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1.  Molecular mechanisms in the selective basal activation of pyrabactin receptor 1: Comparative analysis of mutants 
FEBS Open Bio  2014;4:496-509.
Graphical abstract
•The ABA receptor PYR1 is an inhibitor of type 2 protein phosphatases such as HAB1.•PYR1 mutations affect the dynamics of PYR1–HAB1 and PYR1–PYR1 complexes.•Dynamics are influenced at areas distant from the mutation sites.•Mutations that improve PYR1–HAB1 binding tend to also stabilize PYR1–PYR1.•Results support an hypothesis of HAB1 inhibition competing with PYR1 dimerization.
Pyrabactin receptors (PYR) play a central role in abscisic acid (ABA) signal transduction; they are ABA receptors that inhibit type 2C protein phosphatases (PP2C). Molecular aspects contributing to increased basal activity of PYR against PP2C are studied by molecular dynamics (MD) simulations. An extensive series of MD simulations of the apo-form of mutagenized PYR1 as a homodimer and in complex with homology to ABA-insensitive 1 (HAB1) phosphatase are reported. In order to investigate the detailed molecular mechanisms mediating PYR1 activity, the MD data was analyzed by essential collective dynamics (ECD), a novel approach that allows the identification, with atomic resolution, of persistent dynamic correlations based on relatively short MD trajectories. Employing the ECD method, the effects of select mutations on the structure and dynamics of the PYR1 complexes were investigated and considered in the context of experimentally determined constitutive activities against HAB1. Approaches to rationally design constitutively active PYR1 constructs to increase PP2C inhibition are discussed.
PMCID: PMC4060014  PMID: 24944884
MD, molecular dynamics; ECD, essential collective dynamics; PCA, principal component analysis; WT, wild type; ABA, A8S abscisic acid; PDB, Protein Data Bank; PP2C, phosphatase type 2C; PYR1, pyrabactin resistance 1; PYL, PYR1-like; RCAR, regulatory component of ABA response; CA, constitutively active; HAB1, homology to ABA insensitive 1; PYV, pyrabactin or C16H13BrN2O2S; P2M, N-(pyridin-2-ylmethyl) naphthalene-1-sulfonamide or C16H14N2O2S; Pyrabactin resistance; Abscisic acid signaling; Constitutively active mutations; Molecular dynamics simulations; Essential collective dynamics analysis
2.  Clinical outcomes and resource utilisation in Medicare patients with chronic liver disease: a historical cohort study 
BMJ Open  2014;4(5):e004318.
The aim of this study is to assess recent trends in health resource utilisation and patient outcomes of Medicare beneficiaries with chronic liver disease (CLD).
Liver-related mortality is the 10th leading cause of death in the USA, and hepatitis C virus (HCV) and obesity-related non-alcoholic fatty liver disease are the major causes of CLD. As the US population ages and becomes more obese, the impact of CLD is expected to become more prominent for the Medicare population.
This is a retrospective cohort study of Medicare beneficiaries with a diagnosis of CLD based on inpatient (N=21 576; 14 977 unique patients) and outpatient (N=515 990; 244 196 patients) claims from 2005 to 2010.
Primary and secondary outcome measures
The study outcomes included hospital length of stay (LOS) and inpatient mortality as well as inpatient and outpatient inflation-adjusted payments.
Between 2005 and 2010, there was an annual decrease in LOS of 3.17% for CLD-related hospitalisations. Risk-adjusted in-hospital mortality decreased (OR 0.90, 95% CI 0.87 to 0.94), while short-term postdischarge mortality remained stable (1.00, 0.98 to 1.03). Inpatient per-claim payment increased from $11 769 in 2005 to $12 347 in 2010 (p=0.0006). Similarly, the average yearly payments for outpatient care increased from $366 to $404 (p<0.0001). This change in payment was observed together with a consistent decrease in the proportion of beneficiary-paid amount (25.4–20%, p<0.0001) as opposed to Medicare-paid amount (73.1–80%, p<0.0001). The major predictors of higher outpatient payments were younger age, Asian race or Hispanic ethnicity, living in California, and having more diagnoses and outpatient procedures per claim. The predictors of inpatient spending also included younger age, location and the number of inpatient procedures.
Length of inpatient stay and inpatient mortality among Medicare beneficiaries with CLD decreased, while inpatient and outpatient spending increased.
PMCID: PMC4025451  PMID: 24838722
Qualitative Research
3.  Molecular Mechanisms in the Activation of Abscisic Acid Receptor PYR1 
PLoS Computational Biology  2013;9(6):e1003114.
The pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of abscisic acid (ABA) response (RCAR) proteins comprise a well characterized family of ABA receptors. Recent investigations have revealed two subsets of these receptors that, in the absence of ABA, either form inactive homodimers (PYR1 and PYLs 1–3) or mediate basal inhibition of downstream target type 2C protein phosphatases (PP2Cs; PYLs 4–10) respectively in vitro. Addition of ABA has been shown to release the apo-homodimers yielding ABA-bound monomeric holo-receptors that can interact with PP2Cs; highlighting a competitive-interaction process. Interaction selectivity has been shown to be mediated by subtle structural variations of primary sequence and ligand binding effects. Now, the dynamical contributions of ligand binding on interaction selectivity are investigated through extensive molecular dynamics (MD) simulations of apo and holo-PYR1 in monomeric and dimeric form as well as in complex with a PP2C, homology to ABA insensitive 1 (HAB1). Robust comparative interpretations were enabled by a novel essential collective dynamics approach. In agreement with recent experimental findings, our analysis indicates that ABA-bound PYR1 should efficiently bind to HAB1. However, both ABA-bound and ABA-extracted PYR1-HAB1 constructs have demonstrated notable similarities in their dynamics, suggesting that apo-PYR1 should also be able to make a substantial interaction with PP2Cs, albeit likely with slower complex formation kinetics. Further analysis indicates that both ABA-bound and ABA-free PYR1 in complex with HAB1 exhibit a higher intra-molecular structural stability and stronger inter-molecular dynamic correlations, in comparison with either holo- or apo-PYR1 dimers, supporting a model that includes apo-PYR1 in complex with HAB1. This possibility of a conditional functional apo-PYR1-PP2C complex was validated in vitro. These findings are generally consistent with the competitive-interaction model for PYR1 but highlight dynamical contributions of the PYR1 structure in mediating interaction selectivity suggesting added degrees of complexity in the regulation of the competitive-inhibition.
Author Summary
Protein pyrabactin resistance 1 (PYR1) belongs to a group of PYR1-like (PYL) proteins that regulate plant development and responses to conditions of drought and salinity. Recent studies have reported characterization of their molecular structures as well as elucidation of important aspects of their function; highlighting their roles as receptors for the stress responsive phytohormone, abscisic acid (ABA). However details of the molecular mechanisms regulating their receptor signalling remain enigmatic. In this work, we use molecular dynamics simulations complemented by a sophisticated statistical-mechanical analysis to investigate structural and dynamical properties of PYR1 protein and how its interaction with ABA modifies receptor-protein complex formation. Our results provide detailed insight into how the PYR1-mediated inactivation of its downstream phosphatase target is regulated by homodimer formation and yield new hypotheses, supported by in vitro experiments, for further investigation. Ultimately, this knowledge provides insight into how plants respond to stress, with potential applications in the development of crops with improved growth characteristics and higher stress tolerance.
PMCID: PMC3694813  PMID: 23825939
4.  SML resist processing for high-aspect-ratio and high-sensitivity electron beam lithography 
Nanoscale Research Letters  2013;8(1):139.
A detailed process characterization of SML electron beam resist for high-aspect-ratio nanopatterning at high sensitivity is presented. SML contrast curves were generated for methyl isobutyl ketone (MIBK), MIBK/isopropyl alcohol (IPA) (1:3), IPA/water (7:3), n-amyl acetate, xylene, and xylene/methanol (3:1) developers. Using IPA/water developer, the sensitivity of SML was improved considerably and found to be comparable to benchmark polymethylmethacrylate (PMMA) resist without affecting the aspect ratio performance. Employing 30-keV exposures and ultrasonic IPA/water development, an aspect ratio of 9:1 in 50-nm half-pitch dense grating patterns was achieved representing a greater than two times improvement over PMMA. Through demonstration of 25-nm lift-off features, the pattern transfer performance of SML is also addressed.
PMCID: PMC3617037  PMID: 23531370
SML resist; Electron beam lithography; High-aspect-ratio nanolithography; Nanolithography; Nanofabrication; Lift-off; Preventing pattern collapse; Resists (85.40.Hp); Electron beam lithography (85.40.Hp); Nanolithography (81.16.Nd)
5.  Knowledge-Based Identification of Soluble Biomarkers: Hepatic Fibrosis in NAFLD as an Example 
PLoS ONE  2013;8(2):e56009.
The discovery of biomarkers is often performed using high-throughput proteomics-based platforms and is limited to the molecules recognized by a given set of purified and validated antigens or antibodies. Knowledge-based, or systems biology, approaches that involve the analysis of integrated data, predominantly molecular pathways and networks may infer quantitative changes in the levels of biomolecules not included by the given assay from the levels of the analytes profiled. In this study we attempted to use a knowledge-based approach to predict biomarkers reflecting the changes in underlying protein phosphorylation events using Nonalcoholic Fatty Liver Disease (NAFLD) as a model. Two soluble biomarkers, CCL-2 and FasL, were inferred in silico as relevant to NAFLD pathogenesis. Predictive performance of these biomarkers was studied using serum samples collected from patients with histologically proven NAFLD. Serum levels of both molecules, in combination with clinical and demographic data, were predictive of hepatic fibrosis in a cohort of NAFLD patients. Our study suggests that (1) NASH-specific disruption of the kinase-driven signaling cascades in visceral adipose tissue lead to detectable changes in the levels of soluble molecules released into the bloodstream, and (2) biomarkers discovered in silico could contribute to predictive models for non-malignant chronic diseases.
PMCID: PMC3566090  PMID: 23405244
6.  Gene expression profiles associated with depression in patients with chronic hepatitis C (CH-C) 
Brain and Behavior  2012;2(5):525-531.
The standard treatment for CH-C, pegylated interferon-α and ribavirin (PEG-IFN + RBV), is associated with depression. Recent studies have proposed a new role for cytokines in the pathogenesis of depression. We aimed to assess differential gene expression related to depression in CH-C patients treated with PEG-IFN + RBV. We included 67 CH-C patients being treated with PEG-IFN+RBV. Of the entire study cohort, 22% had pre-existing depression, while another 37% developed new depression in course of the treatment. Pretreatment blood samples were collected into PAXgene™ RNA tubes, the RNAs extracted from peripheral blood mononuclear cells (PBMCs) were used for one step RT-PCR to profile 160 mRNAs. Differentially expressed genes were separated into up- and down-regulated genes according to presence or absence of depression at baseline (pre-existing depression) or following the initiation of treatment (treatment-related depression). The mRNA expression profile associated with any depression and with treatment-related depression included four and six genes, respectively. Our data demonstrate a significant down-regulation of TGF-β1 and the shift of Th1-Th2 cytokine balance in the depression associated with IFN-based treatment of HCV infection. We propose that TGF-β1 plays an important role in the imbalance of Th1/Th2 in patients with CH-C and depression. With further validation, TGF-β1 and other components of Th1/Th2 regulation pathway may provide a future marker for CH-C patients predisposed to depression.
PMCID: PMC3489805  PMID: 23139898
Depression; hepatitis C; interferon; ribavirin; TGFβ1; Th1/Th2 cytokines; treatment
7.  Comparative analysis of essential collective dynamics and NMR-derived flexibility profiles in evolutionarily diverse prion proteins 
Prion  2011;5(3):188-200.
Collective motions on ns-µs time scales are known to have a major impact on protein folding, stability, binding and enzymatic efficiency. It is also believed that these motions may have an important role in the early stages of prion protein misfolding and prion disease. In an effort to accurately characterize these motions and their potential influence on the misfolding and prion disease transmissibility we have conducted a combined analysis of molecular dynamic simulations and NMR-derived flexibility measurements over a diverse range of prion proteins. Using a recently developed numerical formalism, we have analyzed the essential collective dynamics (ECD) for prion proteins from eight different species including human, cow, elk, cat, hamster, chicken, turtle and frog. We also compared the numerical results with flexibility profiles generated by the random coil index (RCI) from NMR chemical shifts. Prion protein backbone flexibility derived from experimental NMR data and from theoretical computations show strong agreement with each other, demonstrating that it is possible to predict the observed RCI profiles employing the numerical ECD formalism. Interestingly, flexibility differences in the loop between second b strand (S2) and the second a helix (HB) appear to distinguish prion proteins from species that are susceptible to prion disease and those that are resistant. Our results show that the different levels of flexibility in the S2-HB loop in various species are predictable via the ECD method, indicating that ECD may be used to identify disease resistant variants of prion proteins, as well as the influence of prion proteins mutations on disease susceptibility or misfolding propensity.
PMCID: PMC3226046  PMID: 21869604
prion proteins structural stability; molecular dynamics simulation; essential collective dynamics; protein dynamic domains; biomolecular NMR; rigid loop
8.  The impact of IL28B genotype on the gene expression profile of patients with chronic hepatitis C treated with pegylated interferon alpha and ribavirin 
Recent studies of CH-C patients have demonstrated a strong association between IL28B CC genotype and sustained virologic response (SVR) after PEG-IFN/RBV treatment. We aimed to assess whether IL28B alleles rs12979860 genotype influences gene expression in response to PEG-IFN/RBV in CH-C patients.
Clinical data and gene expression data were available for 56 patients treated with PEG-IFN/RBV. Whole blood was used to determine IL28B genotypes. Differential expression of 153 human genes was assessed for each treatment time point (Days: 0, 1, 7, 28, 56) and was correlated with IL28B genotype (IL28B C/C or non-C/C) over the course of the PEG-IFN/RBV treatment. Genes with statistically significant changes in their expression at each time point were used as an input for pathway analysis using KEGG Pathway Painter (KPP). Pathways were ranked based on number of gene involved separately per each study cohort.
The most striking difference between the response patterns of patients with IL28B C/C and T* genotypes during treatment, across all pathways, is a sustained pattern of treatment-induced gene expression in patients carrying IL28B C/C. In the case of IL28B T* genotype, pre-activation of genes, the lack of sustained pattern of gene expression or a combination of both were observed. This observation could potentially provide an explanation for the lower rate of SVR observed in these patients. Additionally, when the lists of IL28B genotype-specific genes which were differentially expressed in patients without SVR were compared at their baseline, IRF2 and SOCS1 genes were down-regulated regardless of patients' IL28B genotype. Furthermore, our data suggest that CH-C patients who do not have the SOCS1 gene silenced have a better chance of achieving SVR. Our observations suggest that the action of SOCS1 is independent of IL28B genotype.
IL28B CC genotype patients with CH-C show a sustained treatment-induced gene expression profile which is not seen in non-CC genotype patients. Silencing of SOCS1 is a negative and independent predictor of SVR. These data may provide some mechanistic explanation for higher rate of SVR in IL28B CC patients who are treated with PEG-IFN/RBV.
PMCID: PMC3296607  PMID: 22313623
HCV; Gene Expression; Pathway Analysis; IL28B; SOCS1; IRF2; chronic hepatitis C; HCV treatment
9.  Effects of Temperature on the p53-DNA Binding Interactions and Their Dynamical Behavior: Comparing the Wild Type to the R248Q Mutant 
PLoS ONE  2011;6(11):e27651.
The protein p53 plays an active role in the regulation of cell cycle. In about half of human cancers, the protein is inactivated by mutations located primarily in its DNA-binding domain. Interestingly, a number of these mutations possess temperature-induced DNA-binding characteristics. A striking example is the mutation of Arg248 into glutamine or tryptophan. These mutants are defective for binding to DNA at 310 K although they have been shown to bind specifically to several p53 response elements at sub-physiological temperatures (298–306 K).
Methodology/Principal Findings
This important experimental finding motivated us to examine the effects of temperature on the structure and configuration of R248Q mutant and compare it to the wild type protein. Our aim is to determine how and where structural changes of mutant variants take place due to temperature changes. To answer these questions, we compared the mutant to the wild-type proteins from two different aspects. First, we investigated the systems at the atomistic level through their DNA-binding affinity, hydrogen bond networks and spatial distribution of water molecules. Next, we assessed changes in their long-lived conformational motions at the coarse-grained level through the collective dynamics of their side-chain and backbone atoms separately.
The experimentally observed effect of temperature on the DNA-binding properties of p53 is reproduced. Analysis of atomistic and coarse-grained data reveal that changes in binding are determined by a few key residues and provide a rationale for the mutant-loss of binding at physiological temperatures. The findings can potentially enable a rescue strategy for the mutant structure.
PMCID: PMC3218007  PMID: 22110706
10.  Oxidative stress induced in rat brain by a combination of 3-nitropropionic acid and global ischemia 
In our investigation, we describe the complex model of brain oxidative stress consisted of combination of experimental brain ischemia and energy metabolism violation induced by irreversible inhibitor of mitochondrial succi-nate dehydrogenase, 3-nitropropionate (3-NPA). 3-NPA causes selective degeneration of striatum neurons, which is extremely sensitive to energy deficit. This complex model allows revealing not only biochemical but also neurological symptoms in experimental animals that permits proper estimation of protective effect of different drugs on animal status. Combination of global ischemia induced by 3-vessel occlusion of major arteries supplys rat brain and subsequent 5-day reperfusion with intraperitoneal injection of 3-nitropropionic acid induces vigorous oxidative stress in brain tissues accompanied by evident neurological symptoms in Wistar rats. Such a combination of damaging factors may be considered as a new complex experimental model of brain oxidative stress permitting the evaluation of neuroprotective effect of potential therapeutic agents. Using this model, protective effect of neuropeptide carnosine was demonstrated which is in agreement with previous data.
PMCID: PMC2894649  PMID: 20607040
Brain global ischemia; 3-nitropropionic acid; oxidative stress; carnosine
11.  In silico modelling of hormone response elements 
BMC Bioinformatics  2006;7(Suppl 4):S27.
An important step in understanding the conditions that specify gene expression is the recognition of gene regulatory elements. Due to high diversity of different types of transcription factors and their DNA binding preferences, it is a challenging problem to establish an accurate model for recognition of functional regulatory elements in promoters of eukaryotic genes.
We present a method for precise prediction of a large group of transcription factor binding sites – steroid hormone response elements. We use a large training set of experimentally confirmed steroid hormone response elements, and adapt a sequence-based statistic method of position weight matrix, for identification of the binding sites in the query sequences. To estimate the accuracy level, a table of correspondence of sensitivity vs. specificity values is constructed from a number of independent tests. Furthermore, feed-forward neural network is used for cross-verification of the predicted response elements on genomic sequences.
The proposed method demonstrates high accuracy level, and therefore can be used for prediction of hormone response elements de novo. Experimental results support our analysis by showing significant improvement of the proposed method over previous HRE recognition methods.
PMCID: PMC1780114  PMID: 17217520

Results 1-11 (11)