Semiparametric clustering method for microarray data analysis

Clustering is a major tool for microarray gene expression data analysis. The existing clustering methods fall mainly into two categories: parametric and nonparametric. The parametric methods generally assume a mixture of parametric subdistributions. When the mixture distribution approximately fits the true data generating mechanism, the parametric methods perform well, but not so when there is nonnegligible deviation between them. On the other hand, the nonparametric methods, which usually do not make distributional assumptions, are robust but pay the price for efficiency loss. In an attempt to utilize the known mixture form to increase efficiency, and to free assumptions about the unknown subdistributions to enhance robustness, we propose a semiparametric method for clustering. The proposed approach possesses the form of parametric mixture, with no assumptions to the subdistributions. The subdistributions are estimated nonparametrically, with constraints just being imposed on the modes. An expectation-maximization (EM) algorithm along with a classification step is invoked to cluster the data, and a modified Bayesian information criterion (BIC) is employed to guide the determination of the optimal number of clusters. Simulation studies are conducted to assess the performance and the robustness of the proposed method. The results show that the proposed method yields reasonable partition of the data. As an illustration, the proposed method is applied to a real microarray data set to cluster genes.     

Covariate analysis of viral eradication studies

An important issue arising in therapeutic studies of hepatitis C and HIV is the identification of and adjustment for covariates associated with viral eradication and resistance. Analyses of such data are complicated by the fact that eradication is an occult event that is not directly observable, resulting in unique types of censored observations that do not arise in other competing risks settings. This paper proposes a semiparametric regression model to assess the association between multiple covariates and the eradication/resistance processes. The proposed methods are based on a piecewise proportional hazards model that allows parameters to vary between observation times. We illustrate the methods with data from recent hepatitis C clinical trials.     

Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs.     

The role of research in viral disease eradication and elimination programs: lessons for malaria eradication

By examining the role research has played in eradication or regional elimination initiatives for three viral diseases--smallpox, poliomyelitis, and measles--we derive nine cross-cutting lessons applicable to malaria eradication. In these initiatives, some types of research commenced as the programs began and proceeded in parallel. Basic laboratory, clinical, and field research all contributed notably to progress made in the viral programs. For each program, vaccine was the lynchpin intervention, but as the programs progressed, research was required to improve vaccine formulations, delivery methods, and immunization schedules. Surveillance was fundamental to all three programs, whilst polio eradication also required improved diagnostic methods to identify asymptomatic infections. Molecular characterization of pathogen isolates strengthened surveillance and allowed insights into the geographic source of infections and their spread. Anthropologic, sociologic, and behavioural research were needed to address cultural and religious beliefs to expand community acceptance. The last phases of elimination and eradication became increasingly difficult, as a nil incidence was approached. Any eradication initiative for malaria must incorporate flexible research agendas that can adapt to changing epidemiologic contingencies and allow planning for posteradication scenarios.     

The relationship between virologic and immunologic responses in AIDS clinical research using mixed-effects varying-coefficient models with measurement error

In this article we study the relationship between virologic and immunologic responses in AIDS clinical trials. Since plasma HIV RNA copies (viral load) and CD4+ cell counts are crucial virologic and immunologic markers for HIV infection, it is important to study their relationship during HIV/AIDS treatment. We propose a mixed-effects varying-coefficient model based on an exploratory analysis of data from a clinical trial. Since both viral load and CD4+ cell counts are subject to measurement error, we also consider the measurement error problem in covariates in our model. The regression spline method is proposed for inference for parameters in the proposed model. The regression spline method transforms the unknown nonparametric components into parametric functions. It is relatively simple to implement using readily available software, and parameter inference can be developed from standard parametric models. We apply the proposed models and methods to an AIDS clinical study. From this study, we find an interesting relationship between viral load and CD4+ cell counts during antiviral treatments. Biological interpretations and clinical implications are discussed.     

Widespread occurence of mariner transposons in coastal crabs

Mariner-like elements (MLEs) are ubiquitous DNA mobile elements found in almost all eukaryote genomes. Nevertheless most of the known copies are inactive and the question of the genome invasion by MLEs remains largely hypothetical. We have previously reported the presence of highly homologous copies of MLEs in the genome of phylogenetically distant crustacea living in the same hydrothermal environment suggesting the possibility of horizontal transfer. In order to further support the hypothesis that horizontal transmission of MLEs might occur between crustacean sympatric species, we described here 85 MLE sequences found in the genome of a large spectrum of coastal crab species. The number of the MLEs copies in genomes was variable. Half of these MLEs fit with the irritans subfamily of MLEs whereas the second half grouped in a new subfamily called marmoratus. In addition, a molecular phylogeny of crabs was established by using the 16S information. The comparison between 16S and MLEs based trees reveals their incongruence, and suggests either the existence of horizontal transfer events between phylogenetically distant species, or an ancestral MLE polymorphism followed by different evolution and stochastic loss.     

Directional selection and the site-frequency spectrum.

In this article we explore statistical properties of the maximum-likelihood estimates (MLEs) of the selection and mutation parameters in a Poisson random field population genetics model of directional selection at DNA sites. We derive the asymptotic variances and covariance of the MLEs and explore the power of the likelihood ratio tests (LRT) of neutrality for varying levels of mutation and selection as well as the robustness of the LRT to deviations from the assumption of free recombination among sites. We also discuss the coverage of confidence intervals on the basis of two standard-likelihood methods. We find that the LRT has high power to detect deviations from neutrality and that the maximum-likelihood estimation performs very well when the ancestral states of all mutations in the sample are known. When the ancestral states are not known, the test has high power to detect deviations from neutrality for negative selection but not for positive selection. We also find that the LRT is not robust to deviations from the assumption of independence among sites.     

Assessing antiviral potency of anti-HIV therapies in vivo by comparing viral decay rates in viral dynamic models

A virologic marker, the number of HIV RNA copies or viral load, is currently used to evaluate anti-HIV therapies in AIDS clinical trials. This marker can be used to assess the antiviral potency of therapies, but is easily affected by noncompliance, drug resistance, toxicities, and other factors during the long-term treatment evaluation process. Recently it has been suggested to use viral dynamics to assess the potency of antiviral drugs and therapies, since viral decay rates in viral dynamic models have been shown to be related to the antiviral drug potency directly, and they need a shorter evaluation time. In this paper we first review the two statistical approaches for characterizing HIV dynamics and estimating viral decay rates: the individual nonlinear least squares regression (INLS) method and the population nonlinear mixed-effect model (PMEM) approach. To compare the viral decay rates between two treatment arms, parametric and nonparametric tests, based on the estimates of viral decay rates (the derived variables) from both the INLS and PMEM methods, are proposed and studied. We show, using the concept of exchangeability, that the test based on the empirical Bayes' estimates from the PMEM is valid, powerful and robust. This proposed method is very useful in most practical cases where the INLS-based tests and the general likelihood ratio test may not apply. We validate and compare various tests for finite samples using Monte Carlo simulations. Finally, we apply the proposed tests to an AIDS clinical trial to compare the antiviral potency between a 3-drug combination regimen and a 4-drug combination regimen. The proposed tests provide some significant evidence that the 4-drug regimen is more potent than the 3-drug regimen, while the naive methods fail to give a significant result.*To whom correspondence should be addressed.     

The structure of Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase

Muconate lactonizing enzymes (MLEs) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (NcCMLE) at 2.5 A resolution, with a seven-bladed beta propeller fold. It is related neither to bacterial MLEs nor to other beta propeller enzymes, but is structurally similar to the G protein beta subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and NcCMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. NcCMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.     

Cloning and characterization of mariner-like elements in the soybean aphid, Aphis glycines Matsumura

Soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is currently the most important insect pest of soybean (Glycine max (L.) Merr.) in the United States and causes significant economic damage worldwide, but little is known about the aphid at the molecular level. Mariner-like transposable elements (MLEs) are ubiquitous within the genomes of arthropods and various other invertebrates. In this study, we report the cloning of MLEs from the soybean aphid genome using degenerate PCR primers designed to amplify conserved regions of mariner transposases. Two of the ten sequenced clones (designated as Agmar1 and Agmar2) contained partial but continuous open reading frames, which shared high levels of homology at the protein level with other mariner transposases from insects and other taxa. Phylogenetic analysis revealed Agmar1 to group within the irritans subfamily of MLEs and Agmar2 within the mellifera subfamily. Southern blot analysis and quantitative PCR analysis indicated a low copy number for Agmar1-like elements within the soybean aphid genome. These results suggest the presence of at least two different putative mariner-like transposases encoded by the soybean aphid genome. Both Agmar1 and Agmar2 could play influential roles in the architecture of the soybean aphid genome. Transposable elements are also thought to potentially mediate resistance in insects through changes in gene amplification and mutations in coding sequences. Finally, Agmar1 and Agmar2 may represent useful genetic tools and provide insights on A. glycines adaptation.     

Dose-response curve estimation: a semiparametric mixture approach

In the estimation of a dose-response curve, parametric models are straightforward and efficient but subject to model misspecifications; nonparametric methods are robust but less efficient. As a compromise, we propose a semiparametric approach that combines the advantages of parametric and nonparametric curve estimates. In a mixture form, our estimator takes a weighted average of the parametric and nonparametric curve estimates, in which a higher weight is assigned to the estimate with a better model fit. When the parametric model assumption holds, the semiparametric curve estimate converges to the parametric estimate and thus achieves high efficiency; when the parametric model is misspecified, the semiparametric estimate converges to the nonparametric estimate and remains consistent. We also consider an adaptive weighting scheme to allow the weight to vary according to the local fit of the models. We conduct extensive simulation studies to investigate the performance of the proposed methods and illustrate them with two real examples.     

Fitting nonlinear and constrained generalized estimating equations with optimization software

In this article, we present an estimation approach for solving nonlinear constrained generalized estimating equations that can be implemented using object-oriented software for nonlinear programming, such as nlminb in Splus or fmincon and lsqnonlin in Matlab. We show how standard estimating equation theory includes this method as a special case so that our estimates, when unconstrained, will remain consistent and asymptotically normal. To illustrate this method, we fit a nonlinear dose-response model with nonnegative mixed bound constraints to clustered binary data from a developmental toxicity study. Satisfactory confidence intervals are found using a nonparametric bootstrap method when a common correlation coefficient is assumed for all the dose groups and for some of the dose-specific groups.     

Effect of ULV malathion use in boll weevil (Coleoptera: Curculionidae) eradication on resistance in the tarnished plant bug (Heteroptera: Miridae)

Tarnished plant bugs, Lygus lineolaris (Palisot de Beauvois), from regions 1, 2, and 3 of the boll weevil, Anthonomous grandis Boheman, eradication program in Mississippi were collected from wild hosts and tested for malathion resistance during the spring and fall of 2000 and 2001. Plant bugs were also tested in region 1 in late-July and October of 1999, just before and after multiple applications of ultra-low-volume (ULV) malathion were used for reproduction-diapause control of boll weevils in August and September. Regions 1 (north Delta), 2 (south Delta), and 3 (hills) began boll weevil eradication in 1999, 1998, and 1997, respectively. A glass-vial bioassay was used to determine resistance in plant bugs to malathion by comparing LC50 values against an LC50 value obtained for susceptible plant bugs. Comparison of the LC50 value obtained for plant bugs at a location in the spring was also made with the LC50 value obtained in the fall at the same location. After multiple applications of malathion made for reproduction-diapause boll weevil control in region 1 in August and September, malathion resistance increased by 4.9-, 6.5-, and 20.8-fold in plant bug populations from the three test locations. Results from testing bugs from all three eradication regions were similar. Malathion resistance usually increased significantly from spring to fall and then declined significantly from fall to spring of the next year. Despite reduced use of malathion in all three eradication regions for boll weevils in 2001, resistance to malathion in plant bugs still increased significantly from spring to fall at all test locations in regions 1 and 2 (the Delta). Malathion resistance did not increase significantly in plant bug populations in region 3 (the hills) in 2001 from spring to fall at three of four test locations in this year. Possible causes for the higher malathion resistance found in plant bugs in the Delta are discussed. Overall test results showed that the use of malathion in boll weevil eradication in cotton probably contributed to increases in resistance to malathion in plant bug populations in the eradication areas. However, the expression of this resistance was usually rapidly lost by spring of the following year. Boll weevil eradication did not seem to produce a permanent increase in the expression of malathion resistance in tarnished plant bug populations found in the eradication regions.     

Modeling the Genetic Control of HIV-1 Dynamics After Highly Active Antiretroviral Therapy

The progression of HIV disease has been markedly slowed by the use of highly active antiretroviral therapy (HAART). However, substantial genetic variation was observed to occur among different people in the decay rate of viral loads caused by HAART. The characterization of specific genes involved in HIV dynamics is central to design personalized drugs for the prevention of this disease, but usually cannot be addressed by experimental methods alone rather than require the help of mathematical and statistical methods. A novel statistical model has been recently developed to detect genetic variants that are responsible for the shape of HAART-induced viral decay curves. This model was employed to an HIV/AIDS trial, which led to the identification of a major genetic determinant that triggers an effect on HIV dynamics. This detected major genetic determinant also affects several clinically important parameters, such as half-lives of infected cells and HIV eradication times.Key Words: Hardy-weinberg equilibrium, bi-exponential function, quantitative trait loci, HIV dynamics, functional mapping.     

A nonparametric distance analysis of biochemical genetic data from the Åland Islands,Finland

Biochemical genetic data from 3272 individuals from the Åland Islands, Finland, are analyzed using a recently developed set of nonparametric genetic distance measures. These measures are more robust than the traditional methods used in previous studies of this population. While there was general agreement in the results of the traditional and nonparametric approaches, some important differences were seen. In these cases, the nonparametric methods gave results more congruent with population history, geographic propinquity, and migration patterns. Heterozygosity measures were also calculated for the Åland Islands population and compared to values obtained for Jewish populations. The total heterozygosity values were very similar.     

Isolation and characterization of seventy-nine full-length <Emphasis Type="Italic">mariner</Emphasis>-like transposase genes in the Bambusoideae subfamily

Mariner-like elements (MLEs) are the most diverse and widespread transposable elements, with members of the MLE superfamily found in fungi, plants, ciliates and animals. In a previous study, we characterized 82 MLE transposase gene fragments (average length 383 bp) in 44 bamboo species, indicating that MLEs are widespread, abundant and diverse in the Bambusoideae subfamily. In this study, we isolated 79 full-length MLE transposase genes from 63 bamboo species representing 38 genera in six subtribes mainly found in China. The transposases were highly conserved, mostly uniform in length and contained intact DNA-binding motifs and DD39D catalytic domains with few notable frameshift, indel and nonsense mutations. This suggested the MLEs are probably still mobile, not yet affected by vertical inactivation. A phylogenetic tree of the Bambusoideae subfamily established using ribosomal DNA internal transcribed spacer sequences was incongruent with a second tree based on the MLE transposase genes. This evidence, together with the presence of near-identical MLEs in distantly related species and diverse MLEs in closely related species, indicates that MLEs have evolved in a distinct manner, probably independently of speciation events in the subfamily. The evolution and diversity of MLE transposase genes in the Bambusoideae subfamily is discussed.     

Eradication of Helicobacter pylori in children in Vietnam in relation to antibiotic resistance

Background: Low Helicobacter pylori eradication rates are common in pediatric trials especially in developing countries. The aim of the study was to investigate the role of antibiotic resistance, drug dosage, and administration frequency on treatment outcome for children in Vietnam. Materials and Methods: Antibiotics resistance of H. pylori was analyzed by the Etest in 222 pretreatment isolates from children 3–15 years of age who were originally recruited in a randomized trial with two treatment regiments: lansoprazole with amoxicillin and either clarithromycin (LAC) or metronidazole (LAM) in two weight groups with once‐ or twice‐daily administration. The study design was an observational study embedded in a randomized trial. Results: The overall resistance to clarithromycin, metronidazole, and amoxicillin was 50.9%, 65.3%, and 0.5%, respectively. In LAC, eradication was linked to the strains being susceptible to clarithromycin (78.2% vs 29.3%, p = .0001). Twice‐daily dosage of proton‐pump inhibitor (PPI) and clarithromycin was more effective for eradication than once‐daily dosage for resistant strains (50.0% vs 14.7%, p = .004) and tended to be so also for sensitive strains (87.5% vs 65.2%, p = .051). Exact antibiotic dose per body weight resulted in more eradication for resistant strains (45.3% vs 8.0%, p = .006). These differences were less pronounced for the LAM regimen, with twice‐daily PPI versus once daily for resistant strains resulting in 69.2% and 50.0% eradication (p = .096), respectively. Conclusions: Helicobacter pylori clarithromycin resistance was unexpectedly high in young children in Vietnam. Clarithromycin resistance was an important cause for eradication treatment failure. Twice‐daily administration and exact antibiotic dosing resulted in more eradicated infections when the strains were antibiotic resistant, which has implications for the study design in pediatric H. pylori eradication trials.