Monoclonal antibody (mAb) therapy directed against CD20 is an important tool in the treatment of B cell disorders. However, variable patient response and acquired resistance remain important clinical ...challenges. To identify genetic factors that may influence sensitivity to treatment, the cytotoxic activity of three CD20 mAbs: rituximab; ofatumumab; and obinutuzumab, were screened in high-throughput assays using 680 ethnically diverse lymphoblastoid cell lines (LCLs) followed by a pharmacogenomic assessment. GWAS analysis identified several novel gene candidates. The most significant SNP, rs58600101, in the gene
displayed ethnic stratification, with the variant being significantly more prevalent in the African cohort and resulting in reduced transcript levels as measured by qPCR. Functional validation of
by shRNA-mediated knockdown of MKL1 resulted in a more resistant phenotype. Gene expression analysis identified the developmentally associated
as the most significant gene associated with sensitivity. qPCR among a panel of sensitive and resistant LCLs revealed immunoglobulin class-switching as well as differences in the expression of B cell activation markers. Flow cytometry showed heterogeneity within some cell lines relative to surface Ig isotype with a shift to more IgG
cells among the resistant lines. Pretreatment with prednisolone could partly reverse the resistant phenotype. Results suggest that the efficacy of anti-CD20 mAb therapy may be influenced by B cell developmental status as well as polymorphism in the
gene. A clinical benefit may be achieved by pretreatment with corticosteroids such as prednisolone followed by mAb therapy.
Identifying genetic factors associated with the development of adverse events might allow screening before vaccinia virus administration. Two independent clinical trials of the smallpox vaccine ...(Aventis Pasteur) were conducted in healthy, vaccinia virus-naive adult volunteers. Volunteers were assessed repeatedly for local and systemic adverse events (AEs) associated with the receipt of vaccine and underwent genotyping for 1442 single-nucleotide polymorphisms (SNPs). In the first study, 36 SNPs in 26 genes were associated with systemic AEs (P ≤ .05); these 26 genes were tested in the second study. In the final analysis, 3 SNPs were consistently associated with AEs in both studies. The presence of a nonsynonymous SNP in the methylenetetrahydrofolate reductase (MTHFR) gene was associated with the risk of AE in both trials (odds ratio OR, 2.3 95% confidence interval {CI}1.1–5.2 P = .04 and OR, 4.1 95% CI, 1.4–11.4 P<.01). Two SNPs in the interferon regulatory factor-1 (IRF1) gene were associated with the risk of AE in both sample sets (OR, 3.2 95% CI, 1.1–9.8 P = .03 and OR, 3.0 95% CI, 1.1–8.3 P = .03). Genetic polymorphisms in genes expressing an enzyme previously associated with adverse reactions to a variety of pharmacologic agents (MTHFR) and an immunological transcription factor (IRF1) were associated with AEs after smallpox vaccination in 2 independent study samples.
We present an approach combining alchemical modifications and physical-pathway methods to calculate absolute binding free energies. The employed physical-pathway method is either a stratified ...umbrella sampling to calculate a potential of mean force or nonequilibrium pulling. We devised two basic approaches: the simultaneous approach (S-approach), where, along the physical unbinding pathway, an alchemical transformation of ligand–protein interactions is installed and deinstalled, and the prior-plus-simultaneous approach (PPS-approach), where, prior to the physical-pathway simulation, an alchemical transformation of ligand–protein interactions is installed in the binding site and deinstalled during the physical-pathway simulation. Using a mutant of T4 lysozyme with a benzene ligand as an example, we show that installation and deinstallation of soft-core interactions concurrent with physical ligand unbinding (S-approach) allow successful potential of mean force calculations and nonequilibrium pulling simulations despite the problems posed by the occluded nature of the lysozyme binding pocket. Good agreement between the potential of the mean-force-based S-approach and double decoupling simulations as well as a remarkable efficiency and accuracy of the nonequilibrium-pulling-based S-approach is found. The latter turned out to be more compute-efficient than the potential of mean force calculation by approximately 70%. Furthermore, we illustrate the merits of reducing ligand–protein interactions prior to potential of mean force calculations using the murine double minute homologue protein MDM2 with a p53-derived peptide ligand (PPS-approach). Here, the problem of breaking strong interactions in the binding pocket is transferred to a prior alchemical transformation that reduces the free-energy barrier between the bound and unbound state in the potential of mean force. Besides, disentangling physical ligand displacement from the deinstallation of ligand–protein interactions was seen to allow a more uniform sampling of distance histograms in the umbrella sampling. In the future, physical ligand unbinding combined with simultaneous alchemical modifications may prove useful in the calculation of protein–protein binding free energies, where sampling problems posed by multiple, possibly sticky interactions and potential steric clashes can thus be reduced.
Objectives. This article describes the effects of two specific information and communication technology (ICT) demands (telepressure, technostress creators) on employee well-being, and investigates a ...mediating effect of detachment (Study 1) and moderating effects of technostress inhibitors on the effects of ICT demands on well-being (Study 2) and detachment (Study 3). Methods. Three quantitative studies with employees (Study 1, N = 296; Study 2, N = 142; Study 3, N = 316) were conducted. Results. The results support the negative effect of ICT demands on several well-being indicators. They also show how detachment mediates the effect of telepressure on well-being (Study 1) and how technostress inhibitors moderate the effect of technostress creators on well-being (Study 2). Technostress inhibitors further buffer negative effects of technostress creators on detachment (Study 3). Conclusion. Interventions to reduce negative consequences of ICTs by increasing the level of technostress inhibitors (e.g., technical support) or facilitating employee detachment (e.g., through communication policies) are derived. The findings confirm that general models explaining stress and well-being are applicable to new forms of job demands and extend existing empirical support for the effect of ICT demands on well-being. Future research should investigate the interplay between the studied variables within a moderated mediation model.
The diagnosis and treatment of childhood asthma is complicated by its mechanistically distinct subtypes (endotypes) driven by genetic susceptibility and modulating environmental factors. Clinical ...biomarkers and blood gene expression were collected from a stratified, cross-sectional study of asthmatic and non-asthmatic children from Detroit, MI. This study describes four distinct asthma endotypes identified via a purely data-driven method. Our method was specifically designed to integrate blood gene expression and clinical biomarkers in a way that provides new mechanistic insights regarding the different asthma endotypes. For example, we describe metabolic syndrome-induced systemic inflammation as an associated factor in three of the four asthma endotypes. Context provided by the clinical biomarker data was essential in interpreting gene expression patterns and identifying putative endotypes, which emphasizes the importance of integrated approaches when studying complex disease etiologies. These synthesized patterns of gene expression and clinical markers from our research may lead to development of novel serum-based biomarker panels.
Complex clinical outcomes, such as adverse reaction to vaccination, arise from the concerted interactions among the myriad components of a biological system. Therefore, comprehensive etiological ...models can be developed only through the integrated study of multiple types of experimental data. In this study, we apply this paradigm to high-dimensional genetic and proteomic data collected to elucidate the mechanisms underlying the development of adverse events (AEs) in patients after smallpox vaccination. As vaccination was successful in all of the patients under study, the AE outcomes reported likely represent the result of interactions among immune system components that result in excessive or prolonged immune stimulation. In this study, we examined 1442 genetic variables (single nucleotide polymorphisms) and 108 proteomic variables (serum cytokine concentrations) to model AE risk. To accomplish this daunting analytical task, we employed the Random Forests (RF) method to filter the most important attributes, then we used the selected attributes to build a final decision tree model. This strategy is well suited to integrated analysis, as relevant attributes may be selected from categorical or continuous data. Importantly, RF is a natural approach for studying the type of gene-gene, gene-protein and protein-protein interactions we hypothesize to be involved in the development of clinical AEs. RF importance scores for particular attributes take interactions into account, and there may be interactions across data types. Combining information from previous studies on AEs related to smallpox vaccination with the genetic and proteomic attributes identified by RF, we built a comprehensive model of AE development that includes the cytokines intercellular adhesion molecule-1 (ICAM-1 or CD54), interleukin-10 (IL-10), and colony stimulating factor-3 (CSF-3 or G-CSF) and a genetic polymorphism in the cytokine gene interleukin-4 (IL4). The biological factors included in the model support our hypothesized mechanism for the development of AEs involving prolonged stimulation of inflammatory pathways and an imbalance of normal tissue damage repair pathways. This study shows the utility of RF for such analytical tasks, while both enhancing and reinforcing our working model of AE development after smallpox vaccination.
The vertebrate genome is a result of two rapid and successive rounds of whole genome duplication, referred to as 1R and 2R. Furthermore, teleost fish have undergone a third whole genome duplication ...(3R) specific to their lineage, resulting in the retention of multiple gene paralogs. The more recent 3R event in teleosts provides a unique opportunity to gain insight into how genes evolve through specific evolutionary processes. In this study we compare molecular activities of vitamin D receptors (VDR) from basal species that diverged at key points in vertebrate evolution in order to infer derived and ancestral VDR functions of teleost paralogs. Species include the sea lamprey (Petromyzon marinus), a 1R jawless fish; the little skate (Leucoraja erinacea), a cartilaginous fish that diverged after the 2R event; and the Senegal bichir (Polypterus senegalus), a primitive 2R ray-finned fish. Saturation binding assays and gel mobility shift assays demonstrate high affinity ligand binding and classic DNA binding characteristics of VDR has been conserved across vertebrate evolution. Concentration response curves in transient transfection assays reveal EC50 values in the low nanomolar range, however maximum transactivational efficacy varies significantly between receptor orthologs. Protein-protein interactions were investigated using co-transfection, mammalian 2-hybrid assays, and mutations of coregulator activation domains. We then combined these results with our previous study of VDR paralogs from 3R teleosts into a bioinformatics analysis. Our results suggest that 1, 25D3 acts as a partial agonist in basal species. Furthermore, our bioinformatics analysis suggests that functional differences between VDR orthologs and paralogs are influenced by differential protein interactions with essential coregulator proteins. We speculate that we may be observing a change in the pharmacodynamics relationship between VDR and 1, 25D3 throughout vertebrate evolution that may have been driven by changes in protein-protein interactions between VDR and essential coregulators.
Thousands of untested chemicals in the environment require efficient characterization of carcinogenic potential in humans. A proposed solution is rapid testing of chemicals using in vitro ...high-throughput screening (HTS) assays for targets in pathways linked to disease processes to build models for priority setting and further testing. We describe a model for predicting rodent carcinogenicity based on HTS data from 292 chemicals tested in 672 assays mapping to 455 genes. All data come from the EPA ToxCast project. The model was trained on a subset of 232 chemicals with in vivo rodent carcinogenicity data in the Toxicity Reference Database (ToxRefDB). Individual HTS assays strongly associated with rodent cancers in ToxRefDB were linked to genes, pathways, and hallmark processes documented to be involved in tumor biology and cancer progression. Rodent liver cancer endpoints were linked to well-documented pathways such as peroxisome proliferator-activated receptor signaling and TP53 and novel targets such as PDE5A and PLAUR. Cancer hallmark genes associated with rodent thyroid tumors were found to be linked to human thyroid tumors and autoimmune thyroid disease. A model was developed in which these genes/pathways function as hypothetical enhancers or promoters of rat thyroid tumors, acting secondary to the key initiating event of thyroid hormone disruption. A simple scoring function was generated to identify chemicals with significant in vitro evidence that was predictive of in vivo carcinogenicity in different rat tissues and organs. This scoring function was applied to an external test set of 33 compounds with carcinogenicity classifications from the EPA's Office of Pesticide Programs and successfully (p = 0.024) differentiated between chemicals classified as "possible"/"probable"/"likely" carcinogens and those designated as "not likely" or with "evidence of noncarcinogenicity." This model represents a chemical carcinogenicity prioritization tool supporting targeted testing and functional validation of cancer pathways.
The GROMOS 54A8 force field Reif et al. J. Chem. Theory Comput. 2012, 8, 3705–3723 is the first of its kind to contain nonbonded parameters for charged amino acid side chains that are derived in a ...rigorously thermodynamic fashion, namely a calibration against single-ion hydration free energies. Considering charged moieties in solution, the most decisive signature of the GROMOS 54A8 force field in comparison to its predecessor 54A7 can probably be found in the thermodynamic equilibrium between salt-bridged ion pair formation and hydration. Possible shifts in this equilibrium might crucially affect the properties of electrolyte solutions or/and the stability of (bio)molecules. It is therefore important to investigate the consequences of the altered description of charged oligoatomic species in the GROMOS 54A8 force field. The present study focuses on examining the ability of the GROMOS 54A8 force field to accurately model the structural properties of electrolyte solutions, lipid bilayers, and proteins. It is found that (i) aqueous electrolytes involving oligoatomic species (sodium acetate, methylammonium chloride, guanidinium chloride) reproduce experimental salt activity derivatives for concentrations up to 1.0 m (1.0-molal) very well, and good agreement between simulated and experimental data is also reached for sodium acetate and methylammonium chloride at 2.0 m concentration, while not even qualitative agreement is found for sodium chloride throughout the whole range of examined concentrations, indicating a failure of the GROMOS 54A7 and 54A8 force-field parameter sets to correctly account for the balance between ion–ion and ion–water binding propensities of sodium and chloride ions; (ii) the GROMOS 54A8 force field reproduces the liquid crystalline-like phase of a hydrated DPPC bilayer at a pressure of 1 bar and a temperature of 323 K, the area per lipid being in agreement with experimental data, whereas other structural properties (volume per lipid, bilayer thickness) appear underestimated; (iii) the secondary structure of a range of different proteins simulated with the GROMOS 54A8 force field at pH 7 is maintained and compatible with experimental NMR data, while, as also observed for the GROMOS 54A7 force field, α-helices are slightly overstabilized with respect to 310-helices; (iv) with the GROMOS 54A8 force field, the side chains of arginine, lysine, aspartate, and glutamate residues appear slightly more hydrated and present a slight excess of oppositely-charged solution components in their vicinity, whereas salt-bridge formation properties between charged residues at the protein surface, as assessed by probability distributions of interionic distances, are largely equivalent in the GROMOS 54A7 and 54A8 force-field parameter sets.
High‐throughput screening (HTS) of environmental chemicals is used to identify chemicals with high potential for adverse human health and environmental effects from among the thousands of untested ...chemicals. Predicting physiologically relevant activity with HTS data requires estimating the response of a large number of chemicals across a battery of screening assays based on sparse dose–response data for each chemical‐assay combination. Many standard dose–response methods are inadequate because they treat each curve separately and under‐perform when there are as few as 6–10 observations per curve. We propose a semiparametric Bayesian model that borrows strength across chemicals and assays. Our method directly parametrizes the efficacy and potency of the chemicals as well as the probability of response. We use the ToxCast data from the U.S. Environmental Protection Agency (EPA) as motivation. We demonstrate that our hierarchical method provides more accurate estimates of the probability of response, efficacy, and potency than separate curve estimation in a simulation study. We use our semiparametric method to compare the efficacy of chemicals in the ToxCast data to well‐characterized reference chemicals on estrogen receptor α (ERα) and peroxisome proliferator‐activated receptor γ (PPARγ) assays, then estimate the probability that other chemicals are active at lower concentrations than the reference chemicals.
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