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1.  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.
PMCID: PMC4237593  PMID: 23651994
2.  Network features suggest new hepatocellular carcinoma treatment strategies 
BMC Systems Biology  2014;8:88.
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.
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.
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.
PMCID: PMC4236726  PMID: 25070212
Co-expression network; Pathway network; Robustness; Redundancy; Resistance; Cancer therapy
3.  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.
PMCID: PMC3990727  PMID: 24743880
4.  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.
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.
PMCID: PMC3985390  PMID: 23911655
5.  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.
PMCID: PMC3798392  PMID: 24147080
6.  Differences in National Antiretroviral Prescribing Patterns between Black and White Patients with HIV/AIDS, 1996–2006 
Southern medical journal  2011;104(12):794-800.
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.
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.
Blacks experienced a lag in the use of antiretrovirals at the beginning of the study; this discrepancy dissipated in more recent years.
PMCID: PMC3222681  PMID: 22089356
HIV/AIDS; highly active antiretroviral therapy; racial disparities
7.  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.
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.
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.
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).
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.
PMCID: PMC3423092  PMID: 22170806
pediatric asthma; lung function; bronchodilator response; spirometry; impulse oscillometry
8.  Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment 
BMC Molecular Biology  2012;13:22.
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.
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.
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.
PMCID: PMC3482582  PMID: 22747760
Candida glabrata; Azole resistance gene; Fluconazole; hkgFinder; Housekeeping gene; Reference gene; RT-qPCR
9.  Construct and Compare Gene Coexpression Networks with DAPfinder and DAPview 
BMC Bioinformatics  2011;12:286.
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.
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.
DAPfinder is a new friendly-user tool for reconstruction and comparison of biological networks.
PMCID: PMC3149583  PMID: 21756334
10.  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.
PMCID: PMC2937775  PMID: 20660185
11.  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.
PMCID: PMC2826063  PMID: 20042512
12.  Black race as a predictor of poor health outcomes among a national cohort of HIV/AIDS patients admitted to US hospitals: a cohort study 
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.
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.
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]).
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.
PMCID: PMC2736968  PMID: 19671170

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