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1.  Voriconazole Metabolism, Toxicity, and the Effect of Cytochrome P450 2C19 Genotype 
The Journal of Infectious Diseases  2014;209(12):1941-1948.
Background. Prospective evaluation of the antifungal drug, voriconazole, is needed to determine whether drug toxicity correlates with CYP2C19 genotype or serum concentrations of voriconazole or its metabolites.
Methods. We conducted a prospective study of 95 patients to determine voriconazole toxicity and its relationship to genotype and serum levels of voriconazole and its two metabolites. Efficacy was not evaluated because, in most cases, the drug was given for empirical or prophylactic therapy.
Results. Hallucinations occurred in 16 patients (16.8%), visual changes in 17 (17.9%), photosensitivity in 10 (10.5%), and hepatotoxicity in 6 (6.3%). There was no correlation between photosensitivity or hepatotoxicity and levels of voriconazole or metabolites. Patients with hallucinations had higher average voriconazole levels (4.5 vs 2.5 μg/mL) but with extensive overlap. The recommended oral dose of 200 mg did not provide consistently detectable serum voriconazole levels in adults. CYP2C19 and CYP2C9 genotypes had a minor influence over levels, though the 4 patients homozygous for the 2C19*2 genotype had higher average levels for voriconazole (4.3 vs 2.5 μg/mL) and lower N-oxide levels (1.6 vs 2.5 μg/mL).
Conclusions. CYP2C19 and 2C9 genotypes were not major determinants of voriconazole metabolism. No toxic serum level of voriconazole or its metabolites could be identified.
doi:10.1093/infdis/jiu017
PMCID: PMC4038142  PMID: 24403552
voriconazole; metabolites; CYP2C19; toxicity
2.  Inhibiting the Mammalian Target of Rapamycin Blocks the Development of Experimental Cerebral Malaria 
mBio  2015;6(3):e00725-15.
ABSTRACT
Malaria is an infectious disease caused by parasites of several Plasmodium spp. Cerebral malaria (CM) is a common form of severe malaria resulting in nearly 700,000 deaths each year in Africa alone. At present, there is no adjunctive therapy for CM. Although the mechanisms underlying the pathogenesis of CM are incompletely understood, it is likely that both intrinsic features of the parasite and the human host’s immune response contribute to disease. The kinase mammalian target of rapamycin (mTOR) is a central regulator of immune responses, and drugs that inhibit the mTOR pathway have been shown to be antiparasitic. In a mouse model of CM, experimental CM (ECM), we show that the mTOR inhibitor rapamycin protects against ECM when administered within the first 4 days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood-brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Rapamycin induced marked transcriptional changes in the brains of infected mice, and analysis of transcription profiles predicted that rapamycin blocked leukocyte trafficking to and proliferation in the brain. Remarkably, animals were protected against ECM even though rapamycin treatment significantly increased the inflammatory response induced by infection in both the brain and spleen. These results open a new avenue for the development of highly selective adjunctive therapies for CM by targeting pathways that regulate host and parasite metabolism.
IMPORTANCE
Malaria is a highly prevalent infectious disease caused by parasites of several Plasmodium spp. Malaria is usually uncomplicated and resolves with time; however, in about 1% of cases, almost exclusively among young children, malaria becomes severe and life threatening, resulting in nearly 700,000 deaths each year in Africa alone. Among the most severe complications of Plasmodium falciparum infection is cerebral malaria with a fatality rate of 15 to 20%, despite treatment with antimalarial drugs. Cerebral malaria takes a second toll on African children, leaving survivors at high risk of debilitating neurological defects. At present, we have no effective adjunctive therapies for cerebral malaria, and developing such therapies would have a large impact on saving young lives in Africa. Here we report results that open a new avenue for the development of highly selective adjunctive therapies for cerebral malaria by targeting pathways that regulate host and parasite metabolism.
doi:10.1128/mBio.00725-15
PMCID: PMC4453009  PMID: 26037126
3.  Assurance of neuroattenuation of a live vaccine against West Nile virus: A comprehensive study of neuropathogenesis after infection with chimeric WN/DEN4Δ30 vaccine in comparison to two parental viruses and a surrogate flavivirus reference vaccine 
Vaccine  2014;32(26):3187-3197.
The upsurge of West Nile virus (WNV) human infections in 2012 suggests that the US can expect periodic WNV outbreaks in the future. Availability of safe and effective vaccines against WNV in endemic areas, particularly for aging populations that are at high risk of West Nile neuroinvasive disease (WNND), could be beneficial. WN/DEN4Δ30 is a live, attenuated chimeric vaccine against WNV produced by replacement of the genes encoding the pre-membrane and envelope protein genes of the vaccine virus against dengue virus type 4 (DEN4Δ30) with corresponding sequences derived from a wild type WNV. Following intrathalamic inoculation of nonhuman primates (NHPs), a comprehensive neuropathogenesis study was performed and neurovirulence of WN/DEN4Δ30 vaccine candidate was compared to that of two parental viruses (i.e., WNV and DEN4Δ30), as well as to that of an attenuated flavivirus surrogate reference (i.e., yellow fever YF 17D). Clinical and virological data, as well as results of a semi-quantitative histopathological analysis, demonstrated that WN/DEN4Δ30 vaccine is highly attenuated for the central nervous system (CNS) of NHPs in comparison to a wild type WNV. Importantly, based on the virus replicative ability in the CNS of NHPs and the degree of induced histopathological changes, the level of neuroattenuation of WN/DEN4Δ30 vaccine was similar to that of YF 17D, and therefore within an acceptable range. In addition, we show that the DEN4Δ30 vaccine tested in this study also has a low neurovirulence profile. In summary, our results demonstrate a high level of neuroattenuation of two vaccine candidates, WN/DEN4Δ30 and DEN4Δ30. We also show here a remarkable sensitivity of our WNV-NY99 NHP model, as well as striking resemblance of the observed neuropathology to that seen in human WNND. These results support the use of this NHP model for translational studies of WNV neuropathogenesis and/or testing the effectiveness of vaccines and therapeutic approaches.
doi:10.1016/j.vaccine.2014.04.002
PMCID: PMC4100552  PMID: 24736001
West Nile Virus (WNV); live attenuated WNV vaccine; neuropathogenesis; neurovirulence; nonhuman primates
4.  Malaria-associated atypical memory B cells exhibit markedly reduced B cell receptor signaling and effector function 
eLife  null;4:e07218.
Protective antibodies in Plasmodium falciparum malaria are only acquired after years of repeated infections. Chronic malaria exposure is associated with a large increase in atypical memory B cells (MBCs) that resemble B cells expanded in a variety of persistent viral infections. Understanding the function of atypical MBCs and their relationship to classical MBCs will be critical to developing effective vaccines for malaria and other chronic infections. We show that VH gene repertoires and somatic hypermutation rates of atypical and classical MBCs are indistinguishable indicating a common developmental history. Atypical MBCs express an array of inhibitory receptors and B cell receptor (BCR) signaling is stunted in atypical MBCs resulting in impaired B cell responses including proliferation, cytokine production and antibody secretion. Thus, in response to chronic malaria exposure, atypical MBCs appear to differentiate from classical MBCs becoming refractory to BCR-mediated activation and potentially interfering with the acquisition of malaria immunity.
DOI: http://dx.doi.org/10.7554/eLife.07218.001
eLife digest
The human immune system works to protect individuals from harmful microbes, such as the parasites that cause malaria. One line of defense is to produce a large array of proteins called antibodies that specifically bind to microbes to mark them for destruction by the immune system. The immune system also produces long-lived memory B cells that are able to mount a quicker and more effective antibody response if the microbe enters the body again. This means that most people only become ill with a particular disease the first time they encounter the microbe that causes it.
However, malaria is unusual in that it can take many years of exposure to the parasite that causes it before an individual produces enough antibodies and memory B cells to be protected from the disease. There is also no vaccine that provides effective and long-lasting protection against malaria. Vaccinations rely on stimulating the body's natural defenses, and so understanding more about antibodies and memory B cells in relation to malaria may aid future efforts to develop a vaccine.
Researchers have discovered that many of the memory B cells that accumulate in people who have been exposed to the malaria parasite over long-periods of time are different from the normal memory B cells. But it was not clear what role these ‘atypical’ cells play in immunity to malaria. To address this question, Portugal et al. studied the genetics and activity of B cells collected from children and adults living in Mali who—by living in a region where malaria is common—had been repeatedly exposed to the parasite.
The experiments indicate that atypical and normal memory B cells both develop from the same precursor cells. However, the genes that are active in each cell type are different, resulting in the atypical cells being less able to respond to the parasite than the normal memory B cells. Portugal et al.'s findings suggest that the atypical cells develop from normal memory B cells during long-term exposure to malaria, which may delay the development of immunity to this disease.
Future challenges include understanding what drives the formation of the atypical memory B cells in malaria, and finding out why they are less active than the normal cells. This could aid the development of vaccines and/or therapies that restore their activity in patients.
DOI: http://dx.doi.org/10.7554/eLife.07218.002
doi:10.7554/eLife.07218
PMCID: PMC4444601  PMID: 25955968
atypical memory B cells; Plasmodium falciparum; malaria; human
5.  HIV Malaria Co-Infection Is Associated with Atypical Memory B Cell Expansion and a Reduced Antibody Response to a Broad Array of Plasmodium falciparum Antigens in Rwandan Adults 
PLoS ONE  2015;10(4):e0124412.
HIV infected individuals in malaria endemic areas experience more frequent and severe malaria episodes compared to non HIV infected. This clinical observation has been linked to a deficiency in antibody responses to Plasmodium falciparum antigens; however, prior studies have only focused on the antibody response to <0.5% of P. falciparum proteins. To obtain a broader and less-biased view of the effect of HIV on antibody responses to malaria we compared antibody profiles of HIV positive (HIV+) and negative (HIV-) Rwandan adults with symptomatic malaria using a microarray containing 824 P. falciparum proteins. We also investigated the cellular basis of the antibody response in the two groups by analyzing B and T cell subsets by flow cytometry. Although HIV malaria co-infected individuals generated antibodies to a large number of P. falciparum antigens, including potential vaccine candidates, the breadth and magnitude of their response was reduced compared to HIV- individuals. HIV malaria co-infection was also associated with a higher percentage of atypical memory B cells (MBC) (CD19+CD10-CD21-CD27-) compared to malaria infection alone. Among HIV+ individuals the CD4+ T cell count and HIV viral load only partially explained variability in the breadth of P. falciparum-specific antibody responses. Taken together, these data indicate that HIV malaria co-infection is associated with an expansion of atypical MBCs and a diminished antibody response to a diverse array of P. falciparum antigens, thus offering mechanistic insight into the higher risk of malaria in HIV+ individuals.
doi:10.1371/journal.pone.0124412
PMCID: PMC4415913  PMID: 25928218
6.  Gene network reconstruction reveals cell cycle and antiviral genes as major drivers of cervical cancer 
Nature communications  2013;4:1806.
Although human papillomavirus (HPV) was identified as an etiological factor in cervical cancer, the key human gene drivers of this disease remain unknown. Here we apply an unbiased approach integrating gene expression and chromosomal aberration data. In an independent group of patients, we reconstruct and validate a gene regulatory meta-network, and identify cell cycle and antiviral genes that constitute two major sub-networks up-regulated in tumour samples. These genes are located within the same regions as chromosomal amplifications, most frequently on 3q. We propose a model in which selected chromosomal gains drive activation of antiviral genes contributing to episomal virus elimination, which synergizes with cell cycle dysregulation. These findings may help to explain the paradox of episomal HPV decline in women with invasive cancer who were previously unable to clear the virus.
doi:10.1038/ncomms2693
PMCID: PMC4237593  PMID: 23651994
7.  Network features suggest new hepatocellular carcinoma treatment strategies 
BMC Systems Biology  2014;8:88.
Background
Resistance to therapy remains a major cause of the failure of cancer treatment. A major challenge in cancer therapy is to design treatment strategies that circumvent the higher-level homeostatic functions of the robust cellular network that occurs in resistant cells. There is a lack of understanding of mechanisms responsible for the development of cancer and the basis of therapy-resistance mechanisms. Cellular signaling networks have an underlying architecture guided by universal principles. A robust system, such as cancer, has the fundamental ability to survive toxic anticancer drug treatments or a stressful environment mainly due to its mechanisms of redundancy. Consequently, inhibition of a single component/pathway would probably not constitute a successful cancer therapy.
Results
We developed a computational method to study the mechanisms of redundancy and to predict communications among the various pathways based on network theory, using data from gene expression profiles of hepatocellular carcinoma (HCC) of patients with poor and better prognosis cancers. Our results clearly indicate that immune system pathways tightly regulate most cancer pathways, and when those pathways are targeted by drugs, the network connectivity is dramatically changed. We examined the main HCC targeted treatments that are currently being evaluated in clinical trials. One prediction of our study is that Sorafenib combined with immune system treatments will be a more effective combination strategy than Sorafenib combined with any other targeted drugs.
Conclusions
We developed a computational framework to analyze gene expression data from HCC tumors with varying degrees of responsiveness and non-tumor samples, based on both Gene and Pathway Co-expression Networks. Our hypothesis is that redundancy is one of the major causes of drug resistance, and can be described as a function of the network structure and its properties. From this perspective, we believe that integration of the redundant variables could lead to the development of promising new methodologies to selectively identify and target the most significant resistance mechanisms of HCC. We describe three mechanisms of redundancy based on their levels of generalization and study the possible impact of those redundancy mechanisms on HCC treatments.
doi:10.1186/s12918-014-0088-0
PMCID: PMC4236726  PMID: 25070212
Co-expression network; Pathway network; Robustness; Redundancy; Resistance; Cancer therapy
8.  Exposure-Dependent Control of Malaria-Induced Inflammation in Children 
PLoS Pathogens  2014;10(4):e1004079.
In malaria-naïve individuals, Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, individuals in endemic areas who are repeatedly infected are often asymptomatic and have low levels of iRBCs, even young children. We hypothesized that febrile malaria alters the immune system such that P. falciparum re-exposure results in reduced production of pro-inflammatory cytokines/chemokines and enhanced anti-parasite effector responses compared to responses induced before malaria. To test this hypothesis we used a systems biology approach to analyze PBMCs sampled from healthy children before the six-month malaria season and the same children seven days after treatment of their first febrile malaria episode of the ensuing season. PBMCs were stimulated with iRBC in vitro and various immune parameters were measured. Before the malaria season, children's immune cells responded to iRBCs by producing pro-inflammatory mediators such as IL-1β, IL-6 and IL-8. Following malaria there was a marked shift in the response to iRBCs with the same children's immune cells producing lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines (IL-10, TGF-β). In addition, molecules involved in phagocytosis and activation of adaptive immunity were upregulated after malaria as compared to before. This shift was accompanied by an increase in P. falciparum-specific CD4+Foxp3− T cells that co-produce IL-10, IFN-γ and TNF; however, after the subsequent six-month dry season, a period of markedly reduced malaria transmission, P. falciparum–inducible IL-10 production remained partially upregulated only in children with persistent asymptomatic infections. These findings suggest that in the face of P. falciparum re-exposure, children acquire exposure-dependent P. falciparum–specific immunoregulatory responses that dampen pathogenic inflammation while enhancing anti-parasite effector mechanisms. These data provide mechanistic insight into the observation that P. falciparum–infected children in endemic areas are often afebrile and tend to control parasite replication.
Author Summary
Malaria remains a major cause of disease and death worldwide. When mosquitoes infect people with malaria parasites for the first time, the parasite rapidly multiplies in the blood and the body responds by producing molecules that cause inflammation and fever, and sometimes the infection progresses to life-threatening disease. However, in regions where people are repeatedly infected with malaria parasites, most infections do not cause fever and parasites often do not multiply uncontrollably. For example, in Mali where this study was conducted, children are infected with malaria parasites ≥100 times/year but only get malaria fever ∼2 times/year and often manage to control parasite numbers in the blood. To understand these observations we collected immune cells from the blood of healthy children before the malaria season and 7 days after malaria fever. We simulated malaria infection at these time points by exposing the immune cells to malaria parasites in a test-tube. We found that re-exposing immune cells to parasites after malaria fever results in reduced expression of molecules that cause fever and enhanced expression of molecules involved in parasite killing. These findings help explain how the immune system prevents fever and controls malaria parasite growth in children who are repeatedly infected with malaria parasites.
doi:10.1371/journal.ppat.1004079
PMCID: PMC3990727  PMID: 24743880
9.  Multi-donor Analysis Reveals Structural Elements, Genetic Determinants, and Maturation Pathway for Effective HIV-1 Neutralization by VRCO1-class Antibodies 
Immunity  2013;39(2):245-258.
Summary
Antibodies of the VRC01 class neutralize HIV-1, arise in diverse HIV-1-infected donors, and are potential templates for an effective HIV-1 vaccine. However, the stochastic processes that generate repertoires in each individual of >1012 antibodies make elicitation of specific antibodies uncertain. Here we determine the ontogeny of the VRC01 class by crystallography and next-generation sequencing. Despite antibody-sequence differences exceeding 50%, antibody-gp120 cocrystal structures reveal VRC01-class recognition to be remarkably similar. B cell transcripts indicate that VRC01-class antibodies require few specific genetic elements, suggesting that naive-B cells with VRC01-class features are generated regularly by recombination. Virtually all of these fail to mature, however, with only a few—likely one—ancestor B cell expanding to form a VRC01-class lineage in each donor. Developmental similarities in multiple donors thus reveal the generation of VRC01-class antibodies to be reproducible in principle, thereby providing a framework for attempts to elicit similar antibodies in the general population.
doi:10.1016/j.immuni.2013.04.012
PMCID: PMC3985390  PMID: 23911655
10.  Monkeypox Virus Infection of Rhesus Macaques Induces Massive Expansion of Natural Killer Cells but Suppresses Natural Killer Cell Functions 
PLoS ONE  2013;8(10):e77804.
Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.
doi:10.1371/journal.pone.0077804
PMCID: PMC3798392  PMID: 24147080
11.  Differences in National Antiretroviral Prescribing Patterns between Black and White Patients with HIV/AIDS, 1996–2006 
Southern medical journal  2011;104(12):794-800.
Objectives
The benefit of improved health outcomes for blacks receiving highly active antiretroviral therapy (HAART) lags behind that of whites. This project therefore sought to determine whether the reason for this discrepancy in health outcomes could be attributed to disparities in use of antiretroviral therapy between black and white patients with HIV.
Materials and Methods
The 1996–2006 National Hospital Ambulatory Medical Care Surveys were used to identify hospital outpatient visits that documented antiretrovirals. Patients younger than 18 years, of nonblack or nonwhite race, and lacking documentation of antiretrovirals were excluded. A multivariable logistic regression model was constructed with race as the independent variable and use of HAART as the dependent variable.
Results
Approximately 3 million HIV/AIDS patient visits were evaluated. Blacks were less likely than whites to use HAART and protease inhibitors (odds ratio, 95% CI 0.81 [0.81–0.82] and 0.67 [0.67–0.68], respectively). More blacks than whites used non-nucleoside reverse transcriptase inhibitors (odds ratio, 95% CI 1.18 [1.17–1.18]). In 1996, the crude rates of HAART were relatively low for both black and white cohorts (5% vs 6%). The rise in HAART for blacks appeared to lag behind that of whites for several years, until 2002, when the proportion of blacks receiving HAART slightly exceeded the proportion of whites receiving HAART. In later years, the rates of HAART were similar for blacks and whites (81% vs 82% in 2006). Blacks appeared less likely than whites to use protease inhibitors and more likely than whites to use non-nucleoside reverse transcriptase inhibitors from 2000 to 2004.
Conclusions
Blacks experienced a lag in the use of antiretrovirals at the beginning of the study; this discrepancy dissipated in more recent years.
doi:10.1097/SMJ.0b013e318236c23a
PMCID: PMC3222681  PMID: 22089356
HIV/AIDS; highly active antiretroviral therapy; racial disparities
12.  A Study of the Use of Impulse Oscillometry in the Evaluation of Children With Asthma: Analysis of Lung Parameters, Order Effect, and Utility Compared With Spirometry 
Pediatric pulmonology  2011;47(1):18-26.
Summary
Background
The ability to objectively measure lung function in children is critical in the assessment and treatment of asthma in this age group. We thus determined the effectiveness of impulse oscillometry (IOS) as a non-invasive technique to assess lung function in children and in comparison to spirometry for sensitivity and specificity, testing variability, and the order effect of sequential testing of IOS and spirometry.
Methods
One hundred seventeen children sequentially evaluated in a pediatric clinic and under medical care for disease, were asked to perform IOS and spirometry. The utility of IOS and spirometry in differentiating children that had asthma versus those children who did not was then analyzed.
Results
In the primary analysis (n = 117), bronchodilator response using IOS distinguished asthmatics from non-asthmatics, P = 0.0008 for R10. Receiver–operator characteristic curve (ROC) analysis of R10 bronchodilator response at the best cut-off (–8.6% change) correctly identified 77% of patients with asthma and excluded 76% of non-asthmatics. Amongst those children able to perform spirometry (asthmatics, n = 66; non-asthmatics, n = 16), FEV1 did not reveal a difference between these two groups, while area of reactance (AX) did distinguish these groups (P = 0.0092). Sequential testing of IOS and then spirometry (n = 47) showed a significant decrement in lung function as determined by IOS following performance of spirometry (P = 0.0309).
Conclusion
In the diagnosis and management of children with lung disease, IOS is a non-invasive approach that easily and objectively measures lung impedance and should be considered as both an adjunct, and in some situations, an alternative to standard spirometry.
doi:10.1002/ppul.21507
PMCID: PMC3423092  PMID: 22170806
pediatric asthma; lung function; bronchodilator response; spirometry; impulse oscillometry
13.  Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment 
BMC Molecular Biology  2012;13:22.
Background
The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.
Results
The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1α, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-ΔΔCT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-ΔΔCT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.
Conclusions
We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.
doi:10.1186/1471-2199-13-22
PMCID: PMC3482582  PMID: 22747760
Candida glabrata; Azole resistance gene; Fluconazole; hkgFinder; Housekeeping gene; Reference gene; RT-qPCR
14.  Construct and Compare Gene Coexpression Networks with DAPfinder and DAPview 
BMC Bioinformatics  2011;12:286.
Background
DAPfinder and DAPview are novel BRB-ArrayTools plug-ins to construct gene coexpression networks and identify significant differences in pairwise gene-gene coexpression between two phenotypes.
Results
Each significant difference in gene-gene association represents a Differentially Associated Pair (DAP). Our tools include several choices of filtering methods, gene-gene association metrics, statistical testing methods and multiple comparison adjustments. Network results are easily displayed in Cytoscape. Analyses of glioma experiments and microarray simulations demonstrate the utility of these tools.
Conclusions
DAPfinder is a new friendly-user tool for reconstruction and comparison of biological networks.
doi:10.1186/1471-2105-12-286
PMCID: PMC3149583  PMID: 21756334
15.  Local Conformational Stability of HIV-1 gp120 in Unliganded and CD4-Bound States as Defined by Amide Hydrogen/Deuterium Exchange▿ †  
Journal of Virology  2010;84(19):10311-10321.
The binding reaction of the HIV-1 gp120 envelope glycoprotein to the CD4 receptor involves exceptional changes in enthalpy and entropy. Crystal structures of gp120 in unliganded and various ligand-bound states, meanwhile, reveal an inner domain able to fold into diverse conformations, a structurally invariant outer domain, and, in the CD4-bound state, a bridging sheet minidomain. These studies, however, provide only hints as to the flexibility of each state. Here we use amide hydrogen/deuterium exchange coupled to mass spectrometry to provide quantifications of local conformational stability for HIV-1 gp120 in unliganded and CD4-bound states. On average, unliganded core gp120 displayed >10,000-fold slower exchange of backbone-amide hydrogens than a theoretically unstructured protein of the same composition, with binding by CD4 reducing the rate of gp120 amide exchange a further 10-fold. For the structurally constant CD4, alterations in exchange correlated well with alterations in binding surface (P value = 0.0004). For the structurally variable gp120, however, reductions in flexibility extended outside the binding surface, and regions of expected high structural diversity (inner domain/bridging sheet) displayed roughly 20-fold more rapid exchange in the unliganded state than regions of low diversity (outer domain). Thus, despite an extraordinary reduction in entropy, neither unliganded gp120 nor free CD4 was substantially unstructured, suggesting that most of the diverse conformations that make up the gp120 unliganded state are reasonably ordered. The results provide a framework for understanding how local conformational stability influences entropic change, conformational diversity, and structural rearrangements in the gp120-CD4 binding reaction.
doi:10.1128/JVI.00688-10
PMCID: PMC2937775  PMID: 20660185
16.  Relationship between Antibody 2F5 Neutralization of HIV-1 and Hydrophobicity of Its Heavy Chain Third Complementarity-Determining Region ▿ † ‡  
Journal of Virology  2009;84(6):2955-2962.
The membrane-proximal external region (MPER) of the HIV-1 gp41 transmembrane glycoprotein is the target of the broadly neutralizing antibody 2F5. Prior studies have suggested a two-component mechanism for 2F5-mediated neutralization involving both structure-specific recognition of a gp41 protein epitope and nonspecific interaction with the viral lipid membrane. Here, we mutationally alter a hydrophobic patch on the third complementarity-determining region of the heavy chain (CDR H3) of the 2F5 antibody and assess the abilities of altered 2F5 variants to bind gp41 and to neutralize diverse strains of HIV-1. CDR H3 alterations had little effect on the affinity of 2F5 variants for a peptide corresponding to its gp41 epitope. In contrast, strong effects and a high degree of correlation (P < 0.0001) were found between virus neutralization and CDR H3 hydrophobicity, as defined by predicted free energies of transfer from water to a lipid bilayer interface or to octanol. The effect of CDR H3 hydrophobicity on neutralization was independent of isolate sensitivity to 2F5, and CDR H3 variants with tryptophan substitutions were able to neutralize HIV-1 ∼10-fold more potently than unmodified 2F5. A threshold was observed for increased hydrophobicity of the 2F5 CDR H3 loop beyond which effects on 2F5-mediated neutralization leveled off. Together, the results provide a more complete understanding of the 2F5 mechanism of HIV-1 neutralization and indicate ways to enhance the potency of MPER-directed antibodies.
doi:10.1128/JVI.02257-09
PMCID: PMC2826063  PMID: 20042512
17.  Black race as a predictor of poor health outcomes among a national cohort of HIV/AIDS patients admitted to US hospitals: a cohort study 
Background
In general, the Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) population has begun to experience the benefits of highly active antiretroviral therapy (HAART); unfortunately, these benefits have not extended equally to Blacks in the United States, possibly due to differences in patient comorbidities and demographics. These differences include rates of hepatitis B and C infection, substance use, and socioeconomic status. To investigate the impact of these factors, we compared hospital mortality and length of stay (LOS) between Blacks and Whites with HIV/AIDS while adjusting for differences in these key characteristics.
Methods
The 1996–2006 National Hospital Discharge Surveys were used to identify HIV/AIDS patients admitted to US hospitals. Survey weights were incorporated to provide national estimates. Patients < 18 years of age, those who left against medical advice, those with an unknown discharge disposition and those with a LOS < 1 day were excluded. Patients were stratified into subgroups by race (Black or White). Two multivariable logistic regression models were constructed with race as the independent variable and outcomes (mortality and LOS > 10 days) as the dependent variables. Factors that were significantly different between Blacks and Whites at baseline via bivariable statistical tests were included as covariates.
Results
In the general US population, there are approximately 5 times fewer Blacks than Whites. In the present study, 1.5 million HIV/AIDS hospital discharges were identified and Blacks were 6 times more likely to be hospitalized than Whites. Notably, Blacks had higher rates of substance use (30% vs. 24%; P < 0.001), opportunistic infections (27% vs. 26%; P < 0.001) and cocaine use (13% vs. 5%; P < 0.001). Conversely, fewer Blacks were co-infected with hepatitis C virus (8% vs. 12%; P < 0.001). Hepatitis B virus was relatively infrequent (3% for both groups). Crude mortality rates were similar for both cohorts (5%); however, a greater proportion of Blacks had a LOS > 10 days (21% vs. 19%; P < 0.001). Black race, in the presence of comorbidities, was correlated with a higher odds of LOS > 10 days (OR, 95% CI = 1.20 [1.10–1.30]), but was not significantly correlated with a higher odds of mortality (OR, 95% CI = 1.07 [0.93–1.25]).
Conclusion
Black race is a predictor of LOS > 10 days, but not mortality, among HIV/AIDS patients admitted to US hospitals. It is possible that racial disparities in hospital outcomes may be closing with time.
doi:10.1186/1471-2334-9-127
PMCID: PMC2736968  PMID: 19671170

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