Healthcare resource utilization among patients receiving non-invasive testing for coronary artery disease in an outpatient setting: A cohort study reflecting daily practice trends.

BACKGROUND: Accurate, early diagnosis and the initiation of appropriate treatment is central to reducing the clinical burden of coronary artery disease (CAD); however, real-world evidence characterizing healthcare resource utilization (HCRU) associated with testing for CAD is lacking. METHODS AND RESULTS: Using a non-interventional, retrospective, secondary database analysis, patients aged ≥18 years who underwent outpatient non-invasive cardiac diagnostic testing were identified. The primary objective was to gain an understanding of pre- and post-assessment care pathways and the associated interventions for patients who underwent non-invasive testing for CAD in either an outpatient or emergency department setting. Overall, chest pain was the primary reason for the index visit (54.8%), followed by shortness of breath (23.7%), myocardial infarction (MI), coronary artery disease (CAD) or congestive heart failure (CHF) (3.8%), and other (46.8%); 3.0% of patients had no apparent reason for testing in the last 45 days. Single-photon emission computed tomography (SPECT) was the dominant diagnostic testing modality (40.3%). During the 90-day follow-up, 7.3% (n = 22,083) of patients were diagnosed with CAD; among these patients, 19.4% had repeat diagnostic testing, 26.0% of patients had a revascularization procedure, and 65.6% underwent cardiac catheterization. These rates varied by testing modality. CONCLUSIONS: In this study of a large real-world data sample, variability in the use of non-invasive tests and HCRU were evident. These results may assist efforts to optimize system-wide care/diagnostic pathways and value-based treatment decisions for patients.

Micropropagation and Cryopreservation of Yukon Draba (Draba yukonensis), a Special Concern Plant Species Endemic to Yukon Territory, Canada.

Yukon Draba (Draba yukonensis) is a small, short-lived perennial mustard species that is endemic to southwestern Yukon in Canada. This plant has been categorized as a species of Special Concern. It faces the threat of habitat loss due to natural and man-made causes and a population that is unevenly distributed to a few large and several small subpopulations in the area. It will therefore be judicious to undertake investigations on the conservation of this species to save it from further deterioration which may lead to its extinction. In this study, a protocol was developed for in vitro propagation and cryopreservation of Yukon Draba. The micropropagation protocol was optimized using shoot tips which enabled clonal propagation and in vitro storage of the species. Shoots grew best in the medium containing MS basal salts and had the highest multiplication with the addition of 2 µM 6-benzylaminopurine or 5 µM Kinetin with 3% sucrose. The addition of 10 µM Indole Butyric Acid (IBA) produced the highest number of adventitious roots on the shoots and the longest root length was observed at 2 µM IBA. The rooted plantlets were transferred to greenhouse and the highest survival (87.5%) was observed for the plantlets treated with a lower concentration of IBA (2 µM). Cryopreservation protocol was developed using the droplet-vitrification method for in vitro shoot tips. Two-week-old shoots had the highest survival and regrowth following exposure to plant vitrification solution 3 (PVS3) for 30 min, prior to direct immersion of the droplets into the liquid nitrogen. The optimized protocols for the micropropagation and cryopreservation may be useful for the long-term germplasm conservation and reintroduction of this species in its natural habitat.

Marine sponge derived natural products as inhibitors of mycothiol-S-conjugate amidase.

Marine sponges have potential sources for secondary metabolites and are considered as a drug treasure house. In this work, 3D model of Mycothiol-S-conjugate amidase (Mca) was determined by comparative homology modeling program MODELLER based on the crystal structure of 1-D-myo-inositol 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis as a template. The computed model's energy was minimized and validated to obtain a stable model structure. Stable model was used for docking of nineteen bioactive compounds isolated from marine sponges against Mca using AutoDock 4.2. The docked complexes were validated and enumerated based on the AutoDock Scoring function to pick out the best marine inhibitors based on binding energy. Thus from the entire marine compounds which were docked, we got best one (Arenosclerin E) of them with optimal binding energy -13.11 kcal/mol. Further the best-docked complex was analyzed through Python Molecular Viewer software for their interaction studies. The docked protein - inhibitor complex structure was optimized using molecular dynamics simulation for 5 ps with the CHARMM-22 force field using NAMD incorporated in VMD 1.9.2 and then evaluating the stability of complex structure by calculating RMSD values. Thus from the Complex scoring and binding ability its deciphered that this marine derived compound could be promising inhibitor for Mca as drug target yet pharmacological studies have to confirm it.

Isolation and identification of actinomycetes for production of novel extracellular glutaminase free L-asparaginase.

Over the recent years glutaminase free L-asparaginase has gained more importance due to better therapeutic properties for treatment of acute lymphoblastic leukemia. Actinomycetes are known for L-asparaginase activity. In the current study, 80 actinomycetes were isolated from various soil habitats by serial dilution technique. Presence of L-asparaginase was investigated in a total of 240 actinomycetes by tubed agar method using modified M-9 medium. A total of 165 actinomycetes were found positive for L-asparaginase activity. Among these, 57 actinomycetes producing larger zones of L-asparagine hydrolysis were further screened for their capacity to produce glutaminase-free L-asparaginase. Four L-glutaminase-free actinomycetes were found to be potential L-asparaginase producers. These actinomycetes were identified as Streptomyces cyaneus (SAP 1287, CFS 1560), S. exfoliates (CFS 1557) and S. phaeochromogenes (GS 1573) on the basis of morphological and biochemical identification studies. Maximum L-asparaginase activity (19.2 Uml(-1)) was observed in culture filtrate of S. phaeochromogenes under submerged fermentation. Results indicate that S. phaeochromogenes could be a potential source of glutaminase free L-asparaginase for commercial purpose. To the best of our knowledge, this is the first report on production of glutaminase free L-asparaginase from S. cyaneus, S. exfoliatus and S. phaeochromogenes.

Multisite ion model in concentrated solutions of divalent cations (MgCl2 and CaCl2): osmotic pressure calculations.

Accurate force field parameters for ions are essential for meaningful simulation studies of proteins and nucleic acids. Currently accepted models of ions, especially for divalent ions, do not necessarily reproduce the right physiological behavior of Ca(2+) and Mg(2+) ions. Saxena and Sept (J. Chem. Theor. Comput. 2013, 9, 3538-3542) described a model, called the multisite-ion model, where instead of treating the ions as an isolated sphere, the charge was split into multiple sites with partial charge. This model provided accurate inner shell coordination of the ion with biomolecules and predicted better free energies for proteins and nucleic acids. Here, we expand and refine the multisite model to describe the behavior of divalent ions in concentrated MgCl2 and CaCl2 electrolyte solutions, eliminating the unusual ion-ion pairing and clustering of ions which occurred in the original model. We calibrate and improve the parameters of the multisite model by matching the osmotic pressure of concentrated solutions of MgCl2 to the experimental values and then use these parameters to test the behavior of CaCl2 solutions. We find that the concentrated solutions of both divalent ions exhibit the experimentally observed behavior with correct osmotic pressure, the presence of solvent separated ion pairs instead of direct ion pairs, and no aggregation of ions. The improved multisite model for (Mg(2+) and Ca(2+)) can be used in classical simulations of biomolecules at physiologically relevant salt concentrations.

Multisite Ion Models That Improve Coordination and Free Energy Calculations in Molecular Dynamics Simulations.

Current ion models in molecular mechanics are simple spheres, and their interactions are solely determined from the van der Waals radius of the sphere and the total charge. Here, we introduce a model where we distribute the total charge of the ion into n-dummy centers that are placed in the direction of the coordinating atoms. We have parametrized this model for two divalent cations, Ca(2+) and Mg(2+), and have tested the model's accuracy in a variety of simulations. With this model we are not only able to correctly predict the free energies and selectivity for cation binding sites in proteins and nucleic acids, but we achieve better coordination geometries and can capture more subtle effects such as the exchange of inner shell waters. Additionally, this model does not employ higher-order electrostatics and thus can be easily used with standard force fields.

An epilepsy/dyskinesia-associated mutation enhances BK channel activation by potentiating Ca2+ sensing.

Ca(2+)-activated BK channels modulate neuronal activities, including spike frequency adaptation and synaptic transmission. Previous studies found that Ca(2+)-binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca(2+) binding opens the gate over this distance remains unknown. By studying an Asp-to-Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca(2+) binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca(2+) sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca(2+) binding to channel opening.

Identification of group specific motifs in beta-lactamase family of proteins.

BACKGROUND: Beta-lactamases are one of the most serious threats to public health. In order to combat this threat we need to study the molecular and functional diversity of these enzymes and identify signatures specific to these enzymes. These signatures will enable us to develop inhibitors and diagnostic probes specific to lactamases. The existing classification of beta-lactamases was developed nearly 30 years ago when few lactamases were available. DLact database contain more than 2000 beta-lactamase, which can be used to study the molecular diversity and to identify signatures specific to this family. METHODS: A set of 2020 beta-lactamase proteins available in the DLact database http://59.160.102.202/DLact were classified using graph-based clustering of Best Bi-Directional Hits. Non-redundant (> 90 percent identical) protein sequences from each group were aligned using T-Coffee and annotated using information available in literature. Motifs specific to each group were predicted using PRATT program. RESULTS: The graph-based classification of beta-lactamase proteins resulted in the formation of six groups (Four major groups containing 191, 726, 774 and 73 proteins while two minor groups containing 50 and 8 proteins). Based on the information available in literature, we found that each of the four major groups correspond to the four classes proposed by Ambler. The two minor groups were novel and do not contain molecular signatures of beta-lactamase proteins reported in literature. The group-specific motifs showed high sensitivity (> 70%) and very high specificity (> 90%). The motifs from three groups (corresponding to class A, C and D) had a high level of conservation at DNA as well as protein level whereas the motifs from the fourth group (corresponding to class B) showed conservation at only protein level. CONCLUSION: The graph-based classification of beta-lactamase proteins corresponds with the classification proposed by Ambler, thus there is no need for formulating a new classification. However, further characterization of two small groups may require updating the existing classification scheme. Better sensitivity and specificity of group-specific motifs identified in this study, as compared to PROSITE motifs, and their proximity to the active site indicates that these motifs represents group-specific signature of beta-lactamases and can be further developed into diagnostics and therapeutics.

The basic concepts of molecular modeling.

Molecular modeling techniques have made significant advances in recent years and are becoming essential components of many chemical, physical and biological studies. Here we present three widely used techniques used in the simulation of biomolecular systems: structural and homology modeling, molecular dynamics and molecular docking. For each of these topics we present a brief discussion of the underlying scientific basis of the technique, some simple examples of how the method is commonly applied, and some discussion of the limitations and caveats of which the user should be aware. References for further reading as well as an extensive list of software resources are provided.

Pharmacovigilance: effects of herbal components on human drugs interactions involving cytochrome P450.

Cytochrome P450 (CYP P450) enzymes are a superfamily of mono-oxygenases that are found in all kingdoms of life. The CYP P450 enzymes constitute a large superfamily of haem-thiolate proteins involved in the metabolism of a wide variety of both exogenous and endogenous compounds. The CYP activities have been shown to be involved in numerous interactions especially between drugs and herbal constituents. The majority of serious cases of drug interactions are as a result of the interference of the metabolic clearance of one drug by yet another co-administered drug, food or natural product. Gaining mechanistic knowledge towards such interactions has been accepted as an approach to avoid adverse reactions. The inductions and inhibition of CYP enzymes by natural products in the presence of a prescribed drug has led to adverse effects. Herbal medicines such as St. John's wort (Hypericum perforatum), garlic (Allium sativa), piperine (from Piper sp.), ginseng (Ginseng sp.), gingko (Gingko biloba), soya beans (Glycine max), alfalfa (Medicago sativa) and grape fruit juice show clinical interactions when co-administered with medicines. This review documents the involvement of CYP enzymes in the metabolism of known available drugs and herbal products. We also document the interactions between herbal constituents & CYP enzymes showing potential drug-herb interactions. Data on CYP450 enzymes in activation (i.e. induction or inhibition) with natural constituents is also reviewed.