information or increasing typing ambiguity. When used in concert with appropriate tools to create, exchange, and parse these strings, we anticipate that GL Strings will replace NMDP allele codes for reporting HLA genotypes.
PMCID: PMC3715123  PMID: 23849068
Genotype; GL String; HLA; KIR
9.  Activated MEK cooperates with Ink4a/Arf loss or Akt activation to induce gliomas in vivo 
Oncogene  2010;30(11):1341-1350.
The RAS/RAF mitogen activated protein kinase pathway (MAPK) is highly active in many tumor types including the majority of high-grade gliomas and expression of activated RAS or RAF in neural progenitor cells combined with either AKT activation or Ink4a/Arf loss leads to the development of high-grade gliomas in vivo. This strongly suggests that this pathway is necessary for glioma formation and maintenance. To further define the role of this pathway in the development of high-grade gliomas, we used the established RCAS/TVA glioma mouse model to test the ability of activated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK), a RAF effector, to induce tumors in vivo in the context of activated AKT or Ink4a/Arf loss. While expression of activated MEK alone in neural progenitor cells is not sufficient for tumorigenesis, the combination of activated MEK and AKT or MEK with Ink4a/Arf loss is transforming. The data reveal that activation of the classical RAS/MAPK pathway, which is mediated through MEK, leads to the development of high-grade gliomas in vivo and suggest that MEK may be a relevant target for glioma therapy. To test this, we treated both mouse and human glioma cells with the MEK inhibitor PD0325901. While this treatment induced apoptosis in a significant percentage of the cells, the effect was enhanced by combined treatment with the PI3K/mTOR inhibitor NVP-BEZ235. Our results demonstrate that combined inhibition of MEK and PI3K/mTOR is a rational strategy for the treatment of high-grade gliomas and may be an effective adjuvant therapy for this disease.
PMCID: PMC4109988  PMID: 21057530
MEK; AKT; Ink4a/Arf; glioma; mouse model; targeted therapy
10.  Activated BRAF induces gliomas in mice when combined with Ink4a/Arf loss or Akt activation 
Oncogene  2009;29(3):335-344.
Mutations in receptor tyrosine kinase (RTK) growth factor receptors (EGFR, PDGFR, MET and ERBB2), which result in downstream activation of the RAS/RAF/MEK/ERK mitogen-activated protein kinase (MAPK) pathway and PI(3)K/AKT pathway, are found in almost all high grade gliomas and MAPK signaling is necessary for continued glioma maintenance. In addition, BRAF is mutated in the majority of low grade gliomas and its expression and activity is significantly increased in the majority of high grade gliomas. While the importance of RTKs and RAS signaling in glioma development has been demonstrated, the role of BRAF has yet to be characterized. We evaluated the effect of activated BRAF in glioma formation using the retroviral RCAS/TVA system to transfer genes encoding activated forms of BRAF, KRas, Akt and Cre to Nestin expressing neural progenitor cells in Ink4a/Arf lox/lox mice in vivo. While expression of activated BRAF alone is not sufficient for tumorigenesis, the combination of activated BRAF and Akt or BRAF with Ink4a/Arf loss is transforming. Interestingly, activated BRAF generates gliomas with characteristics similar to activated KRas in the context of Akt but not Ink4a/Arf loss. Our studies demonstrate a role for BRAF activation and signaling in glioma development and as potential target for glioma therapy.
PMCID: PMC4109991  PMID: 19855433
Glioma; BRAF; KRas; AKT; Ink4a; Arf
12.  Falling up the stairs: the equivalent of ‘bashing it with a bible’ for an ACL ganglion cyst of the knee 
BMJ Case Reports  2012;2012:bcr0120125591.
Intra-articular anterior cruciate ligament (ACL) cysts are rare, the pathogenesis remains unknown, with trauma often implicated. Often asymptomatic, incidental MRI findings, 11% produce symptoms such as pain, locking or instability. Treatment of intra-articular ganglia differs from the traditional ‘bash it with a bible’ mantra for ganglia elsewhere with surgical debridement generally indicated for symptomatic cases. This case report describes a 43-year-old male car mechanic who presented with a symptomatic ACL cyst diagnosed on MRI. While waiting for surgery the patient fell up his stairs at home, causing forced hyperflexion of his knee. After an initial sharp pain, within 24 h the patient experienced complete resolution of symptoms. Postfall MRI showed no evidence of the initial lesion, leading to our conclusion that for this patient, a fall up the stairs was the equivalent of ‘bashing it with a bible’ for an ACL ganglion cyst of the knee.
PMCID: PMC3316796  PMID: 22605799
13.  Lassa Fever in Post-Conflict Sierra Leone 
Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the world's highest incidence of the disease.
Methodology/Principal Findings
Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leone's thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects.
Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF.
Author Summary
Lassa fever (LF) is a major public health threat in West Africa. After the violent civil conflict in Sierra Leone (1991 to 2002) ended, the LF research program at Kenema Government Hospital (KGH) was reestablished. Higher CFRs in LF patients were observed compared to studies conducted prior to the civil conflict. The criteria used for defining the stages of LF and differences in sensitivity of the assays used likely account for these differences. LF may be more widespread in Sierra Leone than recognized previously. Peak presentation of LF cases occurs in the dry season, which is consistent with previous studies. Our studies also confirmed reports conducted prior to the civil conflict that indicate that infants, children, young adults, and pregnant women are disproportionately impacted by LF. High fatality rates were observed among both ribavirin treated and untreated patients, which underscores then need for better LF treatments.
PMCID: PMC3961205  PMID: 24651047
14.  The Role of Fear of Movement in Subacute Whiplash-Associated Disorders Grades I and II 
Pain  2012;154(3):393-401.
Fear and avoidance of activity may play a role in fostering disability in whiplash-associated disorders (WAD). This study examined the role of fear following WAD and assessed the effectiveness of three treatments targeting fear. People still symptomatic from WAD Grade I–II injuries approximately 3 months previously (n = 191) completed questionnaires (e.g., Neck Disability Index [NDI]) and were randomized to one of the treatments: (1) informational booklet (IB) describing WAD and the importance of resuming activities; (2) IB + didactic discussions (DD) with clinicians reinforcing the booklet; and (3) IB + imaginal and direct exposure desensitization (ET) to feared activities. DD and ET participants received three 2-hour treatment sessions. Absolute improvements in NDI were in predicted direction (ET = 14.7, DD = 11.9, IB = 9.9). ETs reported significantly less post-treatment pain severity, compared to the IB (M = 1.5 vs. 2.3, p < 0.001, d = 0.6) and DD (M = 1.5 vs. 2.0, p = 0.039, d = 0.6) groups. Reduction in fear was the most important predictor of improvement in NDI (β = 0.30, p < 0.001), followed by reductions in pain (β = 0.20, p = 0.003) and depression (β = 0.18, p = 0.004). The mediational analysis confirmed that fear reduction significantly mediated the effect of treatment group on outcome. Results highlight the importance of fear in individuals with subacute WAD, and suggest the importance of addressing fear, via exposure therapy and/or educational interventions, to improve function.
PMCID: PMC3582794  PMID: 23318127
15.  Topological Analysis of HIV-1 Glycoproteins Expressed In Situ on Virus Surfaces Reveals Tighter Packing but Greater Conformational Flexibility than for Soluble gp120 
Journal of Virology  2013;87(16):9233-9249.
In natural infection, antibodies interact with HIV-1 primarily through nonfunctional forms of envelope glycoproteins (Env), including uncleaved (UNC) gp160 and gp41 stumps. These antigens are important to fully characterize, as they may be decoys that promote nonneutralizing responses and may also be targets for nonneutralizing effector responses. In this study, we compared the antigenic properties of Env expressed in situ on pseudovirion virus-like particle (VLP) surfaces and soluble gp120 using harmonized enzyme-linked immunosorbent assays (ELISAs) and a panel of 51 monoclonal antibodies (MAbs). Only 32 of 46 soluble gp120-reactive MAbs recognized the primary UNC gp160 antigen of VLPs. Indeed, many epitopes were poorly exposed (C1, V2, C1-C4, C4, C4-V3, CD4 induced [CD4i], and PGT group 3) or obscured (C2, C5, and C1-C5) on VLPs. In further studies, VLP Env exhibited an increased degree of inter-MAb competition, the epicenter of which was the base of the V3 loop, where PGT, 2G12, V3, and CD4 binding site specificities competed. UNC gp160 also underwent more drastic soluble CD4 (sCD4)-induced conformational changes than soluble gp120, exposing CD4i, C1-C4, and V2 epitopes. A greater propensity of UNC gp160 to undergo conformational changes was also suggested by the induction of CD4i MAb binding to VLPs by a V3 MAb as well as by soluble CD4. The same effect was not observed for soluble gp120. Taken together, our data suggest that membrane-expressed UNC gp160 exists in a less “triggered” conformational state than soluble gp120 and that MAb binding to UNC gp160 tends to have greater conformational consequences.
PMCID: PMC3754051  PMID: 23740975
16.  Report from the Immunogenomic Data Analysis Working Group (IDAWG) 16th International HLA and Immunogenetics Workshop (IHIW) Project: Immunogenomic Data-Management Methods 
The goal of the IDAWG is to facilitate the consistent analysis of HLA and KIR data, and the sharing of those data among the immunogenomic and larger genomic communities. However, the data-management approaches currently applied by immunogenomic researchers are not widely discussed or reported in the literature, and the effect of different approaches on data-analyses is not known.
With ASHI’s support, the IDAWG developed a forty-five question survey on HLA and KIR data-generation, data-management, and data-analysis practices. Survey questions detailed the loci genotyped, typing systems used, nomenclature versions reported, computer operating systems and software used to manage and transmit data, the approaches applied to resolve HLA ambiguity, and the methods used for basic population-level analyses. Respondents were invited to demonstrate their HLA ambiguity resolution approaches in simulated data sets.By May 2012, 156 respondents from 35 nations had completed the survey . These survey respondents represent a broad sampling of the Immunogenomic community; 52% were European, 30% North American, 10% Asian, 4% South American, and 4% from the Pacific.
The project will continue in conjunction with the 17th Workshop, with the aim of developing community data-sharing standards, ambiguity resolution documentation formats, single-task data-Management tools, and, novel data-analysis methods and applications. While additional project details and plans for the 17th IHIW will be forthcoming, we welcome the input and participation in these projects from the histocompatibility and immunogenetics community.
PMCID: PMC3789600  PMID: 23280068
Meta-analysis; Statistics; HLA; Genetics
17.  Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing 
Nature genetics  2013;45(3):299-303.
While genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS.
PMCID: PMC3901305  PMID: 23396133
18.  Increased plasma levels of soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) in women by moderate exercise and increased plasma levels of VEGF in overweight/obese women 
The incidence of breast cancer is increasing worldwide, and this seems to be related to an increase in lifestyle risk factors, including physical inactivity, and overweight/obesity. We previously reported that exercise induced a circulating angiostatic phenotype characterized by increased sFlt-1 and endostatin and decreased unbound-VEGF in men. However, there is no data on women. The present study determines the following: 1) whether moderate exercise increased sFlt-1 and endostatin and decreased unbound-VEGF in the circulation of adult female volunteers; 2) whether overweight/obese women have a higher plasma level of unbound-VEGF than lean women. 72 African American and Caucasian adult women volunteers aged from 18–44 were enrolled into the exercise study. All the participants walked on a treadmill for 30 minutes at a moderate intensity (55–59% heart rate reserve), and oxygen consumption (VO2) was quantified by utilizing a metabolic cart. We had the blood samples before and immediately after exercise from 63 participants. ELISA assays (R&D Systems) showed that plasma levels of sFlt-1 were 67.8±3.7 pg/ml immediately after exercise (30 minutes), significantly higher than basal levels, 54.5±3.3 pg/ml, before exercise (P < 0.01; n=63). There was no significant difference in the % increase of sFlt-1 levels after exercise between African American and Caucasian (P=0.533) or between lean and overweight/obese women (P=0.892). There was no significant difference in plasma levels of unbound VEGF (35.28±5.47 vs. 35.23±4.96 pg/ml; P=0.99) or endostatin (111.12±5.48 vs. 115.45±7.15 ng/ml; P=0.63) before and after exercise. Basal plasma levels of unbound-VEGF in overweight/obese women were 52.26±9.6 pg/ml, significantly higher than basal levels of unbound-VEGF in lean women, 27.34±4.99 pg/ml (P < 0.05). The results support our hypothesis that exercise-induced plasma levels of sFlt-1 could be an important clinical biomarker to explore the mechanisms of exercise training in reducing breast cancer progression and that VEGF is an important biomarker in obesity and obesity-related cancer progression.
PMCID: PMC3449013  PMID: 22609636
Exercise; Young adult women; Overweight/obese; sFlt-1; Endostatin; VEGF
19.  A high throughput Chromatin ImmunoPrecipitation approach reveals principles of dynamic gene regulation in mammals 
Molecular cell  2012;47(5):10.1016/j.molcel.2012.07.030.
Understanding the principles governing mammalian gene regulation has been hampered by the difficulty in measuring in-vivo binding dynamics of large numbers of transcription factors (TF) to DNA. Here, we develop a high-throughput Chromatin ImmunoPrecipitation (HT-ChIP) method to systematically map protein-DNA interactions. HT-ChIP was applied to define the dynamics of DNA binding by 25 TFs and 4 chromatin marks at 4 time-points following pathogen stimulus of dendritic cells. Analyzing over 180,000 TF-DNA interactions we find that TFs vary substantially in their temporal binding landscapes. This data suggests a model for transcription regulation whereby TF networks are hierarchically organized into cell differentiation factors, factors that bind targets prior to stimulus to prime them for induction, and factors that regulate specific gene programs. Overlaying HT-ChIP data on gene expression dynamics shows that many TF-DNA interactions are established prior to the stimuli, predominantly at immediate-early genes, and identified specific TF ensembles that coordinately regulate gene-induction.
PMCID: PMC3873101  PMID: 22940246
20.  Variation in Hospital Costs, Payments, and Profitabilty for Cardiac Valve Replacement Surgery 
Health Services Research  2011;46(6 Pt 1):1928-1945.
Examine the variation for Medicare and privately insured patients in hospital costs, payments, and contribution margins and their association with characteristics of the patients, hospitals, and hospital markets.
Data Sources
Administrative records for 1,858 patients undergoing cardiac valve replacement surgery were obtained from 37 hospitals in 7 states for 2008.
Study Design
Bivariate and multivariate statistical analyses of costs, payments, and profitability (contribution margin) for Medicare and privately insured patients, adjusting for patient, hospital, and market characteristics.
Data Collection
Integrated Health Care Association, Aspen Health Metrics, American Hospital Association Annual Survey of Hospitals.
Principal Findings
Cardiac valve replacement surgery is an expensive but profitable procedure, with average cost and contribution margin per case of U.S.$38,667 and U.S.$21,967, respectively. Average costs per case for Medicare patients are 16.1 percent higher in concentrated than in competitive local markets after adjusting for patient comorbidities, complications, and other relevant factors (p<.01). Payments per case were 33.2 percent (p<.01) lower from Medicare than from private insurers. The average contribution margin earned by hospitals from Medicare was U.S.$30,986 lower than the margin earned from private insurers (p<.01), after adjusting for patient, hospital, and market characteristics.
Hospitals charge significantly higher prices and earn significantly higher contribution margins from private insurers than from Medicare for patients undergoing cardiac valve replacement.
PMCID: PMC3393019  PMID: 21762141
Hospital costs and prices; Medicare cost shifting; health care reform; prospective payment; insurance
21.  Compression of Structured High-Throughput Sequencing Data 
PLoS ONE  2013;8(11):e79871.
Large biological datasets are being produced at a rapid pace and create substantial storage challenges, particularly in the domain of high-throughput sequencing (HTS). Most approaches currently used to store HTS data are either unable to quickly adapt to the requirements of new sequencing or analysis methods (because they do not support schema evolution), or fail to provide state of the art compression of the datasets. We have devised new approaches to store HTS data that support seamless data schema evolution and compress datasets substantially better than existing approaches. Building on these new approaches, we discuss and demonstrate how a multi-tier data organization can dramatically reduce the storage, computational and network burden of collecting, analyzing, and archiving large sequencing datasets. For instance, we show that spliced RNA-Seq alignments can be stored in less than 4% the size of a BAM file with perfect data fidelity. Compared to the previous compression state of the art, these methods reduce dataset size more than 40% when storing exome, gene expression or DNA methylation datasets. The approaches have been integrated in a comprehensive suite of software tools ( that support common analyses for a range of high-throughput sequencing assays.
PMCID: PMC3832420  PMID: 24260313
22.  Alcohol decreases RhoA activity through a Nitric Oxide (NO)/Cyclic GMP(cGMP)/Protein Kinase G (PKG)-dependent pathway in the airway epithelium 
Alcoholism, clinical and experimental research  2011;35(7):10.1111/j.1530-0277.2011.01463.x.
Alcohol has been shown to have a number of harmful effects on the lung, including increasing the risk of pneumonia and bronchitis. How alcohol increases the risk of these diseases is poorly defined. RhoA is a small guanosine triphosphate (GTP)ase that plays an integral role in many basic functions of airway epithelial cells. It is not known how alcohol affects RhoA activity in the airway epithelium. We hypothesized that brief alcohol exposure modulates RhoA activity in the airway epithelium through a nitric oxide (NO)/Cyclic GMP (cGMP)/Protein Kinase G (PKG) dependent pathway.
Primary airway epithelial cells were cultured and exposed to ethanol at various concentrations and times. The cell layers were harvested and RhoA activity was measured.
Alcohol induced a time- and concentration-dependent decrease in RhoA activity in airway epithelial cells. We were able to block this decrease in activity using Nω-Nitro-l-arginine methyl ester hydrochloride (L-NAME), a nitric oxide synthase (NOS) inhibitor. Likewise, we were able to demonstrate the same decrease in RhoA activation using 0.1µM sodium nitroprusside (SNP), an NO donor. To determine the role of cGMP/PKG, we pretreated the cells with a cGMP antagonist analogue, Rp-8Br-cGMPS. This blocked the decrease in RhoA activity caused by alcohol, suggesting that alcohol exerts its effect on RhoA activity through cGMP/PKG.
Alcohol decreases airway epithelial RhoA activity through an NO/cGMP/PKG- dependent pathway. RhoA activity controls many aspects of basic cellular function, including cell morphology, tight junction formation, and cell cycle progression and gene regulation. Dysregulation of RhoA activity can potentially have several consequences, including dysregulation of inflammation. This may partially explain how alcohol increases the risk of pneumonia and bronchitis.
PMCID: PMC3821532  PMID: 21410486
ethanol; nitric oxide; PKG; cGMP; airway epithelium
23.  Heterogeneity in Neutralization Sensitivities of Viruses Comprising the Simian Immunodeficiency Virus SIVsmE660 Isolate and Vaccine Challenge Stock 
Journal of Virology  2013;87(10):5477-5492.
The sooty mangabey-derived simian immunodeficiency virus (SIV) strain E660 (SIVsmE660) is a genetically heterogeneous, pathogenic isolate that is commonly used as a vaccine challenge strain in the nonhuman primate (NHP) model of human immunodeficiency virus type 1 (HIV-1) infection. Though it is often employed to assess antibody-based vaccine strategies, its sensitivity to antibody-mediated neutralization has not been well characterized. Here, we utilize single-genome sequencing and infectivity assays to analyze the neutralization sensitivity of the uncloned SIVsmE660 isolate, individual viruses comprising the isolate, and transmitted/founder (T/F) viruses arising from low-dose mucosal inoculation of macaques with the isolate. We found that the SIVsmE660 isolate overall was highly sensitive to neutralization by SIV-infected macaque plasma samples (50% inhibitory concentration [IC50] < 10−5) and monoclonal antibodies targeting V3 (IC50 < 0.01 μg/ml), CD4-induced (IC50 < 0.1 μg/ml), CD4 binding site (IC50 ∼ 1 μg/ml), and V4 (IC50, ∼5 μg/ml) epitopes. In comparison, SIVmac251 and SIVmac239 were highly resistant to neutralization by these same antibodies. Differences in neutralization sensitivity between SIVsmE660 and SIVmac251/239 were not dependent on the cell type in which virus was produced or tested. These findings indicate that in comparison to SIVmac251/239 and primary HIV-1 viruses, SIVsmE660 generally exhibits substantially less masking of antigenically conserved Env epitopes. Interestingly, we identified a minor population of viruses (∼10%) in both the SIVsmE660 isolate and T/F viruses arising from it that were substantially more resistant (>1,000-fold) to antibody neutralization and another fraction (∼20%) that was intermediate in neutralization resistance. These findings may explain the variable natural history and variable protection afforded by heterologous Env-based vaccines in rhesus macaques challenged by high-dose versus low-dose SIVsmE660 inoculation regimens.
PMCID: PMC3648171  PMID: 23468494
24.  Akt Signaling Accelerates Tumor Recurrence Following Ras Inhibition in the Context of Ink4a/Arf Loss 
Genes & Cancer  2013;4(11-12):476-485.
Aberrant activation of the RAS signaling pathway contributes to nearly all human cancers, including gliomas. To determine the dependence of high-grade gliomas on this signaling pathway, we developed a doxycycline-regulated KRas glioma mouse model. Using this model we previously demonstrated that inhibition of KRas expression in gliomas induced by activated KRas and Akt results in complete tumor regression. We have also shown that, in the context of Ink4a/Arf loss, abrogation of KRas signaling is sufficient to decrease tumor burden but resistance ensues. In this study, we sought to determine the effect of activated Akt signaling in combination with activated KRas and loss of Ink4a/Arf on the growth and recurrence of brain tumors following suppression of KRas expression. We observed significant tumor formation in Ink4a/Arflox/lox mice injected with retroviruses containing tetracycline responsive element (TRE)-KRas, Tet-off, Akt, and Cre. Abrogation of KRas signaling resulted in significant tumor regression; however, resistance developed after a relatively short latency. Tumor recurrence occurred more rapidly and the tumors were more aggressive in the presence of activated Akt signaling compared with loss of Ink4a/Arf alone suggesting that this pathway contributes to tumor progression in this context.
PMCID: PMC3877666  PMID: 24386508
Akt; Ras; Ink4a/Arf; glioma; mouse
25.  Metabolic and transcriptional response to a high-fat diet in Drosophila melanogaster★ 
Molecular Metabolism  2013;3(1):42-54.
Obesity has dramatically increased in prevalence, making it essential to understand its accompanying metabolic changes. Modeling diet-induced obesity in Drosophila melanogaster (fruit flies), we elucidated transcriptional and metabolic changes in w1118 flies on a high-fat diet (HFD). Mass spectrometry-based metabolomics revealed altered fatty acid, amino acid, and carbohydrate metabolism with HFD. Microarray analysis uncovered transcriptional changes in nitrogen metabolism, including CG9510, homolog of human argininosuccinate lyase (ASL). CG9510 knockdown in flies phenocopied traits observed with HFD, namely increased triglyceride levels and decreased cold tolerance. Restoration of CG9510 expression ameliorated observed negative consequences of HFD. Metabolomic analysis of CG9510 knockdown flies confirmed functional similarity to ASL, regulating the balance of carbon and nitrogen metabolism. In summary, we found that HFD suppresses CG9510 expression, a gene required for proper triglyceride storage and stress tolerance. These results draw an important link between regulation of amino acid metabolism and the response to diet-induced obesity.
PMCID: PMC3929909  PMID: 24567903
AcCoA, acetyl-coenzyme A; arm-GAL4, armadillo-GAL4; ASL, argininosuccinate lyase; BCAA, branch chain amino acid; CAFE, capillary feeder; da-GAL4, daughterless-Gal4; EASE, Expression Analysis Systematic Explorer (DAVID analysis); FAME, fatty acid methyl ester; Fdr, false discovery rate; GC/MS, gas chromatography/mass spectrometry; HFD, high-fat Diet; MeOH, methanol; PCR, polymerase chain reaction; RT-PCR, reverse-transcriptase PCR; TBDMS, tert-butyldimethylsilyl; TCA, tricarboxylic acid; TG, triglyceride; TMS, trimethylsilyl; VDRC, Vienna Drosophila RNAi Center; w1118, white-1118; Argininosuccinate lyase; Lifespan; Metabolism; Obesity; Triglyceride

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