Doubling the Size of the Glucocorticoid Receptor Ligand Binding Pocket by Deacylcortivazol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suino-Powell, Kelly; Xu, Yong; Zhang, Chenghai

A common feature of nuclear receptor ligand binding domains (LBD) is a helical sandwich fold that nests a ligand binding pocket within the bottom half of the domain. Here we report that the ligand pocket of glucocorticoid receptor (GR) can be continuously extended into the top half of the LBD by binding to deacylcortivazol (DAC), an extremely potent glucocorticoid. It has been puzzling for decades why DAC, which contains a phenylpyrazole replacement at the conserved 3-ketone of steroid hormones that are normally required for activation of their cognate receptors, is a potent GR activator. The crystal structure of the GRmore » LBD bound to DAC and the fourth LXXLL motif of steroid receptor coactivator 1 reveals that the GR ligand binding pocket is expanded to a size of 1,070 {angstrom}{sup 3}, effectively doubling the size of the GR dexamethasone-binding pocket of 540 {angstrom}{sup 3} and yet leaving the structure of the coactivator binding site intact. DAC occupies only {approx}50% of the space of the pocket but makes intricate interactions with the receptor around the phenylpyrazole group that accounts for the high-affinity binding of DAC. The dramatic expansion of the DAC-binding pocket thus highlights the conformational adaptability of GR to ligand binding. The new structure also allows docking of various nonsteroidal ligands that cannot be fitted into the previous structures, thus providing a new rational template for drug discovery of steroidal and nonsteroidal glucocorticoids that can be specifically designed to reach the unoccupied space of the expanded pocket.« less

Exploration of the conformational landscape in pregnane X receptor reveals a new binding pocket

PubMed Central

Chandran, Aneesh

2016-01-01

Abstract Ligand‐regulated pregnane X receptor (PXR), a member of the nuclear receptor superfamily, plays a central role in xenobiotic metabolism. Despite its critical role in drug metabolism, PXR activation can lead to adverse drug‐drug interactions and early stage metabolism of drugs. Activated PXR can induce cancer drug resistance and enhance the onset of malignancy. Since promiscuity in ligand binding makes it difficult to develop competitive inhibitors targeting PXR ligand binding pocket (LBP), it is essential to identify allosteric sites for effective PXR antagonism. Here, molecular dynamics (MD) simulation studies unravelled the existence of two different conformational states, namely “expanded” and “contracted”, in apo PXR ligand binding domain (LBD). Ligand binding events shifted this conformational equilibrium and locked the LBD in a single “ligand‐adaptable” conformational state. Ensemble‐based computational solvent mapping identified a transiently open potential small molecule binding pocket between α5 and α8 helices, named “α8 pocket”, whose opening‐closing mechanism directly correlated with the conformational shift in LBD. A virtual hit identified through structure‐based virtual screening against α8 pocket locks the pocket in its open conformation. MD simulations further revealed that the presence of small molecule at allosteric site disrupts the LBD dynamics and locks the LBD in a “tightly‐contracted” conformation. The molecular details provided here could guide new structural studies to understand PXR activation and antagonism. PMID:27515410

Bridging of a substrate between cyclodextrin and an enzyme’s active site pocket triggers a unique mode of inhibition

PubMed Central

Sule, Nitesh V; Ugrinov, Angel; Mallik, Sanku; Srivastava, D. K.

2014-01-01

Background Methionyl-7-amino-4-methylcoumarin (MetAMC) serves as a substrate for the E. coli Methionine aminopeptidase (MetAP) catalyzed reaction, and is routinely used for screening compounds to identify potential antibiotic agents. In pursuit of screening the enzyme’s inhibitors, we observed that 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), utilized to solubilize hydrophobic inhibitors, inhibited the catalytic activity of the enzyme, and such inhibition was not solely due to sequestration of the substrate by HP-β-CD. Methods The mechanistic path for the HP-β-CD mediated inhibition of MetAP was probed by performing a detailed account of steady-state kinetics, ligand binding, X-ray crystallographic, and molecular modeling studies. Results X-ray crystallographic data of the β-cyclodextrin—substrate (β-CD—MetAMC) complex reveal that while the AMC moiety of the substrate is confined within the CD cavity, the methionine moiety protrudes outward. The steady-state kinetic data for inhibition of MetAP by HP-β-CD—MetAMC conform to a model mechanism in which the substrate is “bridged” between HP-β-CD and the enzyme’s active-site pocket, forming HP-β-CD—MetAMC—MetAP as the catalytically inactive ternary complex. Molecular modeling shows that the scissile bond of HP-β-CD-bound MetAMC substrate does not reach within the proximity of the enzyme’s catalytic metal center, and thus the substrate fails to undergo cleavage. Conclusions The data presented herein suggests that the bridging of the substrate between the enzyme and HP-β-CD cavities is facilitated by interaction of their surfaces, and the resulting complex inhibits the enzyme activity. General Significance Due to its potential interaction with physiological proteins via sequestered substrates, caution must be exercised in HP-β-CD mediated delivery of drugs under pathophysiological conditions. PMID:25450177

[Disposable nursing applicator-pocket of indwelling central venous catheter].

PubMed

Wei, Congli; Ma, Chunyuan

2017-11-01

Catheter related infection is the most common complication of central venous catheter, which pathogen mainly originate from the pipe joint and the skin around puncture site. How to prevent catheter infection is an important issue in clinical nursing. The utility model disclosed a "disposable nursing applicator-pocket of indwelling central venous catheter", which is mainly used for the fixation and the protection. The main structure consists of two parts, one is medical applicator to protect the skin around puncture site, and the other is gauze pocket to protect the catheter external connector. When in use, the catheter connector is fitted into the pocket, and then the applicator is applied to cover the puncture point of the skin. Integrated design of medical applicator and gauze pocket was designed to realize double functions of fixation and protection. The disposable nursing applicator-pocket is made of medical absorbent gauze (outer layer) and non-woven fabric (inner layer), which has the characteristics of comfortable, breathable, dust filtered, bacteria filtered, waterproof, antiperspirant and anti-pollution. The utility model has the advantages of simple structure, low cost, simple operation, effective protection, easy realization and popularization.

Nicotinamide Cofactors Suppress Active-Site Labeling of Aldehyde Dehydrogenases.

PubMed

Stiti, Naim; Chandrasekar, Balakumaran; Strubl, Laura; Mohammed, Shabaz; Bartels, Dorothea; van der Hoorn, Renier A L

2016-06-17

Active site labeling by (re)activity-based probes is a powerful chemical proteomic tool to globally map active sites in native proteomes without using substrates. Active site labeling is usually taken as a readout for the active state of the enzyme because labeling reflects the availability and reactivity of active sites, which are hallmarks for enzyme activities. Here, we show that this relationship holds tightly, but we also reveal an important exception to this rule. Labeling of Arabidopsis ALDH3H1 with a chloroacetamide probe occurs at the catalytic Cys, and labeling is suppressed upon nitrosylation and oxidation, and upon treatment with other Cys modifiers. These experiments display a consistent and strong correlation between active site labeling and enzymatic activity. Surprisingly, however, labeling is suppressed by the cofactor NAD(+), and this property is shared with other members of the ALDH superfamily and also detected for unrelated GAPDH enzymes with an unrelated hydantoin-based probe in crude extracts of plant cell cultures. Suppression requires cofactor binding to its binding pocket. Labeling is also suppressed by ALDH modulators that bind at the substrate entrance tunnel, confirming that labeling occurs through the substrate-binding cavity. Our data indicate that cofactor binding adjusts the catalytic Cys into a conformation that reduces the reactivity toward chloroacetamide probes.

Mutation at a strictly conserved, active site tyrosine in the copper amine oxidase leads to uncontrolled oxygenase activity.

PubMed

Chen, Zhi-Wei; Datta, Saumen; Dubois, Jennifer L; Klinman, Judith P; Mathews, F Scott

2010-08-31

The copper amine oxidases carry out two copper-dependent processes: production of their own redox-active cofactor (2,4,5-trihydroxyphenylalanine quinone, TPQ) and the subsequent oxidative deamination of substrate amines. Because the same active site pocket must facilitate both reactions, individual active site residues may serve multiple roles. We have examined the roles of a strictly conserved active site tyrosine Y305 in the copper amine oxidase from Hansenula polymorpha kinetically, spetroscopically (Dubois and Klinman (2006) Biochemistry 45, 3178), and, in the present work, structurally. While the Y305A enzyme is almost identical to the wild type, a novel, highly oxygenated species replaces TPQ in the Y305F active sites. This new structure not only provides the first direct detection of peroxy intermediates in cofactor biogenesis but also indicates the critical control of oxidation chemistry that can be conferred by a single active site residue.

The 2.1Å Crystal Structure of an Acyl-CoA Synthetase from Methanosarcina acetivorans reveals an alternate acyl binding pocket for small branched acyl substrates†,‡

PubMed Central

Shah, Manish B.; Ingram-Smith, Cheryl; Cooper, Leroy L.; Qu, Jun; Meng, Yu; Smith, Kerry S.; Gulick, Andrew M.

2009-01-01

The acyl-AMP forming family of adenylating enzymes catalyze two-step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X-ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl-CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl-CoA synthetase and 4-chlorobenzoate:CoA ligase. Most interestingly, the acyl binding pocket of the new structure is compared with other acyl- and aryl-CoA synthetases. A comparison of the acyl-binding pocket of the acyl-CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl binding pocket. PMID:19544569

AlloPred: prediction of allosteric pockets on proteins using normal mode perturbation analysis.

PubMed

Greener, Joe G; Sternberg, Michael J E

2015-10-23

Despite being hugely important in biological processes, allostery is poorly understood and no universal mechanism has been discovered. Allosteric drugs are a largely unexplored prospect with many potential advantages over orthosteric drugs. Computational methods to predict allosteric sites on proteins are needed to aid the discovery of allosteric drugs, as well as to advance our fundamental understanding of allostery. AlloPred, a novel method to predict allosteric pockets on proteins, was developed. AlloPred uses perturbation of normal modes alongside pocket descriptors in a machine learning approach that ranks the pockets on a protein. AlloPred ranked an allosteric pocket top for 23 out of 40 known allosteric proteins, showing comparable and complementary performance to two existing methods. In 28 of 40 cases an allosteric pocket was ranked first or second. The AlloPred web server, freely available at http://www.sbg.bio.ic.ac.uk/allopred/home, allows visualisation and analysis of predictions. The source code and dataset information are also available from this site. Perturbation of normal modes can enhance our ability to predict allosteric sites on proteins. Computational methods such as AlloPred assist drug discovery efforts by suggesting sites on proteins for further experimental study.

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens.

PubMed

Naz, Sadia; Ngo, Tony; Farooq, Umar; Abagyan, Ruben

2017-01-01

The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis . The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli , two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis . Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The derived pocket

Enthalpic Breakdown of Water Structure on Protein Active-Site Surfaces

PubMed Central

Haider, Kamran; Wickstrom, Lauren; Ramsey, Steven; Gilson, Michael K.; Kurtzman, Tom

2016-01-01

The principles underlying water reorganization around simple non-polar solutes are well understood and provide the framework for classical hydrophobic effect, whereby water molecules structure themselves around solutes so that they maintain favorable energetic contacts with both the solute and with other water molecules. However, for certain solute surface topographies, water molecules, due to their geometry and size, are unable to simultaneously maintain favorable energetic contacts with both the surface and neighboring water molecules. In this study, we analyze the solvation of ligand-binding sites for six structurally diverse proteins using hydration site analysis and measures of local water structure, in order to identify surfaces at which water molecules are unable to structure themselves in a way that maintains favorable enthalpy relative to bulk water. These surfaces are characterized by a high degree of enclosure, weak solute-water interactions, and surface constraints that induce unfavorable pair interactions between neighboring water molecules. Additionally, we find that the solvation of charged side-chains in an active site generally results in favorable enthalpy but can also lead to pair interactions between neighboring water molecules that are significantly unfavorable relative to bulk water. We find that frustrated local structure can occur not only in apolar and weakly polar pockets, where overall enthalpy tends to be unfavorable, but also in charged pockets, where overall water enthalpy tends to be favorable. The characterization of local water structure in these terms may prove useful for evaluating the displacement of water from diverse protein active-site environments. PMID:27169482

An Insight into the Environmental Effects of the Pocket of the Active Site of the Enzyme. Ab initio ONIOM-Molecular Dynamics (MD) Study on Cytosine Deaminase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsubara, Toshiaki; Dupuis, Michel; Aida, Misako

2008-02-01

We applied the ONIOM-molecular dynamics (MD) method to cytosine deaminase to examine the environmental effects of the amino acid residues in the pocket of the active site on the substrate taking account of their thermal motion. The ab initio ONIOM-MD simulations show that the substrate uracil is strongly perturbed by the amino acid residue Ile33, which sandwiches the uracil with His62, through the steric contact due to the thermal motion. As a result, the magnitude of the thermal oscillation of the potential energy and structure of the substrate uracil significantly increases. TM and MA were partly supported by grants frommore » the Ministry of Education, Culture, Sports, Science and Technology of Japan.MD was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, and by the Office of Biological and Environmental Research of the U.S. Department of Energy DOE. Battelle operates Pacific Northwest National Laboratory for DOE.« less

Associations Between Sedentary Behavior, Physical Activity, and Out-of-Pocket Health Care Expenditure: Evidence From Chinese Older Adults.

PubMed

Yu, Hongjun; Schwingel, Andiara

2018-06-12

This study examined the associations between sedentary behavior, physical activity, and health care expenditures among Chinese older adults. We conducted a survey on 4,165 older men and women living in major cities in China. Sedentary behavior and physical activity were measured by the Physical Activity Scale for the Elderly (PASE) questionnaire. Healthcare costs were assessed by self-reported out-of-pocket health care expenditures across outpatient care, inpatient care, medication, and formal caregiver expenses. Sedentary behavior was associated with an increase in annual out-of-pocket health care expenditures by approximately USD$37 for each additional sedentary hour (p < 0.001). Physical activity was associated with a decrease in annual health care expenditures by approximately USD$1.2 for each 1 PASE score (p < 0.001). Physical activity was a less salient indicator of health care expenditure for men than women. Reducing sedentary behavior among older men and women and promoting physical activity, especially among men, may be important strategies to reduce out-of-pocket health care expenditures in China.

Mechanistic differences between methanol and dimethyl ether carbonylation in side pockets and large channels of mordenite.

PubMed

Boronat, Mercedes; Martínez, Cristina; Corma, Avelino

2011-02-21

The activity and selectivity towards carbonylation presented by Brønsted acid sites located inside the 8MR pockets or in the main 12MR channels of mordenite is studied by means of quantum-chemical calculations, and the mechanistic differences between methanol and DME carbonylation are investigated. The selectivity towards carbonylation is higher inside the 8MR pockets, where the competitive formation of DME and hydrocarbons that finally leads to catalyst deactivation is sterically impeded. Moreover, inclusion of dispersion interactions in the calculations leads to agreement between the calculated activation barriers for the rate determining step and the experimentally observed higher reactivity of methoxy groups located inside the 8MR channels.

An Augmented Pocketome: Detection and Analysis of Small-Molecule Binding Pockets in Proteins of Known 3D Structure.

PubMed

Bhagavat, Raghu; Sankar, Santhosh; Srinivasan, Narayanaswamy; Chandra, Nagasuma

2018-03-06

Protein-ligand interactions form the basis of most cellular events. Identifying ligand binding pockets in proteins will greatly facilitate rationalizing and predicting protein function. Ligand binding sites are unknown for many proteins of known three-dimensional (3D) structure, creating a gap in our understanding of protein structure-function relationships. To bridge this gap, we detect pockets in proteins of known 3D structures, using computational techniques. This augmented pocketome (PocketDB) consists of 249,096 pockets, which is about seven times larger than what is currently known. We deduce possible ligand associations for about 46% of the newly identified pockets. The augmented pocketome, when subjected to clustering based on similarities among pockets, yielded 2,161 site types, which are associated with 1,037 ligand types, together providing fold-site-type-ligand-type associations. The PocketDB resource facilitates a structure-based function annotation, delineation of the structural basis of ligand recognition, and provides functional clues for domains of unknown functions, allosteric proteins, and druggable pockets. Copyright © 2018 Elsevier Ltd. All rights reserved.

Analysis of drug binding pockets and repurposing opportunities for twelve essential enzymes of ESKAPE pathogens

PubMed Central

Naz, Sadia; Ngo, Tony; Farooq, Umar

2017-01-01

Background The rapid increase in antibiotic resistance by various bacterial pathogens underlies the significance of developing new therapies and exploring different drug targets. A fraction of bacterial pathogens abbreviated as ESKAPE by the European Center for Disease Prevention and Control have been considered a major threat due to the rise in nosocomial infections. Here, we compared putative drug binding pockets of twelve essential and mostly conserved metabolic enzymes in numerous bacterial pathogens including those of the ESKAPE group and Mycobacterium tuberculosis. The comparative analysis will provide guidelines for the likelihood of transferability of the inhibitors from one species to another. Methods Nine bacterial species including six ESKAPE pathogens, Mycobacterium tuberculosis along with Mycobacterium smegmatis and Eschershia coli, two non-pathogenic bacteria, have been selected for drug binding pocket analysis of twelve essential enzymes. The amino acid sequences were obtained from Uniprot, aligned using ICM v3.8-4a and matched against the Pocketome encyclopedia. We used known co-crystal structures of selected target enzyme orthologs to evaluate the location of their active sites and binding pockets and to calculate a matrix of pairwise sequence identities across each target enzyme across the different species. This was used to generate sequence maps. Results High sequence identity of enzyme binding pockets, derived from experimentally determined co-crystallized structures, was observed among various species. Comparison at both full sequence level and for drug binding pockets of key metabolic enzymes showed that binding pockets are highly conserved (sequence similarity up to 100%) among various ESKAPE pathogens as well as Mycobacterium tuberculosis. Enzymes orthologs having conserved binding sites may have potential to interact with inhibitors in similar way and might be helpful for design of similar class of inhibitors for a particular species. The

PockDrug: A Model for Predicting Pocket Druggability That Overcomes Pocket Estimation Uncertainties.

PubMed

Borrel, Alexandre; Regad, Leslie; Xhaard, Henri; Petitjean, Michel; Camproux, Anne-Claude

2015-04-27

Predicting protein druggability is a key interest in the target identification phase of drug discovery. Here, we assess the pocket estimation methods' influence on druggability predictions by comparing statistical models constructed from pockets estimated using different pocket estimation methods: a proximity of either 4 or 5.5 Å to a cocrystallized ligand or DoGSite and fpocket estimation methods. We developed PockDrug, a robust pocket druggability model that copes with uncertainties in pocket boundaries. It is based on a linear discriminant analysis from a pool of 52 descriptors combined with a selection of the most stable and efficient models using different pocket estimation methods. PockDrug retains the best combinations of three pocket properties which impact druggability: geometry, hydrophobicity, and aromaticity. It results in an average accuracy of 87.9% ± 4.7% using a test set and exhibits higher accuracy (∼5-10%) than previous studies that used an identical apo set. In conclusion, this study confirms the influence of pocket estimation on pocket druggability prediction and proposes PockDrug as a new model that overcomes pocket estimation variability.

Real-Time Ligand Binding Pocket Database Search Using Local Surface Descriptors

PubMed Central

Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

2010-01-01

Due to the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of a particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two dimensional pseudo-Zernike moments or the 3D Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark study employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed. PMID:20455259

Real-time ligand binding pocket database search using local surface descriptors.

PubMed

Chikhi, Rayan; Sael, Lee; Kihara, Daisuke

2010-07-01

Because of the increasing number of structures of unknown function accumulated by ongoing structural genomics projects, there is an urgent need for computational methods for characterizing protein tertiary structures. As functions of many of these proteins are not easily predicted by conventional sequence database searches, a legitimate strategy is to utilize structure information in function characterization. Of particular interest is prediction of ligand binding to a protein, as ligand molecule recognition is a major part of molecular function of proteins. Predicting whether a ligand molecule binds a protein is a complex problem due to the physical nature of protein-ligand interactions and the flexibility of both binding sites and ligand molecules. However, geometric and physicochemical complementarity is observed between the ligand and its binding site in many cases. Therefore, ligand molecules which bind to a local surface site in a protein can be predicted by finding similar local pockets of known binding ligands in the structure database. Here, we present two representations of ligand binding pockets and utilize them for ligand binding prediction by pocket shape comparison. These representations are based on mapping of surface properties of binding pockets, which are compactly described either by the two-dimensional pseudo-Zernike moments or the three-dimensional Zernike descriptors. These compact representations allow a fast real-time pocket searching against a database. Thorough benchmark studies employing two different datasets show that our representations are competitive with the other existing methods. Limitations and potentials of the shape-based methods as well as possible improvements are discussed.

Raf kinase inhibitory protein function is regulated via a flexible pocket and novel phosphorylation-dependent mechanism.

PubMed

Granovsky, Alexey E; Clark, Matthew C; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-03-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Camproux, Anne-Claude; Petitjean, Michel

2017-11-01

We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA-ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9).

Cavity Versus Ligand Shape Descriptors: Application to Urokinase Binding Pockets

PubMed Central

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Camproux, Anne-Claude

2017-01-01

Abstract We analyzed 78 binding pockets of the human urokinase plasminogen activator (uPA) catalytic domain extracted from a data set of crystallized uPA–ligand complexes. These binding pockets were computed with an original geometric method that does NOT involve any arbitrary parameter, such as cutoff distances, angles, and so on. We measured the deviation from convexity of each pocket shape with the pocket convexity index (PCI). We defined a new pocket descriptor called distributional sphericity coefficient (DISC), which indicates to which extent the protein atoms of a given pocket lie on the surface of a sphere. The DISC values were computed with the freeware PCI. The pocket descriptors and their high correspondences with ligand descriptors are crucial for polypharmacology prediction. We found that the protein heavy atoms lining the urokinases binding pockets are either located on the surface of their convex hull or lie close to this surface. We also found that the radii of the urokinases binding pockets and the radii of their ligands are highly correlated (r = 0.9). PMID:28570103

[Impact of volatile sulphur compounds in periodontal pockets on initial periodontal therapy].

PubMed

Li, Xiao-Jun; Dong, Ling-Ling; Kong, Jing-Jia

2008-07-01

To investigate the relationship of volatile sulphur compounds((VSC)levels in periodontal pockets with severity of periodontitis, and the impact of VSC on the result of initial periodontal therapy. Twenty-five patients with chronic periodontitis(CP)(13 males and 12 females with average age of 35) were included in this study. Clinical periodontal parameters, plaque index, probing depth(PD), attachment loss(AL), and bleeding on probing(BOP) were recorded before and 3 months after the initial therapy. VSC levels were measured with a portable monitor in a digital score ranging from 0.0 to 5.0. All of 5 054 sites for 840 teeth were included in this study. Before treatment the percentage of VSC-positive sites was 17.1%, 52.3% and 86.0% for shallow (PD<3 mm), moderate(PD 4-6 mm) and deep (PD>7 mm) pocket, respectively (P<0.001). In most VSC-positive sites the VSC levels were<1.0. Percentage of sites with a high VSC levels was significantly different among three groups (P<0.01). All clinical parameters in VSC-negative sites were reduced significantly following the initial therapy. The reduction of PD and AL in VSC-positive sites by treatment was less marked than that in VSC-negative sites. VSC in periodontal pockets may be a potential indicator for detecting severity of CP and a useful predictor for therapeutic success.

Raf Kinase Inhibitory Protein Function Is Regulated via a Flexible Pocket and Novel Phosphorylation-Dependent Mechanism▿ †

PubMed Central

Granovsky, Alexey E.; Clark, Matthew C.; McElheny, Dan; Heil, Gary; Hong, Jia; Liu, Xuedong; Kim, Youngchang; Joachimiak, Grazyna; Joachimiak, Andrzej; Koide, Shohei; Rosner, Marsha Rich

2009-01-01

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding- and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics. PMID:19103740

Assessment of Drug Binding Potential of Pockets in the NS2B/NS3 Dengue Virus Protein

NASA Astrophysics Data System (ADS)

Amelia, F.; Iryani; Sari, P. Y.; Parikesit, A. A.; Bakri, R.; Toepak, E. P.; Tambunan, U. S. F.

2018-04-01

Every year an endemic dengue fever estimated to affect over 390 million cases in over 128 countries occurs. However, the antigen types which stimulate the human immune response are variable, as a result, neither effective vaccines nor antiviral treatments have been successfully developed for this disease. The NS2B/NS3 protease of the dengue virus (DENV) responsible for viral replication is a potential drug target. The ligand-enzyme binding site determination is a key role in the success of virtual screening of new inhibitors. The NS2B/NS3 protease of DENV (PDB ID: 2FOM) has two pockets consisting of 37 (Pocket 1) and 27 (Pocket 2) amino acid residues in each pocket. In this research, we characterized the amino acid residues for binding sites in NS3/NS2B based on the hydrophobicity, the percentage of charged residues, volume, depth, ΔGbinding, hydrogen bonding and bond length. The hydrophobic percentages of both pockets are high, 59 % (Pocket 1) and 41% (Pocket 2) and the percentage of charged residues in Pocket 1 and 2 are 22% and 48%, and the pocket volume is less than 700 Å3. An interaction analysis using molecular docking showed that interaction between the ligand complex and protein in Pocket 1 is more negative than Pocket 2. As a result, Pocket 1 is the better potential target for a ligand to inhibit the action of NS2B/NS3 DENV.

Circadian periodicity of resistance to ionizing radiation in the pocket mouse.

NASA Technical Reports Server (NTRS)

Lindberg, R. G.; Hayden, P.; Gambino, J. J.

1971-01-01

Investigation of the response of pocket mice to Co 60 irradiation delivered at two times of day - namely, the predicted high and low points of the metabolic rate. The validity of torpor as an assay of the circadian period of body temperature in pocket mice and as a basis for selecting irradiation times is examined. A study is made of the mitotic activity in the pocket mouse intestinal epithelium as an example of a physiological rhythm which might influence radiation sensitivity. The results of tests in which pocket mice were exposed to ionizing radiation at two different times of day are cited. It is found that under the investigated conditions pocket mice irradiated during their metabolically active period (2330 hr) live significantly longer than those irradiated while their metabolic rate is low (0900 hr).

Understanding Cryptic Pocket Formation in Protein Targets by Enhanced Sampling Simulations.

PubMed

Oleinikovas, Vladimiras; Saladino, Giorgio; Cossins, Benjamin P; Gervasio, Francesco L

2016-11-02

Cryptic pockets, that is, sites on protein targets that only become apparent when drugs bind, provide a promising alternative to classical binding sites for drug development. Here, we investigate the nature and dynamical properties of cryptic sites in four pharmacologically relevant targets, while comparing the efficacy of various simulation-based approaches in discovering them. We find that the studied cryptic sites do not correspond to local minima in the computed conformational free energy landscape of the unliganded proteins. They thus promptly close in all of the molecular dynamics simulations performed, irrespective of the force-field used. Temperature-based enhanced sampling approaches, such as Parallel Tempering, do not improve the situation, as the entropic term does not help in the opening of the sites. The use of fragment probes helps, as in long simulations occasionally it leads to the opening and binding to the cryptic sites. Our observed mechanism of cryptic site formation is suggestive of an interplay between two classical mechanisms: induced-fit and conformational selection. Employing this insight, we developed a novel Hamiltonian Replica Exchange-based method "SWISH" (Sampling Water Interfaces through Scaled Hamiltonians), which combined with probes resulted in a promising general approach for cryptic site discovery. We also addressed the issue of "false-positives" and propose a simple approach to distinguish them from druggable cryptic pockets. Our simulations, whose cumulative sampling time was more than 200 μs, help in clarifying the molecular mechanism of pocket formation, providing a solid basis for the choice of an efficient computational method.

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦

PubMed Central

Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert

2008-01-01

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466

What induces pocket openings on protein surface patches involved in protein-protein interactions?

PubMed

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-X(L), IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

What induces pocket openings on protein surface patches involved in protein-protein interactions?

NASA Astrophysics Data System (ADS)

Eyrisch, Susanne; Helms, Volkhard

2009-02-01

We previously showed for the proteins BCL-XL, IL-2, and MDM2 that transient pockets at their protein-protein binding interfaces can be identified by applying the PASS algorithm to molecular dynamics (MD) snapshots. We now investigated which aspects of the natural conformational dynamics of proteins induce the formation of such pockets. The pocket detection protocol was applied to three different conformational ensembles for the same proteins that were extracted from MD simulations of the inhibitor bound crystal conformation in water and the free crystal/NMR structure in water and in methanol. Additional MD simulations studied the impact of backbone mobility. The more efficient CONCOORD or normal mode analysis (NMA) techniques gave significantly smaller pockets than MD simulations, whereas tCONCOORD generated pockets comparable to those observed in MD simulations for two of the three systems. Our findings emphasize the influence of solvent polarity and backbone rearrangements on the formation of pockets on protein surfaces and should be helpful in future generation of transient pockets as putative ligand binding sites at protein-protein interfaces.

Visualisation of variable binding pockets on protein surfaces by probabilistic analysis of related structure sets.

PubMed

Ashford, Paul; Moss, David S; Alex, Alexander; Yeap, Siew K; Povia, Alice; Nobeli, Irene; Williams, Mark A

2012-03-14

Protein structures provide a valuable resource for rational drug design. For a protein with no known ligand, computational tools can predict surface pockets that are of suitable size and shape to accommodate a complementary small-molecule drug. However, pocket prediction against single static structures may miss features of pockets that arise from proteins' dynamic behaviour. In particular, ligand-binding conformations can be observed as transiently populated states of the apo protein, so it is possible to gain insight into ligand-bound forms by considering conformational variation in apo proteins. This variation can be explored by considering sets of related structures: computationally generated conformers, solution NMR ensembles, multiple crystal structures, homologues or homology models. It is non-trivial to compare pockets, either from different programs or across sets of structures. For a single structure, difficulties arise in defining particular pocket's boundaries. For a set of conformationally distinct structures the challenge is how to make reasonable comparisons between them given that a perfect structural alignment is not possible. We have developed a computational method, Provar, that provides a consistent representation of predicted binding pockets across sets of related protein structures. The outputs are probabilities that each atom or residue of the protein borders a predicted pocket. These probabilities can be readily visualised on a protein using existing molecular graphics software. We show how Provar simplifies comparison of the outputs of different pocket prediction algorithms, of pockets across multiple simulated conformations and between homologous structures. We demonstrate the benefits of use of multiple structures for protein-ligand and protein-protein interface analysis on a set of complexes and consider three case studies in detail: i) analysis of a kinase superfamily highlights the conserved occurrence of surface pockets at the active

Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes.

PubMed

Geeraerts, Zachary; Celis, Arianna I; Mayfield, Jeffery A; Lorenz, Megan; Rodgers, Kenton R; DuBois, Jennifer L; Lukat-Rodgers, Gudrun S

2018-03-06

O 2 -evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO 2 - ) to O 2 and Cl - . Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO 2 - , albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10 -9 M, the K D for the KpCld-CN - complex is 2 orders of magnitude smaller than that of DaCld-CN - and indicates an affinity for CN - that is greater than that of most heme proteins. The difference in CN - affinity between Kp- and DaClds is predominantly due to differences in k off . The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p K a of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 396 cm -1 ) and KpCld-CN - (ν Fe-C , 441 cm -1 ; δ FeCN , 356 cm -1 ) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.

Active sites prediction and binding analysis E1-E2 protein human papillomavirus with biphenylsulfonacetic acid

NASA Astrophysics Data System (ADS)

Iryani, I.; Amelia, F.; Iswendi, I.

2018-04-01

Cervix cancer triggered by Human papillomavirus infection is the second cause to woman death in worldwide. The binding site of E1-E2 protein of HPV 16 is not known from a 3-D structure yet, so in this study we address this issue to study the structure of E1-E2 protein from Human papillomavirus type 16 and to find its potential binding sites using biphenylsulfonacetic acid as inhibitor. Swiss model was used for 3D structure prediction and PDB: 2V9P (E1 protein) and 2NNU (E2 protein) having 52.32% and 100% identity respectively was selected as a template. The 3D model structure developed of E1 and E2 in the core and allowed regions were 99.2% and 99.5%. The ligand binding sites were predicted using online server meta pocket 2.0 and MOE 2009.10 was used for docking. E1-and E2 protein of HPV-16 has three potential binding site that can interact with the inhibitors. The Docking biphenylsulfonacetic acid using these binding sites shows that ligand interact with the protein through hydrogen bonds on Lys 403, Arg 410, His 551 in the first pocket, on Tyr 32, Leu 99 in the second pocket, and Lys 558m Lys 517 in the third pocket.

Relocating the Active-Site Lysine in Rhodopsin: 2. Evolutionary Intermediates.

PubMed

Devine, Erin L; Theobald, Douglas L; Oprian, Daniel D

2016-08-30

The visual pigment rhodopsin is a G protein-coupled receptor that covalently binds its retinal chromophore via a Schiff base linkage to an active-site Lys residue in the seventh transmembrane helix. Although this residue is strictly conserved among all type II retinylidene proteins, we found previously that the active-site Lys in bovine rhodopsin (Lys296) can be moved to three other locations (G90K, T94K, S186K) while retaining the ability to form a pigment with retinal and to activate transducin in a light-dependent manner [ Devine et al. ( 2013 ) Proc. Natl. Acad. Sci. USA 110 , 13351 - 13355 ]. Because the active-site Lys is not functionally constrained to be in helix seven, it is possible that it could relocate within the protein, most likely via an evolutionary intermediate with two active-site Lys. Therefore, in this study we characterized potential evolutionary intermediates with two Lys in the active site. Four mutant rhodopsins were prepared in which the original Lys296 was left untouched and a second Lys residue was substituted for G90K, T94K, S186K, or F293K. All four constructs covalently bind 11-cis-retinal, form a pigment, and activate transducin in a light-dependent manner. These results demonstrate that rhodopsin can tolerate a second Lys in the retinal binding pocket and suggest that an evolutionary intermediate with two Lys could allow migration of the Schiff base Lys to a position other than the observed, highly conserved location in the seventh TM helix. From sequence-based searches, we identified two groups of natural opsins, insect UV cones and neuropsins, that contain Lys residues at two positions in their active sites and also have intriguing spectral similarities to the mutant rhodopsins studied here.

Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

PubMed Central

Kandasamy, Suresh K.

2016-01-01

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing. PMID:27872309

Pulp temperature increase during photo-activated disinfection (PAD) of periodontal pockets: an in vitro study.

PubMed

El Yazami, H; Zeinoun, Toni; Bou Saba, S; Lamard, L; Peremans, A; Limme, M; Geerts, S; Lamy, M; Nammour, S

2010-09-01

The capacity of photo-sensitizers, used in combination with laser light to kill micro-organisms has been demonstrated in different studies. Photo-activated disinfection (PAD) has been introduced in periodontology as an aid for disinfection of periodontal pockets. The aim of this study is to verify the harm for dental vitality of the use of PAD in periodontal pockets. Root canals of 24 freshly extracted human teeth where prepared using profiles up to a size of ISO #50 and filled with thermo-conductor paste. A silicon-based false gum was made in which a periodontal pocket was created and filled with photo-sensitizer phenothiazine chloride (phenothiazine-5-ium, 3.7-bis (dimethylamino)-, chloride). The external root surface was irradiated during 60 s with a 660-nm diode laser (output power: 20 mW; power density: 0.090 W/cm(2); Energy density: 5.46 J/cm(2)) using a periodontal tip with a diameter of 1 mm and a length of 7 mm. Temperatures were recorded inside the root canal using a thermocouple. Measurements were recorded every second, starting at 10 s before lasering, during the irradiation and were continued for 150 s after the end of irradiation, and six measurements were done per tooth. An average temperature increase of 0.48 +/- 0.11 degrees C was recorded. Our results demonstrated that pulp temperature increase was lower than 3 degrees C, which is considered to be harmless for pulp injury. Regarding pulp temperature increase, the use of PAD for disinfection of periodontal pockets can be considered as a safe procedure for dental vitality.

Effective virtual screening protocol for CYP2C9 ligands using a screening site constructed from flurbiprofen and S-warfarin pockets

NASA Astrophysics Data System (ADS)

Polgár, Tímea; Menyhárd, Dóra K.; Keserű, György M.

2007-09-01

An effective virtual screening protocol was developed against an extended active site of CYP2C9, which was derived from X-ray structures complexed with flubiprofen and S-warfarin. Virtual screening has been effectively supported by our structure-based pharmacophore model. Importance of hot residues identified by mutation data and structural analysis was first estimated in an enrichment study. Key role of Arg108 and Phe114 in ligand binding was also underlined. Our screening protocol successfully identified 76% of known CYP2C9 ligands in the top 1% of the ranked database resulting 76-fold enrichment relative to random situation. Relevance of the protocol was further confirmed in selectivity studies, when 89% of CYP2C9 ligands were retrieved from a mixture of CYP2C9 and CYP2C8 ligands, while only 22% of CYP2C8 ligands were found applying the structure-based pharmacophore constraints. Moderate discrimination of CYP2C9 ligands from CYP2C18 and CYP2C19 ligands could also be achieved extending the application domain of our virtual screening protocol for the entire CYP2C family. Our findings further demonstrate the existence of an active site comprising of at least two binding pockets and strengthens the need of involvement of protein flexibility in virtual screening.

Novel quinolone chalcones targeting colchicine-binding pocket kill multidrug-resistant cancer cells by inhibiting tubulin activity and MRP1 function.

PubMed

Lindamulage, I Kalhari; Vu, Hai-Yen; Karthikeyan, Chandrabose; Knockleby, James; Lee, Yi-Fang; Trivedi, Piyush; Lee, Hoyun

2017-08-31

Agents targeting colchicine-binding pocket usually show a minimal drug-resistance issue, albeit often associated with high toxicity. Chalcone-based compounds, which may bind to colchicine-binding site, are found in many edible fruits, suggesting that they can be effective drugs with less toxicity. Therefore, we synthesized and examined 24 quinolone chalcone compounds, from which we identified ((E)-3-(3-(2-Methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-17) and ((E)-6-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one) (CTR-20) as promising leads. In particular, CTR-20 was effective against 65 different cancer cell lines originated from 12 different tissues, largely in a cancer cell-specific manner. We found that both CTR-17 and CTR-20 reversibly bind to the colchicine-binding pocket on β-tubulin. Interestingly however, both the CTRs were highly effective against multidrug-resistant cancer cells while colchicine, paclitaxel and vinblastine were not. Our study with CTR-20 showed that it overcomes multidrug-resistance through its ability to impede MRP1 function while maintaining strong inhibition against microtubule activity. Data from mice engrafted with the MDA-MB-231 triple-negative breast cancer cells showed that both CTR-17 and CTR-20 possess strong anticancer activity, alone or in combination with paclitaxel, without causing any notable side effects. Together, our data demonstrates that both the CTRs can be effective and safe drugs against many different cancers, especially against multidrug-resistant tumors.

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

PubMed Central

Corvo, Ileana; Ferraro, Florencia; Merlino, Alicia; Zuberbühler, Kathrin; O'Donoghue, Anthony J.; Pastro, Lucía; Pi-Denis, Natalia; Basika, Tatiana; Roche, Leda; McKerrow, James H.; Craik, Charles S.; Caffrey, Conor R.; Tort, José F.

2018-01-01

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity. PMID:29725596

Computational Assessment of Potassium and Magnesium Ion Binding to a Buried Pocket in GTPase-Associating Center RNA

PubMed Central

2016-01-01

An experimentally well-studied model of RNA tertiary structures is a 58mer rRNA fragment, known as GTPase-associating center (GAC) RNA, in which a highly negative pocket walled by phosphate oxygen atoms is stabilized by a chelated cation. Although such deep pockets with more than one direct phosphate to ion chelation site normally include magnesium, as shown in one GAC crystal structure, another GAC crystal structure and solution experiments suggest potassium at this site. Both crystal structures also depict two magnesium ions directly bound to the phosphate groups comprising this controversial pocket. Here, we used classical molecular dynamics simulations as well as umbrella sampling to investigate the possibility of binding of potassium versus magnesium inside the pocket and to better characterize the chelation of one of the binding magnesium ions outside the pocket. The results support the preference of the pocket to accommodate potassium rather than magnesium and suggest that one of the closely binding magnesium ions can only bind at high magnesium concentrations, such as might be present during crystallization. This work illustrates the complementary utility of molecular modeling approaches with atomic-level detail in resolving discrepancies between conflicting experimental results. PMID:27983843

Computational Assessment of Potassium and Magnesium Ion Binding to a Buried Pocket in GTPase-Associating Center RNA.

PubMed

Hayatshahi, Hamed S; Roe, Daniel R; Galindo-Murillo, Rodrigo; Hall, Kathleen B; Cheatham, Thomas E

2017-01-26

An experimentally well-studied model of RNA tertiary structures is a 58mer rRNA fragment, known as GTPase-associating center (GAC) RNA, in which a highly negative pocket walled by phosphate oxygen atoms is stabilized by a chelated cation. Although such deep pockets with more than one direct phosphate to ion chelation site normally include magnesium, as shown in one GAC crystal structure, another GAC crystal structure and solution experiments suggest potassium at this site. Both crystal structures also depict two magnesium ions directly bound to the phosphate groups comprising this controversial pocket. Here, we used classical molecular dynamics simulations as well as umbrella sampling to investigate the possibility of binding of potassium versus magnesium inside the pocket and to better characterize the chelation of one of the binding magnesium ions outside the pocket. The results support the preference of the pocket to accommodate potassium rather than magnesium and suggest that one of the closely binding magnesium ions can only bind at high magnesium concentrations, such as might be present during crystallization. This work illustrates the complementary utility of molecular modeling approaches with atomic-level detail in resolving discrepancies between conflicting experimental results.

Deconnable self-reading pocket dosimeter containment with self-contained light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, R.L.; Arnold, G.N.; McBride, R.G.

1995-12-31

A container for a self-reading pocket dosimeter includes a transparent tube for receiving the self-reading pocket dosimeter, a light source mounted at one end of the transparent tube, and an eyepiece mounted on an opposite end of the transparent tube for viewing a read-out of the self-reading pocket dosimeter. The container may further include an activation device for selectively supplying power to the light source. The container both protects the dosimeter from being contaminated and provides a light source for viewing the dosimeter.

Deconnable self-reading pocket dosimeter containment with self-contained light

DOEpatents

Stevens, Robyn L.; Arnold, Greg N.; McBride, Ryan G.

1996-01-01

A container for a self-reading pocket dosimeter includes a transparent tube for receiving the self-reading pocket dosimeter, a light source mounted at one end of the transparent tube, and an eyepiece mounted on an opposite end of the transparent tube for viewing a read-out of the self-reading pocket dosimeter. The container may further include an activation device for selectively supplying power to the light source. The container both protects the dosimeter from being contaminated and provides a light source for viewing the dosimeter.

Deconnable self-reading pocket dosimeter containment with self-contained light

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, R.L.; Arnold, G.N.; McBride, R.G.

1996-10-22

A container for a self-reading pocket dosimeter includes a transparent tube for receiving the self-reading pocket dosimeter, a light source mounted at one end of the transparent tube, and an eyepiece mounted on an opposite end of the transparent tube for viewing a read-out of the self-reading pocket dosimeter. The container may further include an activation device for selectively supplying power to the light source. The container both protects the dosimeter from being contaminated and provides a light source for viewing the dosimeter. 4 figs.

A Hidden Active Site in the Potential Drug Target Mycobacterium tuberculosis dUTPase Is Accessible through Small Amplitude Protein Conformational Changes*

PubMed Central

Lopata, Anna; Leveles, Ibolya; Bendes, Ábris Ádám; Viskolcz, Béla; Vértessy, Beáta G.; Jójárt, Balázs; Tóth, Judit

2016-01-01

dUTPases catalyze the hydrolysis of dUTP into dUMP and pyrophosphate to maintain the proper nucleotide pool for DNA metabolism. Recent evidence suggests that dUTPases may also represent a selective drug target in mycobacteria because of the crucial role of these enzymes in maintaining DNA integrity. Nucleotide-hydrolyzing enzymes typically harbor a buried ligand-binding pocket at interdomain or intersubunit clefts, facilitating proper solvent shielding for the catalyzed reaction. The mechanism by which substrate binds this hidden pocket and product is released in dUTPases is unresolved because of conflicting crystallographic and spectroscopic data. We sought to resolve this conflict by using a combination of random acceleration molecular dynamics (RAMD) methodology and structural and biochemical methods to study the dUTPase from Mycobacterium tuberculosis. In particular, the RAMD approach used in this study provided invaluable insights into the nucleotide dissociation process that reconciles all previous experimental observations. Specifically, our data suggest that nucleotide binding takes place as a small stretch of amino acids transiently slides away and partially uncovers the active site. The in silico data further revealed a new dUTPase conformation on the pathway to a relatively open active site. To probe this model, we developed the Trp21 reporter and collected crystallographic, spectroscopic, and kinetic data that confirmed the interaction of Trp21 with the active site shielding C-terminal arm, suggesting that the RAMD method is effective. In summary, our computational simulations and spectroscopic results support the idea that small loop movements in dUTPase allow the shuttlingof the nucleotides between the binding pocket and the solvent. PMID:27815500

Mutational analysis of the active site flap (20s loop) of mandelate racemase.

PubMed

Bourque, Jennifer R; Bearne, Stephen L

2008-01-15

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG

Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

PubMed

Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

2013-05-10

The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-based design, synthesis and crystallization of 2-arylquinazolines as lipid pocket ligands of p38α MAPK

PubMed Central

Bührmann, Mike; Wiedemann, Bianca M.; Müller, Matthias P.; Hardick, Julia; Ecke, Maria

2017-01-01

In protein kinase research, identifying and addressing small molecule binding sites other than the highly conserved ATP-pocket are of intense interest because this line of investigation extends our understanding of kinase function beyond the catalytic phosphotransfer. Such alternative binding sites may be involved in altering the activation state through subtle conformational changes, control cellular enzyme localization, or in mediating and disrupting protein-protein interactions. Small organic molecules that target these less conserved regions might serve as tools for chemical biology research and to probe alternative strategies in targeting protein kinases in disease settings. Here, we present the structure-based design and synthesis of a focused library of 2-arylquinazoline derivatives to target the lipophilic C-terminal binding pocket in p38α MAPK, for which a clear biological function has yet to be identified. The interactions of the ligands with p38α MAPK was analyzed by SPR measurements and validated by protein X-ray crystallography. PMID:28892510

An alternate binding site for PPARγ ligands

PubMed Central

Hughes, Travis S.; Giri, Pankaj Kumar; de Vera, Ian Mitchelle S.; Marciano, David P.; Kuruvilla, Dana S.; Shin, Youseung; Blayo, Anne-Laure; Kamenecka, Theodore M.; Burris, Thomas P.; Griffin, Patrick R.; Kojetin, Douglas J.

2014-01-01

PPARγ is a target for insulin sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes. Synthetic ligands have been designed to mimic endogenous ligand binding to a canonical ligand-binding pocket to hyperactivate PPARγ. Here we reveal that synthetic PPARγ ligands also bind to an alternate site, leading to unique receptor conformational changes that impact coregulator binding, transactivation and target gene expression. Using structure-function studies we show that alternate site binding occurs at pharmacologically relevant ligand concentrations, and is neither blocked by covalently bound synthetic antagonists nor by endogenous ligands indicating non-overlapping binding with the canonical pocket. Alternate site binding likely contributes to PPARγ hyperactivation in vivo, perhaps explaining why PPARγ full and partial or weak agonists display similar adverse effects. These findings expand our understanding of PPARγ activation by ligands and suggest that allosteric modulators could be designed to fine tune PPARγ activity without competing with endogenous ligands. PMID:24705063

Evolution of a designed retro-aldolase leads to complete active site remodeling

PubMed Central

Giger, Lars; Caner, Sami; Obexer, Richard; Kast, Peter; Baker, David; Ban, Nenad; Hilvert, Donald

2013-01-01

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes. PMID:23748672

Visualizing electron pockets in cuprate superconductors

NASA Astrophysics Data System (ADS)

Das, Tanmoy; Markiewicz, R. S.; Bansil, A.; Balatsky, A. V.

2012-06-01

Fingerprints of the electron pocket in cuprates have been obtained only in numerous magnetotransport measurements, but its absence in spectroscopic observations poses a long-standing mystery. We develop a theoretical tool to provide ways to detect electron pockets via spectroscopies including scanning tunneling microscopy (STM) spectra, inelastic neutron scattering (INS), and angle-resolved photoemission spectroscopy (ARPES). We show that the quasiparticle-interference (QPI) pattern, measured by STM, shows an additional seven q vectors associated with the scattering on the electron pocket than that on the hole pocket. Furthermore, the Bogolyubov quasiparticle scatterings of the electron pocket lead to a second magnetic resonance mode in the INS spectra at a higher resonance energy. Finally, we reanalyze some STM, INS, and ARPES experimental data of several cuprates which dictates the direct fingerprints of electron pockets in these systems.

The structure of amylosucrase from Deinococcus radiodurans has an unusual open active-site topology.

PubMed

Skov, Lars K; Pizzut-Serin, Sandra; Remaud-Simeon, Magali; Ernst, Heidi A; Gajhede, Michael; Mirza, Osman

2013-09-01

Amylosucrases (ASes) catalyze the formation of an α-1,4-glucosidic linkage by transferring a glucosyl unit from sucrose onto an acceptor α-1,4-glucan. To date, several ligand-bound crystal structures of wild-type and mutant ASes from Neisseria polysaccharea and Deinococcus geothermalis have been solved. These structures all display a very similar overall conformation with a deep pocket leading to the site for transglucosylation, subsite -1. This has led to speculation on how sucrose enters the active site during glucan elongation. In contrast to previous studies, the AS structure from D. radiodurans presented here has a completely empty -1 subsite. This structure is strikingly different from other AS structures, as an active-site-lining loop comprising residues Leu214-Asn225 is found in a previously unobserved conformation. In addition, a large loop harbouring the conserved active-site residues Asp133 and Tyr136 is disordered. The result of the changed loop conformations is that the active-site topology is radically changed, leaving subsite -1 exposed and partially dismantled. This structure provides novel insights into the dynamics of ASes and comprises the first structural support for an elongation mechanism that involves considerable conformational changes to modulate accessibility to the sucrose-binding site and thereby allows successive cycles of glucosyl-moiety transfer to a growing glucan chain.

Asymmetric interactions in the adenosine-binding pockets of the MS2 coat protein dimer

PubMed Central

Powell, Amy J; Peabody, David S

2001-01-01

Background The X-ray structure of the MS2 coat protein-operator RNA complex reveals the existence of quasi-synmetric interactions of adenosines -4 and -10 in pockets formed on different subunits of the coat protein dimer. Both pockets utilize the same five amino acid residues, namely Val29, Thr45, Ser47, Thr59, and Lys61. We call these sites the adenosine-binding pockets. Results We present here a heterodimer complementation analysis of the contributions of individual A-pocket amino acids to the binding of A-4 and A-10 in different halves of the dimer. Various substitutions of A-pocket residues were introduced into one half of single-chain coat protein heterodimers where they were tested for their abilities to complement Y85H or T91I substitutions (defects in the A-4 and A-10 half-sites, respectively) present in the other dimer half. Conclusions These experiments provide functional tests of interactions predicted from structural analyses, demonstrating the importance of certain amino acid-nucleotide contacts observed in the crystal structure, and showing that others make little or no contribution to the stability of the complex. In summary, Val29 and Lys61 form important stabilizing interactions with both A-4 and A-10. Meanwhile, Ser47 and Thr59 interact primarily with A-10. The important interactions with Thr45 are restricted to A-4. PMID:11504563

Differential recognition of syk-binding sites by each of the two phosphotyrosine-binding pockets of the Vav SH2 domain.

PubMed

Chen, Chih-Hong; Piraner, Dan; Gorenstein, Nina M; Geahlen, Robert L; Beth Post, Carol

2013-11-01

The association of spleen tyrosine kinase (Syk), a central tyrosine kinase in B cell signaling, with Vav SH2 domain is controlled by phosphorylation of two closely spaced tyrosines in Syk linker B: Y342 and Y346. Previous studies established both singly phosphorylated and doubly phosphorylated forms play a role in signaling. The structure of the doubly phosphorylated form identified a new recognition of phosphotyrosine whereby two phosphotyrosines bind simultaneously to the Vav SH2 domain, one in the canonical pTyr pocket and one in the specificity pocket on the opposite side of the central β-sheet. It is unknown if the specificity pocket can bind phosphotyrosine independent of phosphotyrosine binding the pTyr pocket. To address this gap in knowledge, we determined the structure of the complex between Vav1 SH2 and a peptide (SykLB-YpY) modeling the singly phosphorylated-Y346 form of Syk with unphosphorylated Y342. The nuclear magnetic resonance (NMR) data conclusively establish that recognition of phosphotyrosine is swapped between the two pockets; phosphorylated pY346 binds the specificity pocket of Vav1 SH2, and unphosphorylated Y342 occupies what is normally the pTyr binding pocket. Nearly identical changes in chemical shifts occurred upon binding all three forms of singly and doubly phosphorylated peptides; however, somewhat smaller shift perturbations for SykLB-YpY from residues in regions of high internal mobility suggest that internal motions are coupled to binding affinity. The differential recognition that includes this swapped binding of phosphotyrosine to the specificity pocket of Vav SH2 increases the repertoire of possible phosphotyrosine binding by SH2 domains in regulating protein-protein interactions in cellular signaling. Copyright © 2013 Wiley Periodicals, Inc.

Hand, belt, pocket or bag: Practical activity tracking with mobile phones

PubMed Central

Antos, Stephen A.; Albert, Mark V.; Kording, Konrad P.

2013-01-01

For rehabilitation and diagnoses, an understanding of patient activities and movements is important. Modern smartphones have built in accelerometers which promise to enable quantifying minute-by-minute what patients do (e.g. walk or sit). Such a capability could inform recommendations of physical activities and improve medical diagnostics. However, a major problem is that during everyday life, we carry our phone in different ways, e.g. on our belt, in our pocket, in our hand, or in a bag. The recorded accelerations are not only affected by our activities but also by the phone’s location. Here we develop a method to solve this kind of problem, based on the intuition that activities change rarely, and phone locations change even less often. A Hidden Markov Model (HMM) tracks changes across both activities and locations, enabled by a static Support Vector Machine (SVM) classifier that probabilistically identifies activity-location pairs. We find that this approach improves tracking accuracy on healthy subjects as compared to a static classifier alone. The obtained method can be readily applied to patient populations. Our research enables the use of phones as activity tracking devices, without the need of previous approaches to instruct subjects to always carry the phone in the same location. PMID:24091138

Hand, belt, pocket or bag: Practical activity tracking with mobile phones.

PubMed

Antos, Stephen A; Albert, Mark V; Kording, Konrad P

2014-07-15

For rehabilitation and diagnoses, an understanding of patient activities and movements is important. Modern smartphones have built in accelerometers which promise to enable quantifying minute-by-minute what patients do (e.g. walk or sit). Such a capability could inform recommendations of physical activities and improve medical diagnostics. However, a major problem is that during everyday life, we carry our phone in different ways, e.g. on our belt, in our pocket, in our hand, or in a bag. The recorded accelerations are not only affected by our activities but also by the phone's location. Here we develop a method to solve this kind of problem, based on the intuition that activities change rarely, and phone locations change even less often. A hidden Markov model (HMM) tracks changes across both activities and locations, enabled by a static support vector machine (SVM) classifier that probabilistically identifies activity-location pairs. We find that this approach improves tracking accuracy on healthy subjects as compared to a static classifier alone. The obtained method can be readily applied to patient populations. Our research enables the use of phones as activity tracking devices, without the need of previous approaches to instruct subjects to always carry the phone in the same location. Copyright © 2013 Elsevier B.V. All rights reserved.

Common Anesthetic-binding Site for Inhibition of Pentameric Ligand-gated Ion Channels.

PubMed

Kinde, Monica N; Bu, Weiming; Chen, Qiang; Xu, Yan; Eckenhoff, Roderic G; Tang, Pei

2016-03-01

Identifying functionally relevant anesthetic-binding sites in pentameric ligand-gated ion channels (pLGICs) is an important step toward understanding the molecular mechanisms underlying anesthetic action. The anesthetic propofol is known to inhibit cation-conducting pLGICs, including a prokaryotic pLGIC from Erwinia chrysanthemi (ELIC), but the sites responsible for functional inhibition remain undetermined. We photolabeled ELIC with a light-activated derivative of propofol (AziPm) and performed fluorine-19 nuclear magnetic resonance experiments to support propofol binding to a transmembrane domain (TMD) intrasubunit pocket. To differentiate sites responsible for propofol inhibition from those that are functionally irrelevant, we made an ELIC-γ-aminobutyric acid receptor (GABAAR) chimera that replaced the ELIC-TMD with the α1β3GABAAR-TMD and compared functional responses of ELIC-GABAAR and ELIC with propofol modulations. Photolabeling showed multiple AziPm-binding sites in the extracellular domain (ECD) but only one site in the TMD with labeled residues M265 and F308 in the resting state of ELIC. Notably, this TMD site is an intrasubunit pocket that overlaps with binding sites for anesthetics, including propofol, found previously in other pLGICs. Fluorine-19 nuclear magnetic resonance experiments supported propofol binding to this TMD intrasubunit pocket only in the absence of agonist. Functional measurements of ELIC-GABAAR showed propofol potentiation of the agonist-elicited current instead of inhibition observed on ELIC. The distinctly different responses of ELIC and ELIC-GABAAR to propofol support the functional relevance of propofol binding to the TMD. Combining the newly identified TMD intrasubunit pocket in ELIC with equivalent TMD anesthetic sites found previously in other cationic pLGICs, we propose this TMD pocket as a common site for anesthetic inhibition of pLGICs.

Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket

PubMed Central

Su, Yang; Chong, Huihiui; Xiong, Shengwen; Qiao, Yuanyuan; Qiu, Zonglin

2015-01-01

ABSTRACT The peptide drug enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but it easily induces drug resistance, calling for new strategies for developing effective drugs. On the basis of the M-T hook structure, we recently developed highly potent short-peptide HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the conserved gp41 pocket and possess high genetic barriers to resistance. Here, we focused on the selection and characterization of HIV-1 escape mutants of MTSC22, which revealed new resistance pathways and mechanisms. Two mutations (E49K and L57R) located at the inhibitor-binding site and two mutations (N126K and E136G) located at the C-terminal heptad repeat region of gp41 were identified as conferring high resistance either singly or in combination. While E49K reduced the C-terminal binding of inhibitors via an electrostatic repulsion, L57R dramatically disrupted the N-terminal binding of M-T hook structure and pocket-binding domain. Unlike E49K and N126K, which enhanced the stability of the endogenous viral six-helical bundle core (6-HB), L57R and E136G conversely destabilized the 6-HB structure. We also demonstrated that both primary and secondary mutations caused the structural changes in 6-HB and severely impaired the capability for HIV-1 entry. Collectively, our data provide novel insights into the mechanisms of short-peptide fusion inhibitors targeting the gp41 pocket site and help increase our understanding of the structure and function of gp41 and HIV-1 evolution. IMPORTANCE The deep pocket on the N-trimer of HIV-1 gp41 has been considered an ideal drug target because of its high degree of conservation and essential role in viral entry. Short-peptide fusion inhibitors, which contain an M-T hook structure and mainly target the pocket site, show extremely high binding and inhibitory activities as well as high genetic barriers to resistance. In this study, the HIV-1 mutants resistant to MTSC22 were selected and

The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

PubMed

Strotmeier, Jasmin; Gu, Shenyan; Jutzi, Stephan; Mahrhold, Stefan; Zhou, Jie; Pich, Andreas; Eichner, Timo; Bigalke, Hans; Rummel, Andreas; Jin, Rongsheng; Binz, Thomas

2011-07-01

The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. © 2011 Blackwell Publishing Ltd.

Observing the formation of ice and organic crystals in active sites

PubMed Central

Campbell, James M.; Meldrum, Fiona C.; Christenson, Hugo K.

2017-01-01

Heterogeneous nucleation is vital to a wide range of areas as diverse as ice nucleation on atmospheric aerosols and the fabrication of high-performance thin films. There is excellent evidence that surface topography is a key factor in directing crystallization in real systems; however, the mechanisms by which nanoscale pits and pores promote nucleation remain unclear. Here, we use natural cleavage defects on Muscovite mica to investigate the activity of topographical features in the nucleation from vapor of ice and various organic crystals. Direct observation of crystallization within surface pockets using optical microscopy and also interferometry demonstrates that these sharply acute features provide extremely effective nucleation sites and allows us to determine the mechanism by which this occurs. A confined phase is first seen to form along the apex of the wedge and then grows out of the pocket opening to generate a bulk crystal after a threshold saturation has been achieved. Ice nucleation proceeds in a comparable manner, although our resolution is insufficient to directly observe a condensate before the growth of a bulk crystal. These results provide insight into the mechanism of crystal deposition from vapor on real surfaces, where this will ultimately enable us to use topography to control crystal deposition on surfaces. They are also particularly relevant to our understanding of processes such as cirrus cloud formation, where such topographical features are likely candidates for the “active sites” that make clay particles effective nucleants for ice in the atmosphere. PMID:27994140

Pocket Guide to Transportation 2017

DOT National Transportation Integrated Search

2017-05-01

The BTS Pocket Guide to Transportation is a quick reference guide to significant transportation statistics. All the previous seven sections plus a new Major Trends section are included. This year marks the 20th anniversary of the Pocket Guide, which ...

Enzyme/non-enzyme discrimination and prediction of enzyme active site location using charge-based methods.

PubMed

Bate, Paul; Warwicker, Jim

2004-07-02

Calculations of charge interactions complement analysis of a characterised active site, rationalising pH-dependence of activity and transition state stabilisation. Prediction of active site location through large DeltapK(a)s or electrostatic strain is relevant for structural genomics. We report a study of ionisable groups in a set of 20 enzymes, finding that false positives obscure predictive potential. In a larger set of 156 enzymes, peaks in solvent-space electrostatic properties are calculated. Both electric field and potential match well to active site location. The best correlation is found with electrostatic potential calculated from uniform charge density over enzyme volume, rather than from assignment of a standard atom-specific charge set. Studying a shell around each molecule, for 77% of enzymes the potential peak is within that 5% of the shell closest to the active site centre, and 86% within 10%. Active site identification by largest cleft, also with projection onto a shell, gives 58% of enzymes for which the centre of the largest cleft lies within 5% of the active site, and 70% within 10%. Dielectric boundary conditions emphasise clefts in the uniform charge density method, which is suited to recognition of binding pockets embedded within larger clefts. The variation of peak potential with distance from active site, and comparison between enzyme and non-enzyme sets, gives an optimal threshold distinguishing enzyme from non-enzyme. We find that 87% of the enzyme set exceeds the threshold as compared to 29% of the non-enzyme set. Enzyme/non-enzyme homologues, "structural genomics" annotated proteins and catalytic/non-catalytic RNAs are studied in this context.

Structural and Functional Analyses of a Conserved Hydrophobic Pocket of Flavivirus Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

H Dong; L Liu; G Zou

2011-12-31

The flavivirus methyltransferase (MTase) sequentially methylates the N7 and 2'-O positions of the viral RNA cap (GpppA-RNA {yields} m(7)GpppA-RNA {yields} m(7)GpppAm-RNA), using S-adenosyl-l-methionine (AdoMet) as a methyl donor. We report here that sinefungin (SIN), an AdoMet analog, inhibits several flaviviruses through suppression of viral MTase. The crystal structure of West Nile virus MTase in complex with SIN inhibitor at 2.0-{angstrom} resolution revealed a flavivirus-conserved hydrophobic pocket located next to the AdoMet-binding site. The pocket is functionally critical in the viral replication and cap methylations. In addition, the N7 methylation efficiency was found to correlate with the viral replication ability. Thus,more » SIN analogs with modifications that interact with the hydrophobic pocket are potential specific inhibitors of flavivirus MTase.« less

Computational approaches for identification of conserved/unique binding pockets in the A chain of ricin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecale Zhou, C L; Zemla, A T; Roe, D

2005-01-29

Specific and sensitive ligand-based protein detection assays that employ antibodies or small molecules such as peptides, aptamers, or other small molecules require that the corresponding surface region of the protein be accessible and that there be minimal cross-reactivity with non-target proteins. To reduce the time and cost of laboratory screening efforts for diagnostic reagents, we developed new methods for evaluating and selecting protein surface regions for ligand targeting. We devised combined structure- and sequence-based methods for identifying 3D epitopes and binding pockets on the surface of the A chain of ricin that are conserved with respect to a set ofmore » ricin A chains and unique with respect to other proteins. We (1) used structure alignment software to detect structural deviations and extracted from this analysis the residue-residue correspondence, (2) devised a method to compare corresponding residues across sets of ricin structures and structures of closely related proteins, (3) devised a sequence-based approach to determine residue infrequency in local sequence context, and (4) modified a pocket-finding algorithm to identify surface crevices in close proximity to residues determined to be conserved/unique based on our structure- and sequence-based methods. In applying this combined informatics approach to ricin A we identified a conserved/unique pocket in close proximity (but not overlapping) the active site that is suitable for bi-dentate ligand development. These methods are generally applicable to identification of surface epitopes and binding pockets for development of diagnostic reagents, therapeutics, and vaccines.« less

Transient Ligand Docking Sites in Cerebratulus lacteus Mini-Hemoglobin

PubMed Central

Deng, Pengchi; Nienhaus, Karin; Palladino, Pasquale; Olson, John S.; Blouin, George; Moens, Luc; Dewilde, Sylvia; Geuens, Eva; Nienhaus, G. Ulrich

2007-01-01

The monomeric hemoglobin of the nemertean worm Cerebratulus lacteus functions as an oxygen storage protein to maintain neural activity under hypoxic conditions. It shares a large, apolar matrix tunnel with other small hemoglobins, which has been implicated as a potential ligand migration pathway. Here we explore ligand migration and binding within the distal heme pocket, to which the tunnel provides access to ligands from the outside. FTIR/TDS experiments performed at cryogenic temperatures reveal the presence of three transient ligand docking sites within the distal pocket, the primary docking site B on top of pyrrole C and secondary sites C and D. Site C is assigned to a cavity adjacent to the distal portion of the heme pocket, surrounded by the B and E helices. It has an opening to the apolar tunnel and is expected to be on the pathway for ligand entry and exit, whereas site D, circumscribed by TyrB10, GlnE7, and the CD corner, most likely is located on a side pathway of ligand migration. Flash photolysis experiments at ambient temperatures indicate that the rate-limiting step for ligand binding to CerHb is migration through the apolar channel to site C. Movement from C to B and iron-ligand bond formation involve low energy barriers and thus are very rapid processes in the wt protein. PMID:17531406

Structures of BmrR-Drug Complexes Reveal a Rigid Multidrug Binding Pocket And Transcription Activation Through Tyrosine Expulsion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Newberry, K.J.; Huffman, J.L.; Miller, M.C.

2009-05-22

BmrR is a member of the MerR family and a multidrug binding transcription factor that up-regulates the expression of the bmr multidrug efflux transporter gene in response to myriad lipophilic cationic compounds. The structural mechanism by which BmrR binds these chemically and structurally different drugs and subsequently activates transcription is poorly understood. Here, we describe the crystal structures of BmrR bound to rhodamine 6G (R6G) or berberine (Ber) and cognate DNA. These structures reveal each drug stacks against multiple aromatic residues with their positive charges most proximal to the carboxylate group of Glu-253 and that, unlike other multidrug binding pockets,more » that of BmrR is rigid. Substitution of Glu-253 with either alanine (E253A) or glutamine (E253Q) results in unpredictable binding affinities for R6G, Ber, and tetraphenylphosphonium. Moreover, these drug binding studies reveal that the negative charge of Glu-253 is not important for high affinity binding to Ber and tetraphenylphosphonium but plays a more significant, but unpredictable, role in R6G binding. In vitro transcription data show that E253A and E253Q are constitutively active, and structures of the drug-free E253A-DNA and E253Q-DNA complexes support a transcription activation mechanism requiring the expulsion of Tyr-152 from the multidrug binding pocket. In sum, these data delineate the mechanism by which BmrR binds lipophilic, monovalent cationic compounds and suggest the importance of the redundant negative electrostatic nature of this rigid drug binding pocket that can be used to discriminate against molecules that are not substrates of the Bmr multidrug efflux pump.« less

NASA Pocket Statistics

NASA Technical Reports Server (NTRS)

1995-01-01

NASA Pocket Statistics is published for the use of NASA managers and their staff. Included herein is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, and NASA Procurement, Financial, and Manpower data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

NASA Pocket Statistics

NASA Technical Reports Server (NTRS)

1994-01-01

Pocket Statistics is published for the use of NASA managers and their staff. Included herein is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, and NASA Procurement, Financial, and Manpower data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

Tryptophan 80 and leucine 143 are critical for the hydride transfer step of thymidylate synthase by controlling active site access.

PubMed

Fritz, Timothy A; Liu, Lu; Finer-Moore, Janet S; Stroud, Robert M

2002-06-04

Mutant forms of thymidylate synthase (TS) with substitutions at the conserved active site residue, Trp 80, are deficient in the hydride transfer step of the TS reaction. These mutants produce a beta-mercaptoethanol (beta-ME) adduct of the 2'-deoxyuridine-5'-monophosphate (dUMP) exocyclic methylene intermediate. Trp 80 has been proposed to assist hydride transfer by stabilizing a 5,6,7,8-tetrahydrofolate (THF) radical cation intermediate [Barrett, J. E., Lucero, C. M., and Schultz, P. G. (1999) J. Am. Chem. Soc. 121, 7965-7966.] formed after THF changes its binding from the cofactor pocket to a putative alternate site. To understand the molecular basis of hydride transfer deficiency in a mutant in which Trp 80 was changed to Gly, we determined the X-ray structures of this mutant Escherichia coli TS complexed with dUMP and the folate analogue 10-propargyl-5,8-dideazafolate (CB3717) and of the wild-type enzyme complexed with dUMP and THF. The mutant enzyme has a cavity in the active site continuous with bulk solvent. This cavity, sealed from bulk solvent in wild-type TS by Leu 143, would allow nucleophilic attack of beta-ME on the dUMP C5 exocyclic methylene. The structure of the wild-type enzyme/dUMP/THF complex shows that THF is bound in the cofactor binding pocket and is well positioned to transfer hydride to the dUMP exocyclic methylene. Together, these results suggest that THF does not reorient during hydride transfer and indicate that the role of Trp 80 may be to orient Leu 143 to shield the active site from bulk solvent and to optimally position the cofactor for hydride transfer.

Structural and functional insights into the HIV-1 maturation inhibitor binding pocket.

PubMed

Waki, Kayoko; Durell, Stewart R; Soheilian, Ferri; Nagashima, Kunio; Butler, Scott L; Freed, Eric O

2012-01-01

Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for in vitro. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased

Form and deformity: the trouble with Victorian pockets.

PubMed

Matthews, Christopher Todd

2010-01-01

This essay explores the Victorian debate about the place of pockets in men's and women's clothing. By studying the representation of men as naturally pocketed creatures and the general denial of useful pockets to middle-class women, the essay demonstrates the tenacious cultural logic by which men's and women's pockets were imagined to correspond to sexual differences and to index access, or lack thereof, to public mobility and financial agency. Interconnected readings of visual art, essays, and novels show how the common sense about gendered pockets was utilized and promulgated in Victorian narratives. The question of who gets pockets is thus positioned as part of the history of gendered bodies in public space.

Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

PubMed

Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

1989-12-08

The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form

Residues in the Distal Heme Pocket of Arabidopsis Non-Symbiotic Hemoglobins: Implication for Nitrite Reductase Activity

PubMed Central

Kumar, Nitin; Astegno, Alessandra; Chen, Jian; Giorgetti, Alejandro; Dominici, Paola

2016-01-01

It is well-established that plant hemoglobins (Hbs) are involved in nitric oxide (NO) metabolism via NO dioxygenase and/or nitrite reductase activity. The ferrous-deoxy Arabidopsis Hb1 and Hb2 (AHb1 and AHb2) have been shown to reduce nitrite to NO under hypoxia. Here, to test the hypothesis that a six- to five-coordinate heme iron transition might mediate the control of the nitrite reduction rate, we examined distal pocket mutants of AHb1 and AHb2 for nitrite reductase activity, NO production and spectroscopic features. Absorption spectra of AHbs distal histidine mutants showed that AHb1 mutant (H69L) is a stable pentacoordinate high-spin species in both ferrous and ferric states, whereas heme iron in AHb2 mutant (H66L) is hexacoordinated low-spin with Lys69 as the sixth ligand. The bimolecular rate constants for nitrite reduction to NO were 13.3 ± 0.40, 7.3 ± 0.5, 10.6 ± 0.8 and 171.90 ± 9.00 M−1·s−1 for AHb1, AHb2, AHb1 H69L and AHb2 H66L, respectively, at pH 7.4 and 25 °C. Consistent with the reductase activity, the amount of NO detected by chemiluminescence was significantly higher in the AHb2 H66L mutant. Our data indicate that nitrite reductase activity is determined not only by heme coordination, but also by a unique distal heme pocket in each AHb. PMID:27136534

A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

PubMed Central

Naqvi, Tatheer; Warden, Andrew C.; French, Nigel; Sugrue, Elena; Carr, Paul D.; Jackson, Colin J.; Scott, Colin

2014-01-01

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k cat/K M for malathion. X-ray crystal structures for the PTEAr Ser308Leu\\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue. PMID:24721933

Identification of a putative binding site critical for general anesthetic activation of TRPA1.

PubMed

Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

2017-04-04

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

A pocket of variability in Pinus rigida

Treesearch

F. Thomas Ledig; John H. Fryer

1971-01-01

Steady state gene frequencies around a pocket of differential fitness have been formulated by Hanson (1966) in a generalization of the work of Haldane (1948). A pocket of differential fitness would result in a pocket-of-variability, assuming that the radius of the area of contrasting fitness was large in relation to the vagility of the organism. Conversely, the absence...

Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

PubMed

Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

2016-11-01

Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC 50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

PubMed

Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

2013-08-06

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cyclic AMP Inhibits the Activity and Promotes the Acetylation of Acetyl-CoA Synthetase through Competitive Binding to the ATP/AMP Pocket.

PubMed

Han, Xiaobiao; Shen, Liqiang; Wang, Qijun; Cen, Xufeng; Wang, Jin; Wu, Meng; Li, Peng; Zhao, Wei; Zhang, Yu; Zhao, Guoping

2017-01-27

The high-affinity biosynthetic pathway for converting acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed by the central metabolic enzyme acetyl-coenzyme A synthetase (Acs), which is finely regulated both at the transcriptional level via cyclic AMP (cAMP)-driven trans-activation and at the post-translational level via acetylation inhibition. In this study, we discovered that cAMP directly binds to Salmonella enterica Acs (SeAcs) and inhibits its activity in a substrate-competitive manner. In addition, cAMP binding increases SeAcs acetylation by simultaneously promoting Pat-dependent acetylation and inhibiting CobB-dependent deacetylation, resulting in enhanced SeAcs inhibition. A crystal structure study and site-directed mutagenesis analyses confirmed that cAMP binds to the ATP/AMP pocket of SeAcs, and restrains SeAcs in an open conformation. The cAMP contact residues are well conserved from prokaryotes to eukaryotes, suggesting a general regulatory mechanism of cAMP on Acs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pilot Study of Patient and Caregiver Out-of-Pocket Costs of Allogeneic Hematopoietic Cell Transplantation

PubMed Central

Majhail, Navneet S; Rizzo, J Douglas; Hahn, Theresa; Lee, Stephanie J; McCarthy, Philip L; Ammi, Monique; Denzen, Ellen; Drexler, Rebecca; Flesch, Susan; James, Heather; Omondi, Nancy; Pedersen, Tanya L; Murphy, Elizabeth; Pederson, Kate

2012-01-01

Patient/caregiver out-of pocket costs associated with hematopoietic-cell transplantation (HCT) are not well known. We conducted a pilot study to evaluate patient/caregiver out-of-pocket costs in the first 3 months after allogeneic HCT. Thirty patients were enrolled at three sites. Prior to HCT, participants completed a baseline survey regarding household income and insurance coverage. Subsequently, they maintained a paper-based diary to track daily out-of-pocket expenses for the first 3 months after HCT. Telephone interviews were conducted to followup on missing/incomplete diaries and on study completion. Twenty-five patients/caregivers completed the baseline survey. Among these, the median pre-tax household income was $66,500 (range, $30-$375,000) and 48% had to temporarily relocate close to the transplant center. Insurance coverage was managed care plan (56%), Medicaid (20%), Medicare (17%) and other (8%). Twenty-two patients/caregivers completed ≥4 diaries; the median out-of-pocket expenses were $2,440 (range, $199-$13,769). Patients/caregivers who required temporary lodging had higher out-of-pocket expenses compared to those who did not (median, $5,247 vs. $716). Patients/caregivers can incur substantial out-of-pocket costs over the first 3 months, especially if they need to temporarily relocate close to the transplant center. Our study lays the foundation for future research on early and long-term financial impact of allogeneic HCT on patients/caregivers. PMID:23222378

Discovery of novel hydroxamates as highly potent tumor necrosis factor-[alpha] converting enzyme inhibitors. Part II: Optimization of the S3′ pocket

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mazzola Jr., Robert D.; Zhu, Zhaoning; Sinning, Lisa

2010-10-01

A series of cyclopropyl hydroxamic acids were prepared. Many of the compounds displayed picomolar affinity for the TACE enzyme while maintaining good to excellent selectivity profiles versus MMP-1, -2, -3, -7, -14, and ADAM-10. X-ray analysis of an inhibitor in the TACE active site indicated that the molecules bound to the enzyme in the S1{prime}-S3{prime} pocket.

Health care utilization and outpatient, out-of-pocket costs for active convulsive epilepsy in rural northeastern South Africa: a cross-sectional Survey.

PubMed

Wagner, Ryan G; Bertram, Melanie Y; Gómez-Olivé, F Xavier; Tollman, Stephen M; Lindholm, Lars; Newton, Charles R; Hofman, Karen J

2016-06-28

Epilepsy is a common neurological disorder, with over 80 % of cases found in low- and middle-income countries (LMICs). Studies from high-income countries find a significant economic burden associated with epilepsy, yet few studies from LMICs, where out-of-pocket costs for general healthcare can be substantial, have assessed out-of-pocket costs and health care utilization for outpatient epilepsy care. Within an established health and socio-demographic surveillance system in rural South Africa, a questionnaire to assess self-reported health care utilization and time spent traveling to and waiting to be seen at health facilities was administered to 250 individuals, previously diagnosed with active convulsive epilepsy. Epilepsy patients' out-of-pocket, medical and non-medical costs and frequency of outpatient care visits during the previous 12-months were determined. Within the last year, 132 (53 %) individuals reported consulting at a clinic, 162 (65 %) at a hospital and 34 (14 %) with traditional healers for epilepsy care. Sixty-seven percent of individuals reported previously consulting with both biomedical caregivers and traditional healers. Direct outpatient, median costs per visit varied significantly (p < 0.001) between hospital (2010 International dollar ($) 9.08; IQR: $6.41-$12.83) and clinic consultations ($1.74; IQR: $0-$5.58). Traditional healer fees per visit were found to cost $52.36 (IQR: $34.90-$87.26) per visit. Average annual outpatient, clinic and hospital out-of-pocket costs totaled $58.41. Traveling to and from and waiting to be seen by the caregiver at the hospital took significantly longer than at the clinic. Rural South Africans with epilepsy consult with both biomedical caregivers and traditional healers for both epilepsy and non-epilepsy care. Traditional healers were the most expensive mode of care, though utilized less often. While higher out-of-pocket costs were incurred at hospital visits, more people with ACE visited hospitals

Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.

The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putativemore » nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.« less

“Gate-keeper” Residues and Active-Site Rearrangements in DNA Polymerase μ Help Discriminate Non-cognate Nucleotides

PubMed Central

Li, Yunlang; Schlick, Tamar

2013-01-01

Incorporating the cognate instead of non-cognate substrates is crucial for DNA polymerase function. Here we analyze molecular dynamics simulations of DNA polymerase μ (pol μ) bound to different non-cognate incoming nucleotides including A:dCTP, A:dGTP, A(syn):dGTP, A:dATP, A(syn):dATP, T:dCTP, and T:dGTP to study the structure-function relationships involved with aberrant base pairs in the conformational pathway; while a pol μ complex with the A:dTTP base pair is available, no solved non-cognate structures are available. We observe distinct differences of the non-cognate systems compared to the cognate system. Specifically, the motions of active-site residue His329 and Asp330 distort the active site, and Trp436, Gln440, Glu443 and Arg444 tend to tighten the nucleotide-binding pocket when non-cognate nucleotides are bound; the latter effect may further lead to an altered electrostatic potential within the active site. That most of these “gate-keeper” residues are located farther apart from the upstream primer in pol μ, compared to other X family members, also suggests an interesting relation to pol μ's ability to incorporate nucleotides when the upstream primer is not paired. By examining the correlated motions within pol μ complexes, we also observe different patterns of correlations between non-cognate systems and the cognate system, especially decreased interactions between the incoming nucleotides and the nucleotide-binding pocket. Altered correlated motions in non-cognate systems agree with our recently proposed hybrid conformational selection/induced-fit models. Taken together, our studies propose the following order for difficulty of non-cognate system insertions by pol μ: T:dGTP

The GAP arginine finger movement into the catalytic site of Ras increases the activation entropy

PubMed Central

Kötting, Carsten; Kallenbach, Angela; Suveyzdis, Yan; Wittinghofer, Alfred; Gerwert, Klaus

2008-01-01

Members of the Ras superfamily of small G proteins play key roles in signal transduction pathways, which they control by GTP hydrolysis. They are regulated by GTPase activating proteins (GAPs). Mutations that prevent hydrolysis cause severe diseases including cancer. A highly conserved “arginine finger” of GAP is a key residue. Here, we monitor the GTPase reaction of the Ras·RasGAP complex at high temporal and spatial resolution by time-resolved FTIR spectroscopy at 260 K. After triggering the reaction, we observe as the first step a movement of the switch-I region of Ras from the nonsignaling “off” to the signaling “on” state with a rate of 3 s−1. The next step is the movement of the “arginine finger” into the active site of Ras with a rate of k2 = 0.8 s−1. Once the arginine points into the binding pocket, cleavage of GTP is fast and the protein-bound Pi intermediate forms. The switch-I reversal to the “off” state, the release of Pi, and the movement of arginine back into an aqueous environment is observed simultaneously with k3 = 0.1 s−1, the rate-limiting step. Arrhenius plots for the partial reactions show that the activation energy for the cleavage reaction is lowered by favorable positive activation entropy. This seems to indicate that protein-bound structured water molecules are pushed by the “arginine finger” movement out of the binding pocket into the bulk water. The proposed mechanism shows how the high activation barrier for phosphoryl transfer can be reduced by splitting into partial reactions separated by a Pi-intermediate. PMID:18434546

Current switching ratio optimization using dual pocket doping engineering

NASA Astrophysics Data System (ADS)

Dash, Sidhartha; Sahoo, Girija Shankar; Mishra, Guru Prasad

2018-01-01

This paper presents a smart idea to maximize current switching ratio of cylindrical gate tunnel FET (CGT) by growing pocket layers in both source and channel region. The pocket layers positioned in the source and channel of the device provides significant improvement in ON-state and OFF-state current respectively. The dual pocket doped cylindrical gate TFET (DP-CGT) exhibits much superior performance in term of drain current, transconductance and current ratio as compared to conventional CGT, channel pocket doped CGT (CP-CGT) and source pocket doped CGT (SP-CGT). Further, the current ratio has been optimized w.r.t. width and instantaneous position both the pocket layers. The much improved current ratio and low power consumption makes the proposed device suitable for low-power and high speed application. The simulation work of DP-CGT is done using 3D Sentaurus TCAD device simulator from Synopsys.

Alkylated hydroxylamine derivatives eliminate peripheral retinylidene Schiff bases but cannot enter the retinal binding pocket of light-activated rhodopsin.

PubMed

Piechnick, Ronny; Heck, Martin; Sommer, Martha E

2011-08-23

Besides Lys-296 in the binding pocket of opsin, all-trans-retinal forms adducts with peripheral lysine residues and phospholipids, thereby mimicking the spectral and chemical properties of metarhodopsin species. These pseudophotoproducts composed of nonspecific retinylidene Schiff bases have long plagued the investigation of rhodopsin deactivation and identification of decay products. We discovered that, while hydroxylamine can enter the retinal binding pocket of light-activated rhodopsin, the modified hydroxylamine compounds o-methylhydroxylamine (mHA), o-ethylhydroxylamine (eHA), o-tert-butylhydroxylamine (t-bHA), and o-(carboxymethyl)hydroxylamine (cmHA) are excluded. However, the alkylated hydroxylamines react quickly and efficiently with exposed retinylidene Schiff bases to form their respective retinal oximes. We further investigated how t-bHA affects light-activated rhodopsin and its interaction with binding partners. We found that both metarhodopsin II (Meta II) and Meta III are resistant to t-bHA, and neither arrestin nor transducin binding is affected by t-bHA. This discovery suggests that the hypothetical solvent channel that opens in light-activated rhodopsin is extremely stringent with regard to size and/or polarity. We believe that alkylated hydroxylamines will prove to be extremely useful reagents for the investigation of rhodopsin activation and decay mechanisms. Furthermore, the use of alkylated hydroxylamines should not be limited to in vitro studies and could help elucidate visual signal transduction mechanisms in the living cells of the retina. © 2011 American Chemical Society

Hydrophobic pocket targeting probes for enteroviruses

NASA Astrophysics Data System (ADS)

Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

2015-10-01

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content

Estimating Leaf Area Index (LAI) in Vineyards Using the PocketLAI Smart-App.

PubMed

Orlando, Francesca; Movedi, Ermes; Coduto, Davide; Parisi, Simone; Brancadoro, Lucio; Pagani, Valentina; Guarneri, Tommaso; Confalonieri, Roberto

2016-11-26

Estimating leaf area index (LAI) of Vitis vinifera using indirect methods involves some critical issues, related to its discontinuous and non-homogeneous canopy. This study evaluates the smart app PocketLAI and hemispherical photography in vineyards against destructive LAI measurements. Data were collected during six surveys in an experimental site characterized by a high level of heterogeneity among plants, allowing us to explore a wide range of LAI values. During the last survey, the possibility to combine remote sensing data and in-situ PocketLAI estimates (smart scouting) was evaluated. Results showed a good agreement between PocketLAI data and direct measurements, especially for LAI ranging from 0.13 to 1.41 ( R ² = 0.94, RRMSE = 17.27%), whereas the accuracy decreased when an outlying value (vineyard LAI = 2.84) was included ( R ² = 0.77, RRMSE = 43.00%), due to the saturation effect in case of very dense canopies arising from lack of green pruning. The hemispherical photography showed very high values of R ², even in presence of the outlying value ( R ² = 0.94), although it showed a marked and quite constant overestimation error (RRMSE = 99.46%), suggesting the need to introduce a correction factor specific for vineyards. During the smart scouting, PocketLAI showed its reliability to monitor the spatial-temporal variability of vine vigor in cordon-trained systems, and showed a potential for a wide range of applications, also in combination with remote sensing.

Toward a generalized computational workflow for exploiting transient pockets as new targets for small molecule stabilizers: Application to the homogentisate 1,2-dioxygenase mutants at the base of rare disease Alkaptonuria.

PubMed

Bernini, Andrea; Galderisi, Silvia; Spiga, Ottavia; Bernardini, Giulia; Niccolai, Neri; Manetti, Fabrizio; Santucci, Annalisa

2017-10-01

Alkaptonuria (AKU) is an inborn error of metabolism where mutation of homogentisate 1,2-dioxygenase (HGD) gene leads to a deleterious or misfolded product with subsequent loss of enzymatic degradation of homogentisic acid (HGA) whose accumulation in tissues causes ochronosis and degeneration. There is no licensed therapy for AKU. Many missense mutations have been individuated as responsible for quaternary structure disruption of the native hexameric HGD. A new approach to the treatment of AKU is here proposed aiming to totally or partially rescue enzyme activity by targeting of HGD with pharmacological chaperones, i.e. small molecules helping structural stability. Co-factor pockets from oligomeric proteins have already been successfully exploited as targets for such a strategy, but no similar sites are present at HGD surface; hence, transient pockets are here proposed as a target for pharmacological chaperones. Transient pockets are detected along the molecular dynamics trajectory of the protein and filtered down to a set of suitable sites for structural stabilization by mean of biochemical and pharmacological criteria. The result is a computational workflow relevant to other inborn errors of metabolism requiring rescue of oligomeric, misfolded enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli

PubMed Central

Soparkar, Ketaki; Kinana, Alfred D.; Weeks, Jon W.; Morrison, Keith D.; Nikaido, Hiroshi

2015-01-01

ABSTRACT The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions—Y49S, V127A, V127G, D153E, and G288C—mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions—F453C and L486W—were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure

NASA Pocket Statistics: 1997 Edition

NASA Technical Reports Server (NTRS)

1997-01-01

POCKET STATISTICS is published by the NATIONAL AERONAUTICS AND SPACE ADMINISTRATION (NASA). Included in each edition is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, Aeronautics and Space Transportation and NASA Procurement, Financial and Workforce data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. All Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

Hydrophobic pocket targeting probes for enteroviruses.

PubMed

Martikainen, Mari; Salorinne, Kirsi; Lahtinen, Tanja; Malola, Sami; Permi, Perttu; Häkkinen, Hannu; Marjomäki, Varpu

2015-11-07

Visualization and tracking of viruses without compromising their functionality is crucial in order to understand virus targeting to cells and tissues, and to understand the subsequent subcellular steps leading to virus uncoating and replication. Enteroviruses are important human pathogens causing a vast number of acute infections, and are also suggested to contribute to the development of chronic diseases like type I diabetes. Here, we demonstrate a novel method to target site-specifically the hydrophobic pocket of enteroviruses. A probe, a derivative of Pleconaril, was developed and conjugated to various labels that enabled the visualization of enteroviruses under light and electron microscopes. The probe mildly stabilized the virus particle by increasing the melting temperature by 1-3 degrees, and caused a delay in the uncoating of the virus in the cellular endosomes, but could not however inhibit the receptor binding, cellular entry or infectivity of the virus. The hydrophobic pocket binding moiety of the probe was shown to bind to echovirus 1 particle by STD and tr-NOESY NMR methods. Furthermore, binding to echovirus 1 and Coxsackievirus A9, and to a lesser extent to Coxsackie virus B3 was verified by using a gold nanocluster labeled probe by TEM analysis. Molecular modelling suggested that the probe fits the hydrophobic pockets of EV1 and CVA9, but not of CVB3 as expected, correlating well with the variations in the infectivity and stability of the virus particles. EV1 conjugated to the fluorescent dye labeled probe was efficiently internalized into the cells. The virus-fluorescent probe conjugate accumulated in the cytoplasmic endosomes and caused infection starting from 6 hours onwards. Remarkably, before and during the time of replication, the fluorescent probe was seen to leak from the virus-positive endosomes and thus separate from the capsid proteins that were left in the endosomes. These results suggest that, like the physiological hydrophobic content

Australian Vocational Education and Training Statistics. Pocket Guide. Issued 2005

ERIC Educational Resources Information Center

National Centre for Vocational Education Research (NCVER), 2005

2005-01-01

This publication, presented in pocket guide format, contains highlights from the 2004 vocational education and training (VET) statistics collection. It also includes key data on VET, apprentices and trainees' training activity, and information on technical and further education (TAFE) graduates obtained from the 2004 Student Outcomes Survey. Among…

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B.

PubMed

Ozcan, Ahmet; Olmez, Elif Ozkirimli; Alakent, Burak

2013-05-01

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPDclosed ) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPDopen ) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPDclosed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPDopen conformation. When the active site water molecule network that is a part of the free WPDclosed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. Copyright © 2013 Wiley Periodicals, Inc.

Lead isotope ratios in tree bark pockets: an indicator of past air pollution in the Czech Republic.

PubMed

Conkova, M; Kubiznakova, J

2008-10-15

Tree bark pockets were collected at four sites in the Czech Republic with differing levels of lead (Pb) pollution. The samples, spanning 1923-2005, were separated from beech (Fagus sylvatica) and spruce (Picea abies). Elevated Pb content (0.1-42.4 microg g(-1)) reflected air pollution in the city of Prague. The lowest Pb content (0.3-2.6 microg g(-1)) was found at the Kosetice EMEP "background pollution" site. Changes in (206)Pb/(207)Pb and (208)Pb/(206)Pb isotope ratios were in agreement with operation times of the Czech main anthropogenic Pb sources. Shortly after the Second World War, the (206)Pb/(207)Pb isotope ratio in bark pockets decreased from 1.17 to 1.14 and the (208)Pb/(206)Pb isotope ratio increased from 2.12 to 2.16. Two dominant emission sources responsible for these changes, lignite and leaded petrol combustion, contributed to the shifts in Pb isotope ratios. Low-radiogenic petrol Pb ((206)Pb/(207)Pb of 1.11) lead to lower (206)Pb/(207)Pb in bark pockets over time. High-radiogenic lignite-derived Pb ((206)Pb/(207)Pb of 1.18 to 1.19) was detected in areas affected by coal combustion rather than by traffic.

Comparing clinical attachment level and pocket depth for predicting periodontal disease progression in healthy sites of patients with chronic periodontitis using multi-state Markov models.

PubMed

Mdala, Ibrahimu; Olsen, Ingar; Haffajee, Anne D; Socransky, Sigmund S; Thoresen, Magne; de Blasio, Birgitte Freiesleben

2014-09-01

To understand degeneration of healthy sites and identify factors associated with disease progression in patients with chronic periodontitis. Data on healthy sites from 163 American and Swedish subjects were analysed using two-three-state (health, gingivitis, chronic periodontitis) Markov models based on bleeding on probing (BOP), and either clinical attachment level (CAL) + BOP or pocket depth (PD) + BOP. In 2 years, 10% (CAL + BOP) and 3% (PD + BOP) of healthy sites developed chronic periodontitis. On average, healthy sites remained healthy for 32 months before transiting in both models. Most transitions (87-97%) from health were to the gingivitis state. The expected duration of the gingivitis lesion was 4-5 months and sites recovered with a high probability (96-98%). Disease severity as measured by number of sites with CAL/PD > 4 mm at baseline and smoking, were associated with fast progression from health to chronic periodontitis within 6 months as were gingival redness in the PD + BOP model only. With age, the rate of disease progression to gingivitis decreased. Transition probabilities for gingivitis and chronic periodontitis were higher with CAL + BOP than with PD + BOP. Smoking and disease severity were significant predictors for fast progression. © 2014 The Authors. Journal of Clinical Periodontology Published by John Wiley & Sons Ltd.

Comparing clinical attachment level and pocket depth for predicting periodontal disease progression in healthy sites of patients with chronic periodontitis using multi-state Markov models

PubMed Central

Mdala, Ibrahimu; Olsen, Ingar; Haffajee, Anne D; Socransky, Sigmund S; Thoresen, Magne; de Blasio, Birgitte Freiesleben

2014-01-01

Aim To understand degeneration of healthy sites and identify factors associated with disease progression in patients with chronic periodontitis. Material and Methods Data on healthy sites from 163 American and Swedish subjects were analysed using two-three-state (health, gingivitis, chronic periodontitis) Markov models based on bleeding on probing (BOP), and either clinical attachment level (CAL) + BOP or pocket depth (PD) + BOP. Results In 2 years, 10% (CAL + BOP) and 3% (PD + BOP) of healthy sites developed chronic periodontitis. On average, healthy sites remained healthy for 32 months before transiting in both models. Most transitions (87–97%) from health were to the gingivitis state. The expected duration of the gingivitis lesion was 4–5 months and sites recovered with a high probability (96–98%). Disease severity as measured by number of sites with CAL/PD > 4 mm at baseline and smoking, were associated with fast progression from health to chronic periodontitis within 6 months as were gingival redness in the PD + BOP model only. With age, the rate of disease progression to gingivitis decreased. Conclusion Transition probabilities for gingivitis and chronic periodontitis were higher with CAL + BOP than with PD + BOP. Smoking and disease severity were significant predictors for fast progression. PMID:24888705

The acid pocket: a target for treatment in reflux disease?

PubMed

Kahrilas, Peter J; McColl, Kenneth; Fox, Mark; O'Rourke, Lisa; Sifrim, Daniel; Smout, Andre J P M; Boeckxstaens, Guy

2013-07-01

The nadir esophageal pH of reflux observed during pH monitoring in the postprandial period is often more acidic than the concomitant intragastric pH. This paradox prompted the discovery of the "acid pocket", an area of unbuffered gastric acid that accumulates in the proximal stomach after meals and serves as the reservoir for acid reflux in healthy individuals and gastroesophageal reflux disease (GERD) patients. However, there are differentiating features between these populations in the size and position of the acid pocket, with GERD patients predisposed to upward migration of the proximal margin onto the esophageal mucosa, particularly when supine. This upward migration of acid, sometimes referred to as an "acid film", likely contributes to mucosal pathology in the region of the squamocolumnar junction. Furthermore, movement of the acid pocket itself to a supradiaphragmatic location with hiatus hernia increases the propensity for acid reflux by all conventional mechanisms. Consequently, the acid pocket is an attractive target for GERD therapy. It may be targeted in a global way with proton pump inhibitors that attenuate acid pocket development, or with alginate/antacid combinations that colocalize with the acid pocket and displace it distally, thereby demonstrating the potential for selective targeting of the acid pocket in GERD.

Pocketing mechanics of SRM nozzle liner

NASA Technical Reports Server (NTRS)

Verderaime, V. S.

1986-01-01

A systems approach was adopted to study the pocketing phenomena on a solid rocket nozzle liner. The classical thermoelastic analysis was used to identify marginally strained regions on the composite liner erosion surface and at a depth coincident with the peak value of the across ply coefficient of thermal expansion. A failure criterion was introduced which included a thermal term and permitted failure assessment over the charred liner. The method was verified by satisfactory application to a reported related experiment. Liner pocketing mechanism was attributed to very localized material degradation caused during manufacturing process either by reduction of fiber strength and/or by concentration of resin volume fraction. Pocketing scenario over the degraged material was constructed with supporting formulation to predict size of fissures with respect to degraded material size and location in the liner and with burn time. Sensitivities of liner material parameters were determined to influence test programs designed to update mechanical data base of carbon cloth phenolic over the char temperature range.

Displacement of disordered water molecules from hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S₁'-pocket of thermolysin.

PubMed

Englert, L; Biela, A; Zayed, M; Heine, A; Hangauer, D; Klebe, G

2010-11-01

Prerequisite for the design of tight binding protein inhibitors and prediction of their properties is an in-depth understanding of the structural and thermodynamic details of the binding process. A series of closely related phosphonamidates was studied to elucidate the forces underlying their binding affinity to thermolysin. The investigated inhibitors are identical except for the parts penetrating into the hydrophobic S₁'-pocket. A correlation of structural, kinetic and thermodynamic data was carried out by X-ray crystallography, kinetic inhibition assay and isothermal titration calorimetry. Binding affinity increases with larger ligand hydrophobic P₁'-moieties accommodating the S₁'-pocket. Surprisingly, larger P₁'-side chain modifications are accompanied by an increase in the enthalpic contribution to binding. In agreement with other studies, it is suggested that the release of largely disordered waters from an imperfectly hydrated pocket results in an enthalpically favourable integration of these water molecules into bulk water upon inhibitor binding. This enthalpically favourable process contributes more strongly to the binding energetics than the entropy increase resulting from the release of water molecules from the S₁'-pocket or the formation of apolar interactions between protein and inhibitor. Displacement of highly disordered water molecules from a rather imperfectly hydrated and hydrophobic specificity pocket can reveal an enthalpic signature of inhibitor binding. Copyright © 2010 Elsevier B.V. All rights reserved.

Site-Specific Measurement of Water Dynamics in the Substrate Pocket of Ketosteroid Isomerase Using Time-Resolved Vibrational Spectroscopy

PubMed Central

Jha, Santosh Kumar; Ji, Minbiao; Gaffney, Kelly J.; Boxer, Steven G.

2012-01-01

Little is known about the reorganization capacity of water molecules at the active sites of enzymes and how this couples to the catalytic reaction. Here, we study the dynamics of water molecules at the active site of a highly proficient enzyme, Δ5-3-ketosteroid isomerase (KSI), during a light-activated mimic of its catalytic cycle. Photo-excitation of a nitrile containing photo-acid, coumarin183 (C183), mimics the change in charge density that occurs at the active site of KSI during the first step of the catalytic reaction. The nitrile of C183 is exposed to water when bound to the KSI active site, and we used time-resolved vibrational spectroscopy as a site-specific probe to study the solvation dynamics of water molecules in the vicinity of the nitrile. We observed that water molecules at the active site of KSI are highly rigid, during the light-activated catalytic cycle, compared to the solvation dynamics observed in bulk water. Based upon this result we hypothesize that rigid water dipoles at the active site might help in the maintenance of the pre-organized electrostatic environment required for efficient catalysis. The results also demonstrate the utility of nitrile probes in measuring the dynamics of local (H-bonded) water molecules in contrast to the commonly used fluorescence methods which measure the average behavior of primary and subsequent spheres of solvation. PMID:22931297

Spatial Decomposition of Translational Water–Water Correlation Entropy in Binding Pockets

PubMed Central

2015-01-01

A number of computational tools available today compute the thermodynamic properties of water at surfaces and in binding pockets by using inhomogeneous solvation theory (IST) to analyze explicit-solvent simulations. Such methods enable qualitative spatial mappings of both energy and entropy around a solute of interest and can also be applied quantitatively. However, the entropy estimates of existing methods have, to date, been almost entirely limited to the first-order terms in the IST’s entropy expansion. These first-order terms account for localization and orientation of water molecules in the field of the solute but not for the modification of water–water correlations by the solute. Here, we present an extension of the Grid Inhomogeneous Solvation Theory (GIST) approach which accounts for water–water translational correlations. The method involves rewriting the two-point density of water in terms of a conditional density and utilizes the efficient nearest-neighbor entropy estimation approach. Spatial maps of this second order term, for water in and around the synthetic host cucurbit[7]uril and in the binding pocket of the enzyme Factor Xa, reveal mainly negative contributions, indicating solute-induced water–water correlations relative to bulk water; particularly strong signals are obtained for sites at the entrances of cavities or pockets. This second-order term thus enters with the same, negative, sign as the first order translational and orientational terms. Numerical and convergence properties of the methodology are examined. PMID:26636620

Equity in out-of-pocket payment in Chile.

PubMed

Mondaca, Alicia Lorena Núñez; Chi, Chunhuei

2017-05-04

To assess the distribution of financial burden in Chile, with a focus on the burden and progressivity of out-of-pocket payment. Based on the principle of ability to pay, we explore factors that contribute to inequities in the health system finance and issues about the burden of out-of-pocket payment, as well as the progressivity and redistributive effect of out-of-pocket payment in Chile. Our analysis is based on data from the 2006 National Survey on Satisfaction and Out-of-Pocket Payments. Results from this study indicate evidence of inequity, in spite of the progressivity of the healthcare system. Our analysis also identifies relevant policy variables such as education, insurance system, and method of payment that should be taken into consideration in the ongoing debates and research in improving the Chilean system. In order to reduce the detected disparities among income groups, healthcare priorities should target low-income groups. Furthermore, policies should explore changes in the access to education and its impact on equity.

Dosimetric Effects of Air Pockets Around High-Dose Rate Brachytherapy Vaginal Cylinders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, Susan, E-mail: srichardson@radonc.wustl.ed; Palaniswaamy, Geethpriya; Grigsby, Perry W.

2010-09-01

Purpose: Most physicians use a single-channel vaginal cylinder for postoperative endometrial cancer brachytherapy. Recent published data have identified air pockets between the vaginal cylinders and the vaginal mucosa. The purpose of this research was to evaluate the incidence, size, and dosimetric effects of these air pockets. Methods and Materials: 25 patients receiving postoperative vaginal cuff brachytherapy with a high-dose rate vaginal cylinders were enrolled in this prospective data collection study. Patients were treated with 6 fractions of 200 to 400 cGy per fraction prescribed at 5 mm depth. Computed tomography simulation for brachytherapy treatment planning was performed for each fraction.more » The quantity, volume, and dosimetric impact of the air pockets surrounding the cylinder were quantified. Results: In 25 patients, a total of 90 air pockets were present in 150 procedures (60%). Five patients had no air pockets present during any of their treatments. The average number of air pockets per patient was 3.6, with the average total air pocket volume being 0.34 cm{sup 3} (range, 0.01-1.32 cm{sup 3}). The average dose reduction to the vaginal mucosa at the air pocket was 27% (range, 9-58%). Ten patients had no air pockets on their first fraction but air pockets occurred in subsequent fractions. Conclusion: Air pockets between high-dose rate vaginal cylinder applicators and the vaginal mucosa are present in the majority of fractions of therapy, and their presence varies from patient to patient and fraction to fraction. The existence of air pockets results in reduced radiation dose to the vaginal mucosa.« less

Probing the steric requirements of the γ-aminobutyric acid aminotransferase active site with fluorinated analogues of vigabatrin

PubMed Central

Juncosa, Jose I.; Groves, Andrew P.; Xia, Guoyao; Silverman, Richard B.

2012-01-01

We have synthesized three analogues of 4-amino-5-fluorohexanoic acids as potential inactivators of γ-aminobutyric acid aminotransferase (GABA-AT), which were designed to combine the potency of their shorter chain analogue, 4-amino-5-fluoropentanoic acid (AFPA), with the greater enzyme selectivity of the antiepileptic vigabatrin (Sabril®). Unexpectedly, these compounds failed to inactivate or inhibit the enzyme, even at high concentrations. On the basis of molecular modeling studies, we propose that the GABA-AT active site has an accessory binding pocket that accommodates the vinyl group of vigabatrin and the fluoromethyl group of AFPA, but is too narrow to support the extra width of one distal methyl group in the synthesized analogues. PMID:23306054

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

PubMed

Sayer, Christopher; Finnigan, William; Isupov, Michail N; Levisson, Mark; Kengen, Servé W M; van der Oost, John; Harmer, Nicholas J; Littlechild, Jennifer A

2016-05-10

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site

PubMed Central

Sayer, Christopher; Finnigan, William; Isupov, Michail N.; Levisson, Mark; Kengen, Servé W. M.; van der Oost, John; Harmer, Nicholas J.; Littlechild, Jennifer A.

2016-01-01

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions. PMID:27160974

The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

PubMed Central

Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

2009-01-01

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

PubMed

Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

2009-05-01

The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

Implementation in Indonesia of the WHO Pocket Book of Hospital Care for Children.

PubMed

Li, Michelle Y; Puspita, Ratih; Duke, Trevor; Agung, Fransisca H; Hegar, Badriul; Pritasari, Kirana; Weber, Martin W

2014-05-01

Effective implementation of evidence-based practice guidelines has the potential to improve quality of hospital care for children. To achieve this in Indonesia, a locally adapted version of the WHO Pocket Book of Hospital Care for Children was published in 2009. To document implementation of the Pocket Book in Indonesia and to compare uptake in health facilities in which there has been a quality-improvement approach involving audit and feedback with uptake in settings in which there has been only passive dissemination. Indonesian district health offices, district hospitals, health centres with beds, and medical schools were surveyed by telephone, and an online and telephone survey of paediatricians was conducted. Health facilities in four provinces were visited, and key stakeholders were interviewed. Health facilities were assessed on availability of the guidelines, use by staff, and their incorporation into hospital procedures and activities. There was evidence of use of the Pocket Book across Indonesia, despite limited funding for implementation. Its distribution had reached all provinces; 61% (33/54) of health facilities surveyed had a copy of the guidelines. Hospitals involved in a related quality audit were more likely to report use of the guidelines than hospitals exposed to passive dissemination, although this difference was not significant. Of 150 paediatricians sampled, 109 (73%) reported referring to the guidelines in their clinical practice. The guidelines have been incorporated into the postgraduate paediatric curriculum in four of 13 universities sampled. There was encouraging evidence of uptake of the Pocket Book in Indonesia following local adaptation, nationwide mailing distribution and small-scale local implementation activities.

The Role of the Acid Pocket in Gastroesophageal Reflux Disease.

PubMed

Mitchell, David R; Derakhshan, Mohammad H; Robertson, Elaine V; McColl, Kenneth E L

2016-02-01

Gastroesophageal reflux disease is one of the commonest chronic conditions in the western world and its prevalence is increasing worldwide. The discovery of the acid pocket explained the paradox of acid reflux occurring more frequently in the postprandial period despite intragastric acidity being low due to the buffering effect of the meal. The acid pocket was first described in 2001 when it was detected as an area of low pH immediately distal to the cardia using dual pH electrode pull-through studies 15 minutes after a meal. It was hypothesized that there was a local pocket of acid close to the gastroesophageal junction that escapes the buffering effect of the meal, and that this is the source of postprandial acidic reflux. The presence of the acid pocket has been confirmed in other studies using different techniques including high-resolution pHmetry, Bravo capsule, magnetic resonance imaging, and scintigraphy. This review aims to describe what we know about the acid pocket including its length, volume, fluid constituents, and its relationship to the lower esophageal sphincter and squamocolumnar junction. We will discuss the possible mechanisms that lead to the formation of the acid pocket and examine what differences exist in patients who suffer from acid reflux. Treatments for reflux disease that affect the acid pocket will also be discussed.

Pharmacophore screening of the protein data bank for specific binding site chemistry.

PubMed

Campagna-Slater, Valérie; Arrowsmith, Andrew G; Zhao, Yong; Schapira, Matthieu

2010-03-22

A simple computational approach was developed to screen the Protein Data Bank (PDB) for putative pockets possessing a specific binding site chemistry and geometry. The method employs two commonly used 3D screening technologies, namely identification of cavities in protein structures and pharmacophore screening of chemical libraries. For each protein structure, a pocket finding algorithm is used to extract potential binding sites containing the correct types of residues, which are then stored in a large SDF-formatted virtual library; pharmacophore filters describing the desired binding site chemistry and geometry are then applied to screen this virtual library and identify pockets matching the specified structural chemistry. As an example, this approach was used to screen all human protein structures in the PDB and identify sites having chemistry similar to that of known methyl-lysine binding domains that recognize chromatin methylation marks. The selected genes include known readers of the histone code as well as novel binding pockets that may be involved in epigenetic signaling. Putative allosteric sites were identified on the structures of TP53BP1, L3MBTL3, CHEK1, KDM4A, and CREBBP.

Effect of vegetation management for reducing damage to lodgepole pine seedlings from northern pocket gophers

USGS Publications Warehouse

Engeman, Richard M.; Barnes, Victor G.; Anthony, Richard M.; Krupa, Heather W.

1997-01-01

The effects of vegetation management on northern pocket gopher (Thomomys talpoides) activity and damage to lodgepole pine (Pinus contorta) seedlings were studied using 2,4-D herbicide to alter the habitat. Treatments were applied to a large (8.1 ha) treatment unit and observed effects were compared with an untreated control unit of the same size. The greatly reduced forb and grass cover on the treated unit was associated with a corresponding decrease in pocket gopher activity that persisted for 6 years after initial treatment. Times until seedlings first incurred gopher damage and overall survival of seedlings were greatly increased on the treated unit.

Regulation of Drosophila hematopoietic sites by Activin-β from active sensory neurons

PubMed Central

Makhijani, Kalpana; Alexander, Brandy; Rao, Deepti; Petraki, Sophia; Herboso, Leire; Kukar, Katelyn; Batool, Itrat; Wachner, Stephanie; Gold, Katrina S.; Wong, Corinna; O’Connor, Michael B.; Brückner, Katja

2017-01-01

An outstanding question in animal development, tissue homeostasis and disease is how cell populations adapt to sensory inputs. During Drosophila larval development, hematopoietic sites are in direct contact with sensory neuron clusters of the peripheral nervous system (PNS), and blood cells (hemocytes) require the PNS for their survival and recruitment to these microenvironments, known as Hematopoietic Pockets. Here we report that Activin-β, a TGF-β family ligand, is expressed by sensory neurons of the PNS and regulates the proliferation and adhesion of hemocytes. These hemocyte responses depend on PNS activity, as shown by agonist treatment and transient silencing of sensory neurons. Activin-β has a key role in this regulation, which is apparent from reporter expression and mutant analyses. This mechanism of local sensory neurons controlling blood cell adaptation invites evolutionary parallels with vertebrate hematopoietic progenitors and the independent myeloid system of tissue macrophages, whose regulation by local microenvironments remain undefined. PMID:28748922

Community-level effect of the reproductive health vouchers program on out-of-pocket spending on family planning and safe motherhood services in Kenya.

PubMed

Obare, Francis; Warren, Charlotte; Kanya, Lucy; Abuya, Timothy; Bellows, Ben

2015-08-25

Although vouchers can protect individuals in low-income countries from financial catastrophe and impoverishment arising from out-of-pocket expenditures on healthcare, their effectiveness in achieving this goal depends on whether both service and transport costs are subsidized as well as other factors such as service availability in a given locality and community perceptions about the quality of care. This paper examines the community-level effect of the reproductive health vouchers program on out-of-pocket expenditure on family planning, antenatal, delivery and postnatal care services in Kenya. Data are from two rounds of cross-sectional household surveys in voucher and non-voucher sites. The first survey was conducted between May 2010 and July 2011 among 2,933 women aged 15-49 years while the second survey took place between July and October 2012 among 3,094 women of similar age groups. The effect of the program on out-of-pocket expenditure is determined by difference-in-differences estimation. Analysis entails comparison of changes in proportions, means and medians as well as estimation of multivariate linear regression models with interaction terms between indicators for study site (voucher or non-voucher) and period of study (2010-2011 or 2012). There were significantly greater declines in the proportions of women from voucher sites that paid for antenatal, delivery and postnatal care services at health facilities compared to those from non-voucher sites. The changes were also consistent with increased uptake of the safe motherhood voucher in intervention sites over time. There was, however, no significant difference in changes in the proportions of women from voucher and non-voucher sites that paid for family planning services. The results further show that there were significant differences in changes in the amount paid for family planning and antenatal care services by women from voucher compared to those from non-voucher sites. Although there were greater

Practical Pocket PC Application w/Biometric Security

NASA Technical Reports Server (NTRS)

Logan, Julian

2004-01-01

I work in the Flight Software Engineering Branch, where we provide design and development of embedded real-time software applications for flight and supporting ground systems to support the NASA Aeronautics and Space Programs. In addition, this branch evaluates, develops and implements new technologies for embedded real-time systems, and maintains a laboratory for applications of embedded technology. The majority of microchips that are used in modern society have been programmed using embedded technology. These small chips can be found in microwaves, calculators, home security systems, cell phones and more. My assignment this summer entails working with an iPAQ HP 5500 Pocket PC. This top-of-the-line hand-held device is one of the first mobile PC's to introduce biometric security capabilities. Biometric security, in this case a fingerprint authentication system, is on the edge of technology as far as securing information. The benefits of fingerprint authentication are enormous. The most significant of them are that it is extremely difficult to reproduce someone else's fingerprint, and it is equally difficult to lose or forget your own fingerprint as opposed to a password or pin number. One of my goals for this summer is to integrate this technology with another Pocket PC application. The second task for the summer is to develop a simple application that provides an Astronaut EVA (Extravehicular Activity) Log Book capability. The Astronaut EVA Log Book is what an astronaut would use to report the status of field missions, crew physical health, successes, future plans, etc. My goal is to develop a user interface into which these data fields can be entered and stored. The applications that I am developing are created using eMbedded Visual C++ 4.0 with the Pocket PC 2003 Software Development Kit provided by Microsoft.

Equity in out-of-pocket payment in Chile

PubMed Central

Mondaca, Alicia Lorena Núñez; Chi, Chunhuei

2017-01-01

ABSTRACT OBJECTIVE To assess the distribution of financial burden in Chile, with a focus on the burden and progressivity of out-of-pocket payment. METHODS Based on the principle of ability to pay, we explore factors that contribute to inequities in the health system finance and issues about the burden of out-of-pocket payment, as well as the progressivity and redistributive effect of out-of-pocket payment in Chile. Our analysis is based on data from the 2006 National Survey on Satisfaction and Out-of-Pocket Payments. RESULTS Results from this study indicate evidence of inequity, in spite of the progressivity of the healthcare system. Our analysis also identifies relevant policy variables such as education, insurance system, and method of payment that should be taken into consideration in the ongoing debates and research in improving the Chilean system. CONCLUSIONS In order to reduce the detected disparities among income groups, healthcare priorities should target low-income groups. Furthermore, policies should explore changes in the access to education and its impact on equity. PMID:28492762

Magnetic Resonance Imaging Quantification of Fasted State Colonic Liquid Pockets in Healthy Humans.

PubMed

Murray, Kathryn; Hoad, Caroline L; Mudie, Deanna M; Wright, Jeff; Heissam, Khaled; Abrehart, Nichola; Pritchard, Susan E; Al Atwah, Salem; Gowland, Penny A; Garnett, Martin C; Amidon, Gregory E; Spiller, Robin C; Amidon, Gordon L; Marciani, Luca

2017-08-07

The rate and extent of drug dissolution and absorption from solid oral dosage forms is highly dependent on the volume of liquid in the gastrointestinal tract (GIT). However, little is known about the time course of GIT liquid volumes after drinking a glass of water (8 oz), particularly in the colon, which is a targeted site for both locally and systemically acting drug products. Previous magnetic resonance imaging (MRI) studies offered novel insights on GIT liquid distribution in fasted humans in the stomach and small intestine, and showed that freely mobile liquid in the intestine collects in fairly distinct regions or "pockets". Based on this previous pilot data, we hypothesized that (1) it is possible to quantify the time course of the volume and number of liquid pockets in the undisturbed colon of fasted healthy humans following ingestion of 240 mL, using noninvasive MRI methods; (2) the amount of freely mobile water in the fasted human colon is of the order of only a few milliliters. Twelve healthy volunteers fasted overnight and underwent fasted abdominal MRI scans before drinking 240 mL (∼8 fluid ounces) of water. After ingesting the water they were scanned at frequent intervals for 2 h. The images were processed to quantify freely mobile water in the total and regional colon: ascending, transverse, and descending. The fasted colon contained (mean ± SEM) 11 ± 5 pockets of resting liquid with a total volume of 2 ± 1 mL (average). The colonic fluid peaked at 7 ± 4 mL 30 min after the water drink. This peak fluid was distributed in 17 ± 7 separate liquid pockets in the colon. The regional analysis showed that pockets of free fluid were found primarily in the ascending colon. The interindividual variability was very high; the subjects showed a range of number of colonic fluid pockets from 0 to 89 and total colonic freely mobile fluid volume from 0 to 49 mL. This is the first study measuring the time course of the number, regional location, and volume of

Assessment and management of retraction pockets.

PubMed

Alper, Cuneyt; Olszewska, Ewa

2017-02-28

This manuscript intends to review types, pathogenesis, associated risk factors, and potential methods of prevention and treatment of the retraction pockets in adults and children. The importance of retraction pockets (RP) lies in loss of original histological and anatomical structure which is associated with development of ossicular chain erosion, cho¬lesteatoma formation and potentially life threatening complications of cholesteatoma. The trans-mucosal exchange each gas in the middle ear (ME) is towards equalizing its partial pressures with the partial pressure in the environ¬ment. MEs that have abnormalities in the volume and ventilation pathways in the epitympanic may be more suscep¬tible to retraction pockets. Sustained pressure differences and/or inflammation leads to destruction of collagen fibers in the lamina propria. Inflammatory mediators and cytokines lead to release of collagenases result in viscoelastic properties of the lamina propria. The process of changes in the tympanic membrane structure may evolve to the cho¬lesteatoma formation. There are many different staging systems that clinicians prioritize in their decision making in the management of RP. The authors discuss the management possibilities in different clinical situations: RP without and with ongoing or intermittent evidence of Eustachian Tube Dysfunction (ETD), presence of adenoid hypertrophy or re-growth of adenoids, presence or absence of effusion, invisible depth of RP without effusion. invisible depth of RP with effusion, ongoing RP after VT insertion, and finally suspicion of cholesteatoma in a deep RP with ME effusion. A decision algorithm regarding the management of TM retraction and retraction pockets is provided.

Flavonol Activation Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase Domain of IRE1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wiseman, R. Luke; Zhang, Yuhong; Lee, Kenneth P.K.

2010-08-18

Signaling in the most conserved branch of the endoplasmic reticulum (ER) unfolded protein response (UPR) is initiated by sequence-specific cleavage of the HAC1/XBP1 mRNA by the ER stress-induced kinase-endonuclease IRE1. We have discovered that the flavonol quercetin activates yeast IRE1's RNase and potentiates activation by ADP, a natural activating ligand that engages the IRE1 nucleotide-binding cleft. Enzyme kinetics and the structure of a cocrystal of IRE1 complexed with ADP and quercetin reveal engagement by quercetin of an unanticipated ligand-binding pocket at the dimer interface of IRE1's kinase extension nuclease (KEN) domain. Analytical ultracentrifugation and crosslinking studies support the preeminence ofmore » enhanced dimer formation in quercetin's mechanism of action. These findings hint at the existence of endogenous cytoplasmic ligands that may function alongside stress signals from the ER lumen to modulate IRE1 activity and at the potential for the development of drugs that modify UPR signaling from this unanticipated site.« less

Validity of a Smartphone-Based Fall Detection Application on Different Phones Worn on a Belt or in a Trouser Pocket.

PubMed

Vermeulen, Joan; Willard, Sarah; Aguiar, Bruno; De Witte, Luc P

2015-01-01

The objective of this study was to evaluate the sensitivity and specificity of a smartphone-based fall detection application when different smartphone models are worn on a belt or in a trouser pocket. Eight healthy adults aged between 18 and 24 years old simulated 10 different types of true falls, 5 different types of falls with recovery, and 11 daily activities, five consecutive times. Participants wore one smartphone in a pocket that was attached to their belt and another one in their trouser pocket. All smartphones were equipped with a built-in accelerometer and the fall detection application. Four participants tested the application on a Samsung S3 and four tested the application on a Samsung S3 mini. Sensitivity scores were .75 (Samsung S3 belt), .88 (Samsung S3 mini trouser pocket), and .90 (Samsung S3 mini belt/Samsung S3 trouser pocket). Specificity scores were .87 (Samsung S3 trouser pocket), .91 (Samsung S3 mini trouser pocket), .97 (Samsung S3 belt), and .99 (Samsung S3 mini belt). These results suggest that an application on a smartphone can generate valid fall alarms when worn on a belt or in a trouser pocket. However, sensitivity should be improved before implementation of the application in practice.

A fragment-based approach leading to the discovery of a novel binding site and the selective CK2 inhibitor CAM4066.

PubMed

De Fusco, Claudia; Brear, Paul; Iegre, Jessica; Georgiou, Kathy Hadje; Sore, Hannah F; Hyvönen, Marko; Spring, David R

2017-07-01

Recently we reported the discovery of a potent and selective CK2α inhibitor CAM4066. This compound inhibits CK2 activity by exploiting a pocket located outside the ATP binding site (αD pocket). Here we describe in detail the journey that led to the discovery of CAM4066 using the challenging fragment linking strategy. Specifically, we aimed to develop inhibitors by linking a high-affinity fragment anchored in the αD site to a weakly binding warhead fragment occupying the ATP site. Moreover, we describe the remarkable impact that molecular modelling had on the development of this novel chemical tool. The work described herein shows potential for the development of a novel class of CK2 inhibitors. Copyright © 2017. Published by Elsevier Ltd.

Molecular dynamics simulation of the last step of a catalytic cycle: product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis.

PubMed

Choutko, Alexandra; van Gunsteren, Wilfred F

2012-11-01

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site. Copyright © 2012 The Protein Society.

Coastal geomorphological study of pocket beaches in Crete, with the use of planview indices.

NASA Astrophysics Data System (ADS)

Alexandrakis, George; Karditsa, Aikaterini; Poulos, Serafim; Kampanis, Nikos

2013-04-01

The formation of pocket beaches is a result of a large number of processes and mechanisms that vary on space and time scales. This study aims in defining the planform characteristics of pocket beaches in Crete Isl. and to determine their sheltering effect, embaymentization and their status of equilibrium. Thus, data from 30 pocket beaches along the coastline of Crete, with different geomorphological and hydrodynamical setting, were collected. Planform parameters were applied and coastal planview indices from the bibliography were applied. The parameters included: length and orientation of the headlands between the pocket beach; length between the bay entrance and the center of the beach; lengths of the i) embayed shoreline, ii) embayed beach, iii) beach segment located at the shadow of a headland; linear distance and orientation between the edges of the embayed beach; direction of the incident wave energy flux; wave crest obliquity to the control line; beach area, maximum beach width and headland orientation and river/ torrent catchment areas in beach zones that an active river system existed (Bowman et al.2009). For the morphological mapping of the study areas, 1:5000 orthophoto maps were used. Wave regime has been calculated with the use of prognostic equations and utilising local wind data (mean annual frequency of wind speed and direction), provided by the Wind and Wave Atlas of the Eastern Mediterranean Sea. The diffraction and refraction of the waves has been simulated with the use of numerical models. The study shows that Cretan pocket beaches display a wide range of indentation, suggesting that is the result of several parameters that include tectonics, coastal hydrodynamics and river catchment areas. The more indented bays are, the shorter their beaches become, while low-indented pocket beaches are the widest and the longest ones. Beaches with headland with large length appear to be more protected and receive smaller amount of wave energy. Most of the

Similarities among receptor pockets and among compounds: analysis and application to in silico ligand screening.

PubMed

Fukunishi, Yoshifumi; Mikami, Yoshiaki; Nakamura, Haruki

2005-09-01

We developed a new method to evaluate the distances and similarities between receptor pockets or chemical compounds based on a multi-receptor versus multi-ligand docking affinity matrix. The receptors were classified by a cluster analysis based on calculations of the distance between receptor pockets. A set of low homologous receptors that bind a similar compound could be classified into one cluster. Based on this line of reasoning, we proposed a new in silico screening method. According to this method, compounds in a database were docked to multiple targets. The new docking score was a slightly modified version of the multiple active site correction (MASC) score. Receptors that were at a set distance from the target receptor were not included in the analysis, and the modified MASC scores were calculated for the selected receptors. The choice of the receptors is important to achieve a good screening result, and our clustering of receptors is useful to this purpose. This method was applied to the analysis of a set of 132 receptors and 132 compounds, and the results demonstrated that this method achieves a high hit ratio, as compared to that of a uniform sampling, using a receptor-ligand docking program, Sievgene, which was newly developed with a good docking performance yielding 50.8% of the reconstructed complexes at a distance of less than 2 A RMSD.

Pockets of Rural Prosperity

ERIC Educational Resources Information Center

Center for Rural Pennsylvania, 2004

2004-01-01

Pennsylvania's rural areas are often characterized as having lower incomes and lower housing values than urban areas. This characterization is not universally accurate, however, since there are some impressive pockets of wealth within rural Pennsylvania. To highlight the diversity of wealth among Pennsylvania's rural municipalities, the Center for…

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

PubMed Central

Wang, Xue; Zhao, Kun; Kirberger, Michael; Wong, Hing; Chen, Guantao; Yang, Jenny J

2010-01-01

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca2+-binding pockets of holoproteins to identify such pockets, ignoring possible Ca2+-induced conformational change. In this article, we first report our progress in the analysis of Ca2+-induced conformational changes followed by improved prediction of Ca2+-binding sites in the large group of Ca2+-binding proteins that exhibit only localized conformational changes. The MUGSR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUGSR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUGSR was applied to both X-ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca2+-binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein. PMID:20512971

Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

PubMed

Gray, K A; Dutton, P L; Daldal, F

1994-01-25

Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

3. DOWNHILL SIDE AND END ELEVATION OF COAL POCKETS, WITH ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

3. DOWNHILL SIDE AND END ELEVATION OF COAL POCKETS, WITH RETAINING WALL IN BACKGROUND. VIEW LOOKING NORTHEAST. - Honesdale Coal Pockets, Main & Commercial Streets, between 700 & 800 blocks, Honesdale, Wayne County, PA

Evaluation of the turbine pocket spirometer.

PubMed Central

Gunawardena, K A; Houston, K; Smith, A P

1987-01-01

A compact electronic spirometer, the turbine pocket spirometer, which measures the FEV1, forced vital capacity (FVC), and peak expiratory flow (PEF) in a single expiration, was compared with the Vitalograph and the Wright peak flow meter in 99 subjects (FEV1 range 0.40-5.50 litres; FVC 0.58-6.48 l; PEF 40-650 l min-1). The mean differences between the machines were small--0.05 l for FEV1, 0.05 l for FVC, and 11.6 l min-1 for PEF, with the limits of agreement at +/- 0.25 l, +/- 0.48 l, and +/- 52.2 l min-1 respectively. The wide limits of agreement for the PEF comparison were probably because of the difference in the technique of blowing: a fast, long blow was used for the pocket spirometer and a short, sharp one for the Wright peak flow meter. The FEV1 and FVC showed a proportional bias of around 4-5% in favour of the Vitalograph. The repeatability coefficient for the pocket spirometer FEV1 was 0.18 l, for FVC 0.22 l, and for PEF 31 l min-1. These compared well with the repeatability coefficients of the Vitalograph and the Wright peak flow meter, which gave values of 0.18 l, 0.28 l, and 27 l min-1 respectively. At flow rates of over 600 l min-1 the resistance of the pocket spirometer marginally exceeded the American Thoracic Society recommendations. The machine is easy to operate and portable, and less expensive than the Vitalograph and Wright peak flow meter combined. It can be recommended for general use. Images PMID:3686460

Structure of Bacillus subtilis γ-glutamyltranspeptidase in complex with acivicin: diversity of the binding mode of a classical and electrophilic active-site-directed glutamate analogue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ida, Tomoyo; Suzuki, Hideyuki; Fukuyama, Keiichi

2014-02-01

The binding modes of acivicin, a classical and an electrophilic active-site-directed glutamate analogue, to bacterial γ-glutamyltranspeptidases were found to be diverse. γ-Glutamyltranspeptidase (GGT) is an enzyme that plays a central role in glutathione metabolism, and acivicin is a classical inhibitor of GGT. Here, the structure of acivicin bound to Bacillus subtilis GGT determined by X-ray crystallography to 1.8 Å resolution is presented, in which it binds to the active site in a similar manner to that in Helicobacter pylori GGT, but in a different binding mode to that in Escherichia coli GGT. In B. subtilis GGT, acivicin is bound covalentlymore » through its C3 atom with sp{sup 2} hybridization to Thr403 O{sup γ}, the catalytic nucleophile of the enzyme. The results show that acivicin-binding sites are common, but the binding manners and orientations of its five-membered dihydroisoxazole ring are diverse in the binding pockets of GGTs.« less

Disruption of key NADH-binding pocket residues of the Mycobacterium tuberculosis InhA affects DD-CoA binding ability.

PubMed

Shaw, Daniel J; Robb, Kirsty; Vetter, Beatrice V; Tong, Madeline; Molle, Virginie; Hunt, Neil T; Hoskisson, Paul A

2017-07-05

Tuberculosis (TB) is a global health problem that affects over 10 million people. There is an urgent need to develop novel antimicrobial therapies to combat TB. To achieve this, a thorough understanding of key validated drug targets is required. The enoyl reductase InhA, responsible for synthesis of essential mycolic acids in the mycobacterial cell wall, is the target for the frontline anti-TB drug isoniazid. To better understand the activity of this protein a series of mutants, targeted to the NADH co-factor binding pocket were created. Residues P193 and W222 comprise a series of hydrophobic residues surrounding the cofactor binding site and mutation of both residues negatively affect InhA function. Construction of an M155A mutant of InhA results in increased affinity for NADH and DD-CoA turnover but with a reduction in V max for DD-CoA, impairing overall activity. This suggests that NADH-binding geometry of InhA likely permits long-range interactions between residues in the NADH-binding pocket to facilitate substrate turnover in the DD-CoA binding region of the protein. Understanding the precise details of substrate binding and turnover in InhA and how this may affect protein-protein interactions may facilitate the development of improved inhibitors enabling the development of novel anti-TB drugs.

Compensated electron and hole pockets in an underdoped high- Tc superconductor

NASA Astrophysics Data System (ADS)

Sebastian, Suchitra E.; Harrison, N.; Goddard, P. A.; Altarawneh, M. M.; Mielke, C. H.; Liang, Ruixing; Bonn, D. A.; Hardy, W. N.; Andersen, O. K.; Lonzarich, G. G.

2010-06-01

We report quantum oscillations in the underdoped high-temperature superconductor YBa2Cu3O6+x over a wide range in magnetic field 28≤μ0H≤85T corresponding to ≈12 oscillations, enabling the Fermi surface topology to be mapped to high resolution. As earlier reported by Sebastian [Nature (London) 454, 200 (2008)10.1038/nature07095], we find a Fermi surface comprising multiple pockets, as revealed by the additional distinct quantum oscillation frequencies and harmonics reported in this work. We find the originally reported broad low-frequency Fourier peak at ≈535T to be clearly resolved into three separate peaks at ≈460 , ≈532 , and ≈602T , in reasonable agreement with the reported frequencies of Audouard [Phys. Rev. Lett. 103, 157003 (2009)10.1103/PhysRevLett.103.157003]. However, our increased resolution and angle-resolved measurements identify these frequencies to originate from two similarly sized pockets with greatly contrasting degrees of interlayer corrugation. The spectrally dominant frequency originates from a pocket (denoted α ) that is almost ideally two-dimensional in form (exhibiting negligible interlayer corrugation). In contrast, the newly resolved weaker adjacent spectral features originate from a deeply corrugated pocket (denoted γ ). On comparison with band structure, the d -wave symmetry of the interlayer dispersion locates the minimally corrugated α pocket at the “nodal” point knodal=(π/2,π/2) , and the significantly corrugated γ pocket at the “antinodal” point kantinodal=(π,0) within the Brillouin zone. The differently corrugated pockets at different locations indicate creation by translational symmetry breaking—a spin-density wave has been suggested from the suppression of Zeeman splitting for the spectrally dominant pocket. In a broken-translational symmetry scenario, symmetry points to the nodal (α) pocket corresponding to holes, with the weaker antinodal (γ) pocket corresponding to electrons—likely responsible

Pocket Guide to Transportation 2012

DOT National Transportation Integrated Search

2012-01-01

The Bureau of Transportation Statistics (BTS) of the Research and Innovative Technology Administration produces the Pocket Guide to Transportation as a compact resource that provides snapshots of the U.S. transportation system and highlights major tr...

Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae.

PubMed

Daniel, Bastian; Wallner, Silvia; Steiner, Barbara; Oberdorfer, Gustav; Kumar, Prashant; van der Graaff, Eric; Roitsch, Thomas; Sensen, Christoph W; Gruber, Karl; Macheroux, Peter

2016-01-01

Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively. The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been structurally and biochemically characterized thus far. The most salient and distinguishing features of the active site found in AtBBE-like 28 are a mono-covalent linkage of a histidine to the 8α-position of the flavin-isoalloxazine ring and the lack of a second covalent linkage to the 6-position, owing to the replacement of a cysteine with a histidine. In addition, the structure reveals the interaction of a glutamic acid (Glu426) with an aspartic acid (Asp369) at the active site, which appear to share a proton. This arrangement leads to the delocalization of a negative charge at the active site that may be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation of reduced enzyme by dioxygen. A T-DNA insertional mutant line for AtBBE-like 28 results in a phenotype, that is characterized by reduced biomass and lower salt stress tolerance. Multiple sequence analysis showed that the active site composition found in AtBBE-like 28 is only present in the Brassicaceae, suggesting that it plays a specific role in the metabolism of this plant family.

1. Photocopy of photograph. ORIGINAL CANAL COAL POCKETS Source: Delaware ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

1. Photocopy of photograph. ORIGINAL CANAL COAL POCKETS Source: Delaware and Hudson Railroad and Canal, by Wayne County Historical Society. - Honesdale Coal Pockets, Main & Commercial Streets, between 700 & 800 blocks, Honesdale, Wayne County, PA

The Subgingival Microbiome of Periodontal Pockets With Different Probing Depths in Chronic and Aggressive Periodontitis: A Pilot Study.

PubMed

Shi, Meng; Wei, Yiping; Hu, Wenjie; Nie, Yong; Wu, Xiaolei; Lu, Ruifang

2018-01-01

Periodontitis is a kind of infectious disease initiated by colonization of subgingival periodontal pathogens, which cause destruction of tooth-supporting tissues, and is a predominant threat to oral health as the most common cause of loss of teeth. The aim of this pilot study was to characterize the subgingival bacterial biodiversity of periodontal pockets with different probing depths in patients with different forms of periodontitis. Twenty-one subgingival plaque samples were collected from three patients with chronic periodontitis (ChP), three patients with aggressive periodontitis (AgP) and three periodontally healthy subjects (PH). Each patient with periodontitis was sampled at three sites, at different probing depths (PDs, one each at 4 mm, 5-6 mm, and ≥ 7 mm). Using 16S rRNA gene high-throughput sequencing and bioinformatic analysis, we found that subgingival communities in health and periodontitis samples largely differed. Meanwhile, Acholeplasma, Fretibacterium, Porphyromonas, Peptococcus, Treponema_2, Defluviitaleaceae_UCG_011, Filifactor , and Mycoplasma increased with the deepening of the pockets in ChP, whilst only Corynebacterium was negatively associated with PD. In AgP, Corynebacterium and Klebsiella were positively associated with PD, while Serratia, Pseudoramibacter, Defluviitaleaceae_UCG_011 , and Desulfobulbus were negatively associated with PD. And among these two groups, Corynebacterium shifted differently. Moreover, in subgingival plaque, the unweighted UniFrac distances between samples from pockets with different PD in the same patients were significantly lower than those from pockets within the same PD category from different patients. This study demonstrated the shift of the subgingival microbiome in individual teeth sites during disease development. Within the limitation of the relative small sample size, this pilot study shed new light on the dynamic relationship between the extent of periodontal destruction and the subgingival

Pocket guide to transportation, 1999

DOT National Transportation Integrated Search

1998-12-01

Statistics published in this Pocket Guide to Transportation come from many different sources. Some statistics are based on samples and are subject to sampling variability. Statistics may also be subject to omissions and errors in reporting, recording...

Pocket guide to transportation, 1998

DOT National Transportation Integrated Search

1998-12-01

The statistics in this Pocket Guide to Transportation were compiled by the Bureau of Transportation Statistics from multiple sources. The guide is divided into four sections: (1) transportation system extent and use, (2) transportation and the econom...

Pocket guide to transportation, 2009

DOT National Transportation Integrated Search

2009-01-01

Statistics published in this Pocket Guide to Transportation come from many different sources. Some statistics are based on samples and are subject to sampling variability. Statistics may also be subject to omissions and errors in reporting, recording...

Pocket guide to transportation, 2013.

DOT National Transportation Integrated Search

2013-01-01

Abstract Statistics published in this Pocket Guide to Transportation come from many different sources. Some statistics are based on samples and are subject to sampling variability. Statistics may also be subject to omissions and errors in reporting, ...

Pocket guide to transportation, 2010

DOT National Transportation Integrated Search

2010-01-01

Statistics published in this Pocket Guide to Transportation come from many different sources. Some statistics are based on samples and are subject to sampling variability. Statistics may also be subject to omissions and errors in reporting, recording...

The structure of the Mycobacterium smegmatis trehalose synthase reveals an unusual active site configuration and acarbose-binding mode†

PubMed Central

Caner, Sami; Nguyen, Nham; Aguda, Adeleke; Zhang, Ran; Pan, Yuan T; Withers, Stephen G; Brayer, Gary D

2013-01-01

Trehalose synthase (TreS) catalyzes the reversible conversion of maltose into trehalose in mycobacteria as one of three biosynthetic pathways to this nonreducing disaccharide. Given the importance of trehalose to survival of mycobacteria, there has been considerable interest in understanding the enzymes involved in its production; indeed the structures of the key enzymes in the other two pathways have already been determined. Herein, we present the first structure of TreS from Mycobacterium smegmatis, thereby providing insights into the catalytic machinery involved in this intriguing intramolecular reaction. This structure, which is of interest both mechanistically and as a potential pharmaceutical target, reveals a narrow and enclosed active site pocket within which intramolecular substrate rearrangements can occur. We also present the structure of a complex of TreS with acarbose, revealing a hitherto unsuspected oligosaccharide-binding site within the C-terminal domain. This may well provide an anchor point for the association of TreS with glycogen, thereby enhancing its role in glycogen biosynthesis and degradation. PMID:23735230

Subperiosteal and intraosseous connective tissue grafts for pocket reduction: a 9- to 13-year retrospective case series report.

PubMed

Nelson, S W

2001-10-01

Recent histological evidence has documented that grafted palatal connective tissue is capable of forming a new attachment to previously exposed roots in the treatment of gingival recession. No clinical studies have tested the ability of connective tissue that has been implanted beneath the periosteum into periodontal osseous defects to reduce probing depth and increase clinical attachment levels. This study reports the long-term clinical effect of subperiosteal and intraosseous connective tissue grafts on deep periodontal pockets. Connective tissue (CT) grafts were placed in 32 periodontal pockets on 27 patients. Grafts were classified into 3 groups. Type I grafts had 50% or more vascular surface contact and were < or = 2.5 mm thick. Type II grafts had 50% or more contact but were > 2.5 mm thick, and Type III grafts had less than 50% vascular contact regardless of thickness. Twelve of 14 Type I sites, 9 of 15 Type II sites, and 3 of 3 Type III sites were analyzed 9 to 13 years following treatment. Clinical attachment level change differed significantly between the graft types on survivor teeth (P < 0.05): Type III had 2 mm loss (95% confidence interval [CI]: 0.4 to 3.6), while Type II and Type I grafts had a 2.7 mm gain (95% CI: 2.0 to 3.4) and 4.3 mm gain (95% CI: 3.3 to 5.2), respectively. Similar substantial differences were presented for changes in probing depth and recession. This long-term (9 to 13 years) retrospective case-series analysis suggests substantial improvements in periodontal clinical measures for Type I CT grafts in deep periodontal pockets. Randomized trials are required to evaluate this promising procedure.

Pocket Guide to Transportation 2016

DOT National Transportation Integrated Search

2016-01-01

The 2016 Pocket Guide to Transportation is a compilation of statistics that provides key information on the U.S. transportation system and highlights major trends. Intended as a compact reference, it supports the Bureau of Transportation Statistics m...

Pocket Guide to Transportation 2015

DOT National Transportation Integrated Search

2015-01-01

The 2015 Pocket Guide to Transportation is a compilation of statistics that provide key information and highlight major trends on the U.S. transportation system. Intended as a compact reference, it supports the Bureau of Transportation Statistics mis...

Pocket Guide to Transportation 2014

DOT National Transportation Integrated Search

2014-01-01

The 2014 Pocket Guide to Transportation is a compilation of statistics related to the performance and impact of the U.S. transportation system. Intended as a compact reference, it supports the Bureau of Transportation Statistics mission to create,...

Pocket Guide to Transportation 2018

DOT National Transportation Integrated Search

2018-01-01

The 2018 BTS Pocket Guide to Transportation is a quick reference guide that provides transportation statistics at your fingertips. It provides key information and highlights major trends on the U.S. transportation system. This year features a new and...

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

PubMed Central

Roosild, Tarmo P.; Castronovo, Samantha

2010-01-01

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications. PMID:20856879

Epitympanum volume and tympanic isthmus area in temporal bones with retraction pockets.

PubMed

Monsanto, Rafael da Costa; Pauna, Henrique Furlan; Kaya, Serdar; Hızlı, Ömer; Kwon, Geeyoun; Paparella, Michael M; Cureoglu, Sebahattin

2016-11-01

To compare the volume of the epitympanic space, as well as the area of the tympanic isthmus, in human temporal bones with retraction pockets to those with chronic otitis media without retraction pockets and to those with neither condition. Comparative human temporal bone study. We generated a three-dimensional model of the bony epitympanum and measured the epitympanic space. We also compared the area of the tympanic isthmus. The mean total volume of the epitympanum was 40.55 ± 7.14 mm 3 in the retraction pocket group, 50.03 ± 8.49 mm 3 in the chronic otitis media group, and 48.03 ± 9.16 mm 3 in the neither condition group. The mean volume of the anterior, lateral, and medial compartments in temporal bones in the retraction pocket group was significantly smaller than in the two control groups (P < 0.05). Total epitympanic volume was also significantly smaller in the retraction pocket group than in both control groups (P < 0.05). The mean area of the tympanic isthmus was significantly smaller in the retraction pocket group (8.11 ± 2.44 mm 2 ) than in the chronic otitis media group (9.82 ± 2.06 mm 2 ) or the neither condition group (10.66 ± 1.78 mm 2 ) (P < 0.05). Our data indicate that temporal bones with retraction pockets have a smaller volume bony epitympanum and a smaller tympanic isthmus area as compared with temporal bones from both control groups. The smaller volume tympanic isthmus in the retraction pocket group may suggest that a blockage in the aeration pathways to the epitympanum could create dysventilation, resulting in negative pressure and ultimately in retraction pockets and cholesteatomas. NA Laryngoscope, 126:E369-E374, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

Histological analysis of retraction pocket pars tensa of tympanic membrane in children.

PubMed

Urík, M; Hurník, P; Žiak, D; Machač, J; Šlapák, I; Motyka, O; Vaculová, J; Dvořáčková, J

2016-07-01

Histological and histochemical analysis of retraction pocket of pars tensa of tympanic membrane in children. Identification of morphological abnormalities in comparison with a healthy tympanic membrane as it is described in standard textbook. Identification of signs typical for cholesteatoma and support for a retraction theory of cholesteatoma formation. A prospective study analysing 31 samples of retraction pockets taken during surgery. University Hospital, Children's Medical Centre Samples of retraction pockets were processed by a standard process for light microscopy, stained by haematoxylin-eosin. Van Gieson's stain was used for differential staining of collagen, Verhoeff's stain for elastic fibre tissues, Alcian blue for acidic polysaccharides and PAS (Periodic Acid Schiff) method for basement membrane polysaccharides. The following findings were observed in the samples of retraction pockets: hyperkeratosis (100%), hypervascularisations (100%), subepithelial fragmented elastic fibres (96%), myxoid changes (87%), subepithelial inflammatory infiltration (84%), rete pegs (71%), papilomatosis (71%), intraepithelial inflammatory cellularizations, (48%), intraepithelial spongiosis (16%) and parakeratosis (3%). No basement membrane continuity interruptions were observed. Thickness of retraction pocket, thickness of epidermis, occurrence of rete pegs and frequency of fragmented elastic fibres was higher in a Grade III stage RP than Grade II stage RP (according to Charachon). Morphological abnormalities in the structure of retraction pockets in comparison with a healthy tympanic membrane were described. The changes are typical for a structure of cholesteatoma (these changes are common in matrix and perimatrix), supporting retraction theory of its origin. Our observations show that it is inflammation that probably plays a key role in the pathogenesis of retraction pocket. The frequency of some of the changes increases with the stage of retraction pocket (II

X-ray crystal structure of plasmin with tranexamic acid-derived active site inhibitors.

PubMed

Law, Ruby H P; Wu, Guojie; Leung, Eleanor W W; Hidaka, Koushi; Quek, Adam J; Caradoc-Davies, Tom T; Jeevarajah, Devadharshini; Conroy, Paul J; Kirby, Nigel M; Norton, Raymond S; Tsuda, Yuko; Whisstock, James C

2017-05-09

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO ( trans -4-aminomethylcyclohexanecarbonyl-l-tyrosine- n -octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3' subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S' subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders.

PockDrug-Server: a new web server for predicting pocket druggability on holo and apo proteins

PubMed Central

Hussein, Hiba Abi; Borrel, Alexandre; Geneix, Colette; Petitjean, Michel; Regad, Leslie; Camproux, Anne-Claude

2015-01-01

Predicting protein pocket's ability to bind drug-like molecules with high affinity, i.e. druggability, is of major interest in the target identification phase of drug discovery. Therefore, pocket druggability investigations represent a key step of compound clinical progression projects. Currently computational druggability prediction models are attached to one unique pocket estimation method despite pocket estimation uncertainties. In this paper, we propose ‘PockDrug-Server’ to predict pocket druggability, efficient on both (i) estimated pockets guided by the ligand proximity (extracted by proximity to a ligand from a holo protein structure) and (ii) estimated pockets based solely on protein structure information (based on amino atoms that form the surface of potential binding cavities). PockDrug-Server provides consistent druggability results using different pocket estimation methods. It is robust with respect to pocket boundary and estimation uncertainties, thus efficient using apo pockets that are challenging to estimate. It clearly distinguishes druggable from less druggable pockets using different estimation methods and outperformed recent druggability models for apo pockets. It can be carried out from one or a set of apo/holo proteins using different pocket estimation methods proposed by our web server or from any pocket previously estimated by the user. PockDrug-Server is publicly available at: http://pockdrug.rpbs.univ-paris-diderot.fr. PMID:25956651

PockDrug-Server: a new web server for predicting pocket druggability on holo and apo proteins.

PubMed

Hussein, Hiba Abi; Borrel, Alexandre; Geneix, Colette; Petitjean, Michel; Regad, Leslie; Camproux, Anne-Claude

2015-07-01

Predicting protein pocket's ability to bind drug-like molecules with high affinity, i.e. druggability, is of major interest in the target identification phase of drug discovery. Therefore, pocket druggability investigations represent a key step of compound clinical progression projects. Currently computational druggability prediction models are attached to one unique pocket estimation method despite pocket estimation uncertainties. In this paper, we propose 'PockDrug-Server' to predict pocket druggability, efficient on both (i) estimated pockets guided by the ligand proximity (extracted by proximity to a ligand from a holo protein structure) and (ii) estimated pockets based solely on protein structure information (based on amino atoms that form the surface of potential binding cavities). PockDrug-Server provides consistent druggability results using different pocket estimation methods. It is robust with respect to pocket boundary and estimation uncertainties, thus efficient using apo pockets that are challenging to estimate. It clearly distinguishes druggable from less druggable pockets using different estimation methods and outperformed recent druggability models for apo pockets. It can be carried out from one or a set of apo/holo proteins using different pocket estimation methods proposed by our web server or from any pocket previously estimated by the user. PockDrug-Server is publicly available at: http://pockdrug.rpbs.univ-paris-diderot.fr. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Frequency of "Pocket" Hematoma in Patients Receiving Vitamin K Antagonist and Antiplatelet Therapy at the Time of Pacemaker or Cardioverter Defibrillator Implantation (from the POCKET Study).

PubMed

Malagù, Michele; Trevisan, Filippo; Scalone, Antonella; Marcantoni, Lina; Sammarco, Giuseppe; Bertini, Matteo

2017-04-01

In patients undergoing cardiac device implantation, anticoagulant and antiplatelet therapy are associated with an increased risk of pocket hematoma. In case of vitamin K antagonist therapy, a strategy of continued warfarin with no heparin bridge showed a reduction of pocket hematoma. Evidence regarding antiplatelet therapy management is limited. This is a single-center observational study which reflects our systematic approach to the problem. In 2012, we proposed an improved management protocol for anticoagulant and antiplatelet therapy (no-bridge protocol) based on individual thromboembolic risk stratification, noninterruption of oral anticoagulation, no bridge with heparin and elastic adherence compression bandage. The primary end point was the incidence of clinically significant pocket hematoma in the first 30 days after implantation. A total of 1,035 patients were enrolled, of whom 522 received the standard management and 513 the new protocol. The primary end point occurred in 34 patients of the standard management group and 8 patients of the no-bridge protocol group (6.5% vs 1.6%, p <0.001). Patients in the standard management group had a higher incidence of pocket infections (2.3% vs 0.6%, p = 0.02), lead dislodgements (4.8% vs 2.1%, p = 0.02), and thromboembolic events (1.3% vs 0.0%, p <0.01). On a multivariate analysis, heparin and coronary artery disease were independent predictors of pocket hematoma (relative risk [RR] 3.48, 95% confidence interval [CI] 1.55 to 7.83 and RR 2.43, 95% CI 1.25 to 4.76, respectively), whereas the no-bridge protocol was associated with a reduction of pocket hematoma (RR 0.33, 95% CI 0.14 to 0.76). New anticoagulant and antiplatelet therapy management protocol was associated with a reduced incidence of clinically significant pocket hematomas, thromboembolic events, pocket infections, and lead dislodgements. Copyright © 2017 Elsevier Inc. All rights reserved.

Use of a pocket compression device for the prevention and treatment of pocket hematoma after pacemaker and defibrillator implantation (STOP-HEMATOMA-I).

PubMed

Turagam, Mohit K; Nagarajan, Darbhamulla V; Bartus, Krzysztof; Makkar, Akash; Swarup, Vijay

2017-08-01

Pocket hematoma is a recognized complication after placement of cardiac implantable electronic devices and is associated with increased device infection, length of hospitalization, and morbidity especially with uninterrupted antiplatelet agents and anticoagulants. We assessed the use of a post-surgical vest to decrease the incidence of pocket hematoma in patients undergoing device implantation with uninterrupted antiplatelet and anticoagulants. In this observational study, a vest was used by 20 consecutive patients who were compared to 20 age-, gender-, procedure-matched patients who received standard care. All patients were continued on antiplatelet and anticoagulants in the perioperative period. The pocket was assessed at post procedure day 0, 2, and 7, respectively. There were no significant differences in the baseline characteristics between both groups. Baseline mean international normalized ratio (INR) was significantly higher in the vest group when compared with the control group (2.7 ± 0.4 vs. 2.2 ± 0.3 = <0.001). The incidence of pocket hematoma was significantly lower in the vest group than the control group (0 vs 30%, p = 0.02) at the end of 7 days. Control group had a total of six hematomas with one patient requiring evacuation and blood transfusion. The vest group had three hematomas on day 2 that resolved by day 7. The risk of moderate or large pocket hematoma is significantly reduced with the use of this vest in high-risk patients undergoing implantable devices on uninterrupted antiplatelet and anticoagulants.

Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

PubMed

Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

2017-01-27

We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R 2  = 0.49). However, the addition of the active-site waters resulted in significant improvement (R 2  = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

PubMed

Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket

Insights into an Original Pocket-Ligand Pair Classification: A Promising Tool for Ligand Profile Prediction

PubMed Central

Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A.; Villoutreix, Bruno O.; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket

Guiding periodontal pocket recolonization: a proof of concept.

PubMed

Teughels, W; Newman, M G; Coucke, W; Haffajee, A D; Van Der Mei, H C; Haake, S Kinder; Schepers, E; Cassiman, J-J; Van Eldere, J; van Steenberghe, D; Quirynen, M

2007-11-01

The complexity of the periodontal microbiota resembles that of the gastro-intestinal tract, where infectious diseases are treatable via probiotics. In the oropharyngeal region, probiotic or replacement therapies have shown some benefit in the prevention of dental caries, otitis media, and pharyngitis, but their effectiveness in the treatment of periodontitis is unknown. Therefore, this study addressed the hypothesis that the application of selected beneficial bacteria, as an adjunct to scaling and root planing, would inhibit the periodontopathogen recolonization of periodontal pockets. Analysis of the data showed, in a beagle dog model, that when beneficial bacteria were applied in periodontal pockets adjunctively after root planing, subgingival recolonization of periodontopathogens was delayed and reduced, as was the degree of inflammation, at a clinically significant level. The study confirmed the hypothesis and provides a proof of concept for a guided pocket recolonization (GPR) approach in the treatment of periodontitis.

Conformational flexibility related to enzyme activity: evidence for a dynamic active-site gatekeeper function of Tyr(215) in Aerococcus viridans lactate oxidase.

PubMed

Stoisser, Thomas; Brunsteiner, Michael; Wilson, David K; Nidetzky, Bernd

2016-06-15

L-Lactate oxidase (LOX) belongs to a large family of flavoenzymes that catalyze oxidation of α-hydroxy acids. How in these enzymes the protein structure controls reactivity presents an important but elusive problem. LOX contains a prominent tyrosine in the substrate binding pocket (Tyr(215) in Aerococcus viridans LOX) that is partially responsible for securing a flexible loop which sequesters the active site. To characterize the role of Tyr(215), effects of substitutions of the tyrosine (Y215F, Y215H) were analyzed kinetically, crystallographically and by molecular dynamics simulations. Enzyme variants showed slowed flavin reduction and oxidation by up to 33-fold. Pyruvate release was also decelerated and in Y215F, it was the slowest step overall. A 2.6-Å crystal structure of Y215F in complex with pyruvate shows the hydrogen bond between the phenolic hydroxyl and the keto oxygen in pyruvate is replaced with a potentially stronger hydrophobic interaction between the phenylalanine and the methyl group of pyruvate. Residues 200 through 215 or 216 appear to be disordered in two of the eight monomers in the asymmetric unit suggesting that they function as a lid controlling substrate entry and product exit from the active site. Substitutions of Tyr(215) can thus lead to a kinetic bottleneck in product release.

Do medical out-of-pocket expenses thrust families into poverty?

PubMed

O'Hara, Brett

2004-02-01

This paper estimates the impact of medical out-of-pocket expenses on families' well-being using the Survey of Income and Program Participation. Medical out-of-pocket expenses include the out-of-pocket costs from medical services and the family's share of health insurance premiums. Demographic characteristics, insurance status, and medical usage of the family are analyzed to determine which characteristics are most likely to impoverish a family. Families impoverished because of medical out-of-pocket expenses are far more likely to have older heads of the family, at least one family member in poor health, or some adults without health insurance. Families without at least one person who worked full time for the entire year were also likely to be impoverished. However, children in the family had little effect on the probability that the family became impoverished. This odd result is probably due to the high correlation between parental health insurance coverage and the health insurance coverage of their children.

Statistical Profiling of One Promiscuous Protein Binding Site: Illustrated by Urokinase Catalytic Domain.

PubMed

Cerisier, Natacha; Regad, Leslie; Triki, Dhoha; Petitjean, Michel; Flatters, Delphine; Camproux, Anne-Claude

2017-10-01

While recent literature focuses on drug promiscuity, the characterization of promiscuous binding sites (ability to bind several ligands) remains to be explored. Here, we present a proteochemometric modeling approach to analyze diverse ligands and corresponding multiple binding sub-pockets associated with one promiscuous binding site to characterize protein-ligand recognition. We analyze both geometrical and physicochemical profile correspondences. This approach was applied to examine the well-studied druggable urokinase catalytic domain inhibitor binding site, which results in a large number of complex structures bound to various ligands. This approach emphasizes the importance of jointly characterizing pocket and ligand spaces to explore the impact of ligand diversity on sub-pocket properties and to establish their main profile correspondences. This work supports an interest in mining available 3D holo structures associated with a promiscuous binding site to explore its main protein-ligand recognition tendency. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

POVME 2.0: An Enhanced Tool for Determining Pocket Shape and Volume Characteristics

PubMed Central

2015-01-01

Analysis of macromolecular/small-molecule binding pockets can provide important insights into molecular recognition and receptor dynamics. Since its release in 2011, the POVME (POcket Volume MEasurer) algorithm has been widely adopted as a simple-to-use tool for measuring and characterizing pocket volumes and shapes. We here present POVME 2.0, which is an order of magnitude faster, has improved accuracy, includes a graphical user interface, and can produce volumetric density maps for improved pocket analysis. To demonstrate the utility of the algorithm, we use it to analyze the binding pocket of RNA editing ligase 1 from the unicellular parasite Trypanosoma brucei, the etiological agent of African sleeping sickness. The POVME analysis characterizes the full dynamics of a potentially druggable transient binding pocket and so may guide future antitrypanosomal drug-discovery efforts. We are hopeful that this new version will be a useful tool for the computational- and medicinal-chemist community. PMID:25400521

Out-of-pocket health care expenditures, by insurance status, 2007-10.

PubMed

Catlin, Mary K; Poisal, John A; Cowan, Cathy A

2015-01-01

Out-of-pocket health care spending in the United States totaled $306.2 billion in 2010 and represented 11.8 percent of total national health expenditures, according to the Centers for Medicare and Medicaid Services' National Health Expenditure Accounts. Spending by people with employer-sponsored health insurance and those covered by Medicare accounted for over 80 percent of total out-of-pocket spending. People without comprehensive medical coverage accounted for less than 8 percent of all out-of-pocket expenditures in 2010. Between 2007 and 2010 per person out-of-pocket spending grew most rapidly for people primarily covered by employer-sponsored insurance and declined for people primarily covered by Medicare and those without coverage. Project HOPE—The People-to-People Health Foundation, Inc.

NASA Pocket Statistics

NASA Technical Reports Server (NTRS)

1996-01-01

This booklet of pocket statistics includes the 1996 NASA Major Launch Record, NASA Procurement, Financial, and Workforce data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Luanch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

Accommodation of GDP-Linked Sugars in the Active Site of GDP-Perosamine Synthase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, Paul D.; Carney, Amanda E.; Holden, Hazel M.

2009-01-12

Perosamine (4-amino-4,6-dideoxy-d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue,more » which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 {angstrom} resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K{sub m} for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k{sub cat} was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy-d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.« less

NOTE: Cell-phone interference with pocket dosimeters

NASA Astrophysics Data System (ADS)

Djajaputra, David; Nehru, Ramasamy; Bruch, Philip M.; Ayyangar, Komanduri M.; Raman, Natarajan V.; Enke, Charles A.

2005-05-01

Accurate reporting of personal dose is required by regulation for hospital personnel that work with radioactive material. Pocket dosimeters are commonly used for monitoring this personal dose. We show that operating a cell phone in the vicinity of a pocket dosimeter can introduce large and erroneous readings of the dosimeter. This note reports a systematic study of this electromagnetic interference. We found that simple practical measures are enough to mitigate this problem, such as increasing the distance between the cell phone and the dosimeter or shielding the dosimeter, while maintaining its sensitivity to ionizing radiation, by placing it inside a common anti-static bag.

Uncovering the determinants of a highly perturbed tyrosine pKa in the active site of ketosteroid isomerase.

PubMed

Schwans, Jason P; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

2013-11-05

Within the idiosyncratic enzyme active-site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near charged groups or within hydrophobic pockets, Tyr57 of a Pseudomonas putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semisynthesis, (13)C NMR spectroscopy, absorbance spectroscopy, and X-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from the introduction of nearby anionic groups, arises from accompanying active-site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen-bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen-bond donors, two Tyr side chains, and a water molecule positioned by other side chains and by a water-mediated hydrogen-bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural

Uncovering the Determinants of a Highly Perturbed Tyrosine pKa in the Active Site of Ketosteroid Isomerase†

PubMed Central

Schwans, Jason P.; Sunden, Fanny; Gonzalez, Ana; Tsai, Yingssu; Herschlag, Daniel

2013-01-01

Within the idiosyncratic enzyme active site environment, side chain and ligand pKa values can be profoundly perturbed relative to their values in aqueous solution. Whereas structural inspection of systems has often attributed perturbed pKa values to dominant contributions from placement near to charged groups or within hydrophobic pockets, Tyr57 of a P. putida ketosteroid isomerase (KSI) mutant, suggested to have a pKa perturbed by nearly 4 units to 6.3, is situated within a solvent-exposed active site devoid of cationic side chains, metal ions, or cofactors. Extensive comparisons among 45 variants with mutations in and around the KSI active site, along with protein semi-synthesis, 13C NMR spectroscopy, absorbance spectroscopy, and x-ray crystallography, was used to unravel the basis for this perturbed Tyr pKa. The results suggest that the origin of large energetic perturbations are more complex than suggested by visual inspection. For example, the introduction of positively charged residues near Tyr57 raises its pKa rather than lowers it; this effect, and part of the increase in the Tyr pKa from introduction of nearby anionic groups arise from accompanying active site structural rearrangements. Other mutations with large effects also cause structural perturbations or appear to displace a structured water molecule that is part of a stabilizing hydrogen bond network. Our results lead to a model in which three hydrogen bonds are donated to the stabilized ionized Tyr, with these hydrogen bond donors, two Tyr side chains and a water molecule, positioned by other side chains and by a water-mediated hydrogen bond network. These results support the notion that large energetic effects are often the consequence of multiple stabilizing interactions, rather than a single dominant interaction. Most generally, this work provides a case study for how extensive and comprehensive comparisons via site-directed mutagenesis in a tight feedback loop with structural analysis can

Planview Geometry and morphological characteristics of pocket beaches on the Catalan coast (Spain)

NASA Astrophysics Data System (ADS)

Bowman, D.; Guillén, J.; López, L.; Pellegrino, V.

2009-07-01

Coastal planform studies are a relevant initial stage before launching detailed dynamic field experiments. The aim of this study is to define the planform characteristics of 72 Catalan pocket beaches, natural and man-made, and to determine their sheltering effect, embaymentization and their status of equilibrium. Planform measurements were performed on SIGPAC, 1:5000 orthophoto sets and wave climate was provided by Puertos del Estado (Wana model). Planform parameters were applied and coastal planview indexes were determined. The study shows that the Catalan pocket beaches display a wide range of indentation, suggesting that no single structural, tectonic or morphological control dominates their planform. The man-made pocket beaches typically display indentations which are smaller than those shown by natural pocket beaches. Headland spacing and beach area are positively correlated. The more indented bays are, the shorter their beaches become. Low-indented pocket beaches are the widest and the longest ones. Deep indentation contributes towards beach protection and energy dissipation which counteracts rip efficiency and inhibits the formation of mega-rips. Pocket beaches often show gradual and moderate alongshore changes in texture and beach morphology. One third of the Catalan pocket beaches are "sediment starved", i.e., 60% and more of their embayed shorelines are deprived of beach sediments. Examination of the status of equilibrium demonstrates that most of the Catalan pocket beaches are in an unstable mode, with indentation ratios that are unrelated to the wave obliquity.

Morphological alterations of periodontal pocket epithelium following Nd:YAG laser irradiation.

PubMed

Ting, Chun-Chan; Fukuda, Mitsuo; Watanabe, Tomohisa; Sanaoka, Atsushi; Mitani, Akio; Noguchi, Toshihide

2014-12-01

The purpose of this in vivo study was to examine morphologic alterations in the periodontal pocket epithelium with presence or absence of clinical inflammation following the use of the Neodymium: Yttrium-Aluminum-Garnet (Nd:YAG) laser irradiation. Subgingival Nd:YAG laser irradiation has been proposed as an alternative technique for treatment of chronic periodontitis. Several published studies have reported the clinical outcomes of such treatment. Twenty patients, diagnosed with moderate chronic periodontitis, were selected for the study. A total of 32 sites was identified and divided into a control (n=18) and laser-treated test groups (n=14). Probing depth (PD) and bleeding on probing (BOP) were recorded for all sites. Test sites were irradiated with an Nd:YAG laser using parameters of 2 W, 200 mJ pulse energy, and 10 pps delivered through a 320 μm diameter tip. Total laser treatment time ranged from 1 to 2 min. Following treatment, all specimens were harvested via biopsy and processed for scanning electron microscopy (SEM) and histologic examination. Control group specimens, depending upon initial PD, exhibited either a relatively smooth and intact epithelium with little desquamation (PD≤3 mm), or increasing degrees of epithelial desquamation and leukocytic infiltration at a PD of ≥4 mm. In the laser-treated test group, the specimens with PD≤3 mm that were BOP negative (-) exhibited a thin layer of epithelium that was disrupted. In the specimens with initial PD of ≥4 mm, complete removal of the epithelium whose extent and degree were increasing, was observed in the inflamed portion, while epithelium remained in the uninflamed portion. The SEM and histologic findings demonstrated the feasibility of ablating pocket epithelium with an Nd:YAG laser irradiation using parameters of 2 W of power (200 mJ, 10 pps). Furthermore, the presence or absence of clinical inflammation appeared to have an impact on the degree of laser

Enceladus' near-surface CO2 gas pockets and surface frost deposits

NASA Astrophysics Data System (ADS)

Matson, Dennis L.; Davies, Ashley Gerard; Johnson, Torrence V.; Combe, Jean-Philippe; McCord, Thomas B.; Radebaugh, Jani; Singh, Sandeep

2018-03-01

Solid CO2 surface deposits were reported in Enceladus' South Polar Region by Brown et al. (2006). They noted that such volatile deposits are temporary and posited ongoing replenishment. We present a model for this replenishment by expanding on the Matson et al. (2012) model of subsurface heat and chemical transport in Enceladus. Our model explains the distributions of both CO2 frost and complexed CO2 clathrate hydrate as seen in the Cassini Visual and Infrared Mapping Spectrometer (VIMS) data. We trace the journey of CO2 from a subsurface ocean. The ocean-water circulation model of Matson et al. (2012) brings water up to near the surface where gas exsolves to form bubbles. Some of the CO2 bubbles are trapped and form pockets of gas in recesses at the bottom of the uppermost ice layer. When fissures break open these pockets, the CO2 gas is vented. Gas pocket venting is episodic compared to the more or less continuous eruptive plumes, emanating from the "tiger stripes", that are supported by plume chambers. Two styles of gas pocket venting are considered: (1) seeps, and (2) blowouts. The presence of CO2 frost patches suggests that the pocket gas slowly seeped through fractured, cold ice and when some of the gas reached the surface it was cold enough to condense (i.e., T ∼70 to ∼119 K). If the fissure opening is large, a blowout occurs. The rapid escape of gas and drop in pocket pressure causes water in the pocket to boil and create many small aerosol droplets of seawater. These may be carried along by the erupting gas. Electrically charged droplets can couple to the magnetosphere, and be dragged away from Enceladus. Most of the CO2 blowout gas escapes from Enceladus and the remainder is distributed globally. However, CO2 trapped in a clathrate structure does not escape. It is much heavier and slower moving than the CO2 gas. Its motion is ballistic and has an average range of about 17 km. Thus, it contributes to deposits in the vicinity of the vent. Local heat

Effect of baclofen on the acid pocket at the gastroesophageal junction.

PubMed

Scarpellini, E; Boecxstaens, V; Farré, R; Bisschops, R; Dewulf, D; Gasbarrini, A; Pauwels, A; Blondeau, K; Tack, J

2015-07-01

Previous studies established that a pocket of highly acidic gastric juice is present postprandially at the gastroesophageal junction in man. The GABA-B agonist baclofen inhibits postprandial reflux events through its effects on the lower esophageal sphincter (LES). The aim of the current study was to investigate whether baclofen would affect the location and the extent of the postprandial acid pocket in healthy volunteers. Twelve healthy volunteers underwent acid pocket studies on two different occasions, at least 1 week apart. LES position was determined preprandially with pull-through manometry. Dual pH electrode and manometry probe stepwise pull-through (1 cm/minute, LES-10 to +5 cm) was performed at 30-minute intervals for 150 minutes, with administration of placebo or baclofen 40 mg after the first and ingestion of a liquid meal after the second pull-through. After placebo, a significant drop in intragastric gastric pH was present at the gastroesophageal junction after the meal, reflecting the acid pocket, and this was associated with a drop in LES pressure. Baclofen did not affect the presence of the acid pocket, but prevented the postprandial drop in LES pressure, and the extent of the acid pocket above the upper margin of the manometrically located LES was significantly decreased by baclofen (1.6 ± 0.7 vs. 0.3 ± 0.4 cm at 60 minutes, 2.2 ± 0.6 vs. 0.2 ± 0.6 at 90 minutes, and 1.5 ± 0.5 vs. 0.7 ± 0.7 cm at 120 minutes, all P < 0.05). Baclofen does not alter the intragastric acid pocket, but limits its extension into the distal esophagus, probably through an increase in postprandial LES pressure. © 2014 International Society for Diseases of the Esophagus.

A three-dimensional construction of the active site (region 507-749) of human neutral endopeptidase (EC.3.4.24.11).

PubMed

Tiraboschi, G; Jullian, N; Thery, V; Antonczak, S; Fournie-Zaluski, M C; Roques, B P

1999-02-01

A three-dimensional model of the 507-749 region of neutral endopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integrating the results of secondary structure predictions and sequence homologies with the bacterial endopeptidase thermolysin. Additional data were extracted from the structure of two other metalloproteases, astacin and stromelysin. The resulting model accounts for the main biological properties of NEP and has been used to describe the environment close to the zinc atom defining the catalytic site. The analysis of several thiol inhibitors, complexed in the model active site, revealed the presence of a large hydrophobic pocket at the S1' subsite level. This is supported by the nature of the constitutive amino acids. The computed energies of bound inhibitors correspond with the relative affinities of the stereoisomers of benzofused macrocycle derivatives of thiorphan. The model could be used to facilitate the design of new NEP inhibitors, as illustrated in the paper.

Pocket ECG electrode

NASA Technical Reports Server (NTRS)

Lund, Gordon F. (Inventor)

1982-01-01

A low-noise electrode suited for sensing electrocardiograms when chronically and subcutaneously implanted in a free-ranging subject. The electrode comprises a pocket-shaped electrically conductive member with a single entrance adapted to receive body fluids. The exterior of the member and the entrance region is coated with electrical insulation so that the only electrolyte/electrode interface is within the member remote from artifact-generating tissue. Cloth straps are bonded to the member to permit the electrode to be sutured to tissue and to provide electrical lead flexure relief.

Pocket ECG electrode

NASA Technical Reports Server (NTRS)

Lund, G. F. (Inventor)

1980-01-01

A low noise electrode suited for sensing electrocardiograms when chronically and subcutaneously implanted in a free ranging subject is described. The electrode comprises a pocket shaped electrically conductive member with a single entrance adapted to receive body fluids. The exterior of the member and the entrance region is coated with electrical insulation so that the only electrolyte/electrode interface is within the member, remote from artifact-generating tissue. Cloth straps are bonded to the member to permit the electrode to be sutured to tissue and to provide electrical lead flexure relief.

Cryptic binding sites on proteins: definition, detection, and druggability.

PubMed

Vajda, Sandor; Beglov, Dmitri; Wakefield, Amanda E; Egbert, Megan; Whitty, Adrian

2018-05-22

Many proteins in their unbound structures lack surface pockets appropriately sized for drug binding. Hence, a variety of experimental and computational tools have been developed for the identification of cryptic sites that are not evident in the unbound protein but form upon ligand binding, and can provide tractable drug target sites. The goal of this review is to discuss the definition, detection, and druggability of such sites, and their potential value for drug discovery. Novel methods based on molecular dynamics simulations are particularly promising and yield a large number of transient pockets, but it has been shown that only a minority of such sites are generally capable of binding ligands with substantial affinity. Based on recent studies, current methodology can be improved by combining molecular dynamics with fragment docking and machine learning approaches. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

PubMed Central

Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

2009-01-01

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

PubMed

Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

2009-11-13

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

Out-of-pocket payments for health care in Serbia.

PubMed

Arsenijevic, Jelena; Pavlova, Milena; Groot, Wim

2015-10-01

This study focuses on out-of-pocket payments for health care in Serbia. In contrast to previous studies, we distinguish three types of out-of-pocket patient payments: official co-payments, informal (under-the-table) payments and payments for "bought and brought goods" (i.e. payments for health care goods brought by the patient to the health care facility). We analyse the probability and intensity of three different types of out-of-pocket patient payments in the public health care sector in Serbia and their distribution among different population groups. We use data from the Serbian Living Standard Measures Study carried out in 2007. Out-of-pocket patients payments for both outpatient and inpatient health care are included. The data are analysed using regression analysis. The majority of health care users report official co-payments (84.7%) and payments for "bought and brought goods" (61.1%), whereas only 5.7% health care users declare that they have paid informally. Regarding the regression results, users with an income below the poverty line, those from rural areas and who are not married are more likely to report payments for "bought and brought goods, while young and more educated users are more likely to report informal patient payments. Overall, the three types of out-of-pocket payments are not correlated. Payments for "bought and brought goods" take the highest share of the total annual household budget. Serbian policymakers need to consider different strategies to deal with informal payments and to eliminate the practice of "bought and brought goods". Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

A salt bridge turns off the foot-pocket in class-II HDACs.

PubMed

Zhou, Jingwei; Yang, Zuolong; Zhang, Fan; Luo, Hai-Bin; Li, Min; Wu, Ruibo

2016-08-21

Histone Deacetylases (HDACs) are promising anticancer targets and several selective inhibitors have been created based on the architectural differences of foot-pockets among HDACs. However, the "gate-keeper" of foot-pockets is still controversial. Herein, it is for the first time revealed that a conserved R-E salt bridge plays a critical role in keeping foot-pockets closed in class-II HDACs by computational simulations. This finding is further substantiated by our mutagenesis experiments.

Engineers demonstrate the pocket rocket

NASA Technical Reports Server (NTRS)

1996-01-01

Part of Stennis Space Center's mission with its traveling exhibits is to educate the younger generation on how propulsion systems work. A popular tool is the 'pocket rocket,' which demonstrates how a hybrid rocket works. A hybrid rocket is a cross breed between a solid fuel rocket and a liquid fuel rocket.

X-ray crystal structure of plasmin with tranexamic acid–derived active site inhibitors

PubMed Central

Wu, Guojie; Leung, Eleanor W. W.; Hidaka, Koushi; Quek, Adam J.; Caradoc-Davies, Tom T.; Jeevarajah, Devadharshini; Kirby, Nigel M.; Norton, Raymond S.; Tsuda, Yuko; Whisstock, James C.

2017-01-01

The zymogen protease plasminogen and its active form plasmin perform key roles in blood clot dissolution, tissue remodeling, cell migration, and bacterial pathogenesis. Dysregulation of the plasminogen/plasmin system results in life-threatening hemorrhagic disorders or thrombotic vascular occlusion. Accordingly, inhibitors of this system are clinically important. Currently, tranexamic acid (TXA), a molecule that prevents plasminogen activation through blocking recruitment to target substrates, is the most widely used inhibitor for the plasminogen/plasmin system in therapeutics. However, TXA lacks efficacy on the active form of plasmin. Thus, there is a need to develop specific inhibitors that target the protease active site. Here we report the crystal structures of plasmin in complex with the novel YO (trans-4-aminomethylcyclohexanecarbonyl-l-tyrosine-n-octylamide) class of small molecule inhibitors. We found that these inhibitors form key interactions with the S1 and S3′ subsites of the catalytic cleft. Here, the TXA moiety of the YO compounds inserts into the primary (S1) specificity pocket, suggesting that TXA itself may function as a weak plasmin inhibitor, a hypothesis supported by subsequent biochemical and biophysical analyses. Mutational studies reveal that F587 of the S′ subsite plays a key role in mediating the inhibitor interaction. Taken together, these data provide a foundation for the future development of small molecule inhibitors to specifically regulate plasmin function in a range of diseases and disorders. PMID:29296720

Free enthalpies of replacing water molecules in protein binding pockets.

PubMed

Riniker, Sereina; Barandun, Luzi J; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F

2012-12-01

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH(3) group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH(3) at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

Free enthalpies of replacing water molecules in protein binding pockets

NASA Astrophysics Data System (ADS)

Riniker, Sereina; Barandun, Luzi J.; Diederich, François; Krämer, Oliver; Steffen, Andreas; van Gunsteren, Wilfred F.

2012-12-01

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH3 group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH3 at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.

Cigarette smoking in Chinese adolescents: importance of controlling the amount of pocket money.

PubMed

Ma, J; Zhu, J; Li, N; He, Y; Cai, Y; Qiao, Y; Redmon, P; Wang, Z

2013-07-01

To estimate the proportion of smokers that could potentially have been prevented from smoking by limiting the amount of pocket money received by Chinese adolescents. Cross-sectional study. Current smoking, ever smoking and the amount of pocket money were determined through self-administered questionnaires among 12,708 adolescents (aged 12-18 years) from 21 schools in Shanghai, China. Adjusted odds ratios for current smoking ranged from 2.0 [95% confidence interval (CI) 1.5-2.7] for adolescents receiving 200-399 Reminbin (RMB)/month as pocket money to 6.5 (95% CI 3.3-12.7) for those receiving ≥1000 RMB/month, compared with those receiving <200 RMB/month. The crude population-attributable risk percentage (PAR%) due to higher pocket money (≥200 RMB/month) for current smoking was 50.4% (95% CI 42.2-57.4), and adjusted PAR% was 43.3% (95% CI 30.7-53.1). Approximately half of current smokers may have been prevented from smoking if pocket money was limited to <200 RMB/month among Chinese adolescents. An even larger proportion could have been prevented from smoking if pocket money was reduced further. It is recommended that future intervention programmes should target parents to reduce the amount of pocket money in China. Copyright © 2013 The Royal Society for Public Health. Published by Elsevier Ltd. All rights reserved.

24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

Code of Federal Regulations, 2011 CFR

2011-04-01

... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

Code of Federal Regulations, 2013 CFR

2013-04-01

... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

Code of Federal Regulations, 2012 CFR

2012-04-01

... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

Code of Federal Regulations, 2014 CFR

2014-04-01

... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

24 CFR 570.466 - Additional application submission requirements for Pockets of Poverty-employment opportunities.

Code of Federal Regulations, 2010 CFR

2010-04-01

... requirements for Pockets of Poverty-employment opportunities. 570.466 Section 570.466 Housing and Urban... application submission requirements for Pockets of Poverty—employment opportunities. Applicants for Action Grants under the Pockets of Poverty provision must describe the number and, to the extent possible, the...

Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

PubMed Central

Fürst, Oliver; Nichols, Colin G.; Lamoureux, Guillaume; D’Avanzo, Nazzareno

2014-01-01

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K+ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket. PMID:25517146

Proton pump inhibitors reduce the size and acidity of the acid pocket in the stomach.

PubMed

Rohof, Wout O; Bennink, Roelof J; Boeckxstaens, Guy E

2014-07-01

The gastric acid pocket is believed to be the reservoir from which acid reflux events originate. Little is known about how changes in position, size, and acidity of the acid pocket contribute to the therapeutic effect of proton pump inhibitors (PPIs) in patients with gastroesophageal reflux disease (GERD). Thirty-six patients with GERD (18 not taking PPIs, 18 taking PPIs; 19 men; age, 55 ± 2.1 y) were analyzed by concurrent high-resolution manometry and pH-impedance monitoring after a standardized meal. The acid pocket was visualized using scintigraphy after intravenous administration of (99m)technetium-pertechnetate. The size of the acid pocket was measured and its position was determined, relative to the diaphragm, using radionuclide markers on a high-resolution manometry catheter. At the end of the study, the acid pocket was aspirated, and its pH level was measured. The number of reflux episodes was comparable between patients on and off PPIs, but the number of acid reflux episodes was reduced significantly in patients on PPIs. In patients on PPIs, the acid pocket was smaller and more frequently located below the diaphragm. The mean pH of the acid pocket was significantly lower in patients not taking PPIs (n = 6) than in those who were (n = 16) (0.9; range, 0.7-1.2 vs 4.0; range, 1.6-5.9; P < .001). The pH of acid pockets correlated significantly with the lowest pH values measured for refluxate (r = 0.72; P < .01). Based on analyses of acid pockets in patients with GERD, the acid pocket appears to be a reservoir from which reflux occurs when patients are receiving PPIs. PPIs might affect the size, acidity, or position of the acid pocket, which contributes to the efficacy in patients with GERD. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Reynolds number scaling of pocket events in the viscous sublayer

NASA Astrophysics Data System (ADS)

Metzger, M.; Fershtut, A.; Kunkel, C.; Klewicki, J.

2017-12-01

Recent findings [X. Wu et al., Proc. Natl. Acad. Sci. USA 114, E5292 (2017), 10.1073/pnas.1704671114] reinforce earlier assertions [e.g., R. Falco, Philos. Trans. R. Soc. London A 336, 103 (1991), 10.1098/rsta.1991.0069] that the sublayer pocket motions play a distinctly important role in near-wall dynamics. In the present study, smoke visualization and axial velocity measurements are combined in order to establish the scaling behavior of pocket events in the viscous sublayer of the turbulent boundary layer. In doing so, an identical analysis methodology is employed over an extensive range of friction Reynolds numbers 388 ≤δ+≤2.2 ×105 . Both the pocket width W and time interval between pocket events T increase logarithmically with Reynolds number when normalized by viscous units. Normalization of W and T by the Taylor microscales evaluated at a wall-normal location of about 100 viscous units, however, appears to successfully remove this Reynolds-number dependence. The present results are discussed in the context of motion formation owing to the three dimensionalization of the near-wall vorticity field and, concomitantly, the recurring perturbation of the viscous sublayer.

Identification of protein-ligand binding sites by the level-set variational implicit-solvent approach.

PubMed

Guo, Zuojun; Li, Bo; Cheng, Li-Tien; Zhou, Shenggao; McCammon, J Andrew; Che, Jianwei

2015-02-10

Protein–ligand binding is a key biological process at the molecular level. The identification and characterization of small-molecule binding sites on therapeutically relevant proteins have tremendous implications for target evaluation and rational drug design. In this work, we used the recently developed level-set variational implicit-solvent model (VISM) with the Coulomb field approximation (CFA) to locate and characterize potential protein–small-molecule binding sites. We applied our method to a data set of 515 protein–ligand complexes and found that 96.9% of the cocrystallized ligands bind to the VISM-CFA-identified pockets and that 71.8% of the identified pockets are occupied by cocrystallized ligands. For 228 tight-binding protein–ligand complexes (i.e, complexes with experimental pKd values larger than 6), 99.1% of the cocrystallized ligands are in the VISM-CFA-identified pockets. In addition, it was found that the ligand binding orientations are consistent with the hydrophilic and hydrophobic descriptions provided by VISM. Quantitative characterization of binding pockets with topological and physicochemical parameters was used to assess the “ligandability” of the pockets. The results illustrate the key interactions between ligands and receptors and can be very informative for rational drug design.

Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.

PubMed

Mathias, Jordan D; Ran, Yong; Carter, Jeffery D; Fanucci, Gail E

2009-09-02

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

Predicting the effectiveness of viscoelastic damping pockets in beams

NASA Astrophysics Data System (ADS)

Butler, Nigel D.; Oyadiji, S. O.

2005-05-01

This paper looks at the use of viscoelastic damping pockets in the suppression of structural vibration. These are in the form of cavities filled with a viscoelastic material. The benefits and uses of these designed-in damping treatments are highlighted. The vibration responses of viscoelastically-damped beams are predicted using the finite element method. A series of cantilevered beams are considered and the damping performance of several configurations of designed-in dampers are predicted and compared to that of a traditional CLD treatment. It is shown that the effectiveness of the damping pockets and sinks depends on their location and size with respect to the highly stressed regions of the beams. Although there is a practical limit on the sizes of the geometrical features that can be designed-in, it is shown that if located correctly the damping pockets and sinks can be more effective at suppressing structural vibration than traditional CLD treatments.

Ethics pocket cards: an educational tool for busy clinicians.

PubMed

Volpe, Rebecca L; Levi, Benjamin H; Blackhall, George F; Green, Michael J

2014-01-01

The adage "an ounce of prevention is worth a pound of cure" is widely used in healthcare settings and can be applied to the work of institutional clinical ethics committees. The model of clinical ethics consultation, however, is inherently reactive: a crisis or question emerges, and ethics experts are called to help. In an effort to employ a proactive component to the model of clinical ethics consultation (as well as to standardize our educational interventions), we developed ethics pocket cards. The purpose of this article is to: (1) describe the rationale for using ethics pocket cards, (2) provide examples of our cards, and (3) begin a dialogue about the potential uses of ethics pocket cards. In doing so, we hope to explore how such portable, economical devices can advance the goals of ethics consultation as well as the educational aims of ethics committees. Copyright 2014 The Journal of Clinical Ethics. All rights reserved.

Laser supported reduction of specific microorganisms in the periodontal pocket with the aid of an Er,Cr:YSGG laser: a pilot study.

PubMed

Gutknecht, N; Van Betteray, C; Ozturan, S; Vanweersch, L; Franzen, R

2015-01-01

The aim of this study was to evaluate the effectiveness of a radial firing tip of an Er,Cr:YSGG laser as an adjunct to a nonsurgical periodontal treatment. Twelve patients with chronic or aggressive periodontitis were treated by conventional periodontal treatment using ultrasonic devices and hand instruments and, additionally, in two quadrants with an Er,Cr:YSGG laser. A new radial firing tip (RFPT 14-5, Biolase) was used with 1.5 W, 30 Hz, 11% air, 20% water, and pulse duration 140 μs. Microbiological smears were taken before treatment, one day after lasing, and three and six months after lasing. Pocket depths of all periodontal sites were measured before and six months after treatment. The total bacterial load of Prevotella intermedia, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, Porphyromonas gingivalis, and Aggregatibacter actinomycetemcomitans inside the pocket was reduced significantly throughout the whole examination time. Greater pocket depth reductions were observed in all groups. There was a slight higher reduction of pocket depth in the lased group after six months. These results support the thesis that Er,Cr:YSGG laser supported periodontal treatment leads to a significant reduction of periopathogenes and thereby helps the maintenance of periodontal health.

Drug-like density: a method of quantifying the "bindability" of a protein target based on a very large set of pockets and drug-like ligands from the Protein Data Bank.

PubMed

Sheridan, Robert P; Maiorov, Vladimir N; Holloway, M Katharine; Cornell, Wendy D; Gao, Ying-Duo

2010-11-22

One approach to estimating the "chemical tractability" of a candidate protein target where we know the atomic resolution structure is to examine the physical properties of potential binding sites. A number of other workers have addressed this issue. We characterize ~290,000 "pockets" from ~42,000 protein crystal structures in terms of a three parameter "pocket space": volume, buriedness, and hydrophobicity. A metric DLID (drug-like density) measures how likely a pocket is to bind a drug-like molecule. This is calculated from the count of other pockets in its local neighborhood in pocket space that contain drug-like cocrystallized ligands and the count of total pockets in the neighborhood. Surprisingly, despite being defined locally, a global trend in DLID can be predicted by a simple linear regression on log(volume), buriedness, and hydrophobicity. Two levels of simplification are necessary to relate the DLID of individual pockets to "targets": taking the best DLID per Protein Data Bank (PDB) entry (because any given crystal structure can have many pockets), and taking the median DLID over all PDB entries for the same target (because different crystal structures of the same protein can vary because of artifacts and real conformational changes). We can show that median DLIDs for targets that are detectably homologous in sequence are reasonably similar and that median DLIDs correlate with the "druggability" estimate of Cheng et al. (Nature Biotechnology 2007, 25, 71-75).

Pockets of Hope: How Students and Teachers Change the World. Series in Language and Ideology.

ERIC Educational Resources Information Center

de los Reyes, Eileen; Gozemba, Patricia A.

This book presents case studies of six "pockets of hope," democratic educational projects in which empowered students and their teachers actively engage with relevant academic and social problems. Although seemingly very different, the projects share a core commitment to democratic pedagogy and demonstrate practices consistent with…

Competitive Inhibition Mechanism of Acetylcholinesterase without Catalytic Active Site Interaction: Study on Functionalized C60 Nanoparticles via in Vitro and in Silico Assays.

PubMed

Liu, Yanyan; Yan, Bing; Winkler, David A; Fu, Jianjie; Zhang, Aiqian

2017-06-07

Acetylcholinesterase (AChE) activity regulation by chemical agents or, potentially, nanomaterials is important for both toxicology and pharmacology. Competitive inhibition via direct catalytic active sites (CAS) binding or noncompetitive inhibition through interference with substrate and product entering and exiting has been recognized previously as an AChE-inhibition mechanism for bespoke nanomaterials. The competitive inhibition by peripheral anionic site (PAS) interaction without CAS binding remains unexplored. Here, we proposed and verified the occurrence of a presumed competitive inhibition of AChE without CAS binding for hydrophobically functionalized C 60 nanoparticles (NPs) by employing both experimental and computational methods. The kinetic inhibition analysis distinguished six competitive inhibitors, probably targeting the PAS, from the pristine and hydrophilically modified C 60 NPs. A simple quantitative nanostructure-activity relationship (QNAR) model relating the pocket accessible length of substituent to inhibition capacity was then established to reveal how the geometry of the surface group decides the NP difference in AChE inhibition. Molecular docking identified the PAS as the potential binding site interacting with the NPs via a T-shaped plug-in mode. Specifically, the fullerene core covered the enzyme gorge as a lid through π-π stacking with Tyr72 and Trp286 in the PAS, while the hydrophobic ligands on the fullerene surface inserted into the AChE active site to provide further stability for the complexes. The modeling predicted that inhibition would be severely compromised by Tyr72 and Trp286 deletions, and the subsequent site-directed mutagenesis experiments proved this prediction. Our results demonstrate AChE competitive inhibition of NPs without CAS participation to gain further understanding of both the neurotoxicity and the curative effect of NPs.

Dynamic water behaviour due to one trapped air pocket in a laboratory pipeline apparatus

NASA Astrophysics Data System (ADS)

Bergant, A.; Karadžić, U.; Tijsseling, A.

2016-11-01

Trapped air pockets may cause severe operational problems in hydropower and water supply systems. A locally isolated air pocket creates distinct amplitude, shape and timing of pressure pulses. This paper investigates dynamic behaviour of a single trapped air pocket. The air pocket is incorporated as a boundary condition into the discrete gas cavity model (DGCM). DGCM allows small gas cavities to form at computational sections in the method of characteristics (MOC). The growth of the pocket and gas cavities is described by the water hammer compatibility equation(s), the continuity equation for the cavity volume, and the equation of state of an ideal gas. Isentropic behaviour is assumed for the trapped gas pocket and an isothermal bath for small gas cavities. Experimental investigations have been performed in a laboratory pipeline apparatus. The apparatus consists of an upstream end high-pressure tank, a horizontal steel pipeline (total length 55.37 m, inner diameter 18 mm), four valve units positioned along the pipeline including the end points, and a downstream end tank. A trapped air pocket is captured between two ball valves at the downstream end of the pipeline. The transient event is initiated by rapid opening of the upstream end valve; the downstream end valve stays closed during the event. Predicted and measured results for a few typical cases are compared and discussed.

Accelerometer's position independent physical activity recognition system for long-term activity monitoring in the elderly.

PubMed

Khan, Adil Mehmood; Lee, Young-Koo; Lee, Sungyoung; Kim, Tae-Seong

2010-12-01

Mobility is a good indicator of health status and thus objective mobility data could be used to assess the health status of elderly patients. Accelerometry has emerged as an effective means for long-term physical activity monitoring in the elderly. However, the output of an accelerometer varies at different positions on a subject's body, even for the same activity, resulting in high within-class variance. Existing accelerometer-based activity recognition systems thus require firm attachment of the sensor to a subject's body. This requirement makes them impractical for long-term activity monitoring during unsupervised free-living as it forces subjects into a fixed life pattern and impede their daily activities. Therefore, we introduce a novel single-triaxial-accelerometer-based activity recognition system that reduces the high within-class variance significantly and allows subjects to carry the sensor freely in any pocket without its firm attachment. We validated our system using seven activities: resting (lying/sitting/standing), walking, walking-upstairs, walking-downstairs, running, cycling, and vacuuming, recorded from five positions: chest pocket, front left trousers pocket, front right trousers pocket, rear trousers pocket, and inner jacket pocket. Its simplicity, ability to perform activities unimpeded, and an average recognition accuracy of 94% make our system a practical solution for continuous long-term activity monitoring in the elderly.

A pocket model for aluminum agglomeration in composite propellants

NASA Technical Reports Server (NTRS)

Cohen, N. S.

1981-01-01

This paper presents a model for the purpose of estimating the fraction of aluminum powder that will form agglomerates at the surface of deflagrating composite propellants. The basic idea is that the fraction agglomerated depends upon the amount of aluminum that melts within effective binder pocket volumes framed by oxidizer particles. The effective pocket depends upon the ability of ammonium perchlorate modals to encapsulate the aluminum and provide a local temperature sufficient to ignite the aluminum. Model results are discussed in the light of data showing effects of propellant formulation variables and pressure.

MSPocket: an orientation-independent algorithm for the detection of ligand binding pockets.

PubMed

Zhu, Hongbo; Pisabarro, M Teresa

2011-02-01

Identification of ligand binding pockets on proteins is crucial for the characterization of protein functions. It provides valuable information for protein-ligand docking and rational engineering of small molecules that regulate protein functions. A major number of current prediction algorithms of ligand binding pockets are based on cubic grid representation of proteins and, thus, the results are often protein orientation dependent. We present the MSPocket program for detecting pockets on the solvent excluded surface of proteins. The core algorithm of the MSPocket approach does not use any cubic grid system to represent proteins and is therefore independent of protein orientations. We demonstrate that MSPocket is able to achieve an accuracy of 75% in predicting ligand binding pockets on a test dataset used for evaluating several existing methods. The accuracy is 92% if the top three predictions are considered. Comparison to one of the recently published best performing methods shows that MSPocket reaches similar performance with the additional feature of being protein orientation independent. Interestingly, some of the predictions are different, meaning that the two methods can be considered complementary and combined to achieve better prediction accuracy. MSPocket also provides a graphical user interface for interactive investigation of the predicted ligand binding pockets. In addition, we show that overlap criterion is a better strategy for the evaluation of predicted ligand binding pockets than the single point distance criterion. The MSPocket source code can be downloaded from http://appserver.biotec.tu-dresden.de/MSPocket/. MSPocket is also available as a PyMOL plugin with a graphical user interface.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed

Iversen, L F; Brzozowski, M; Hastrup, S; Hubbard, R; Kastrup, J S; Larsen, I K; Naerum, L; Nørskov-Lauridsen, L; Rasmussen, P B; Thim, L; Wiberg, F C; Lundgren, K

1997-05-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

PubMed Central

Iversen, L. F.; Brzozowski, M.; Hastrup, S.; Hubbard, R.; Kastrup, J. S.; Larsen, I. K.; Naerum, L.; Nørskov-Lauridsen, L.; Rasmussen, P. B.; Thim, L.; Wiberg, F. C.; Lundgren, K.

1997-01-01

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand. PMID:9144768

Science and Technology Pocket Data Book.

ERIC Educational Resources Information Center

National Science Foundation, Washington, DC. Div. of Science Resources Studies.

This pocket guide contains a collection of graphed data, available in 1994, on science and technology funding patterns within the United States, public attitudes toward science and technology, and international trends in science and technology. Sections contain: (1) national research and development (R&D) funding patterns; (2) academic R&D…

X-ray crystal structure of divalent metal-activated ß-xyloisdase, RS223BX

USDA-ARS?s Scientific Manuscript database

We report the first X-ray structure of a glycoside hydrolase family 43 ß-xylosidase, RS223BX, which is strongly activated by the addition of divalent metal cations. The 2.69 Å structure reveals that the Ca2+ cation is located at the back of the active site pocket. The Ca2+ coordinates to H274 to sta...

The effect of hillslope angle on pocket gopher (Thomomys bottae) burrow geometry.

PubMed

Seabloom, E W; Reichman, O J; Gabet, E J

2000-10-01

generated by pocket gopher activity was invariant to hillslope. This relationship is in contrast to the most common model of soil movement generated by purely physical processes.

Neurology diagnostics security and terminal adaptation for PocketNeuro project.

PubMed

Chemak, C; Bouhlel, M-S; Lapayre, J-C

2008-09-01

This paper presents new approaches of medical information security and terminal mobile phone adaptation for the PocketNeuro project. The latter term refers to a project created for the management of neurological diseases. It consists of transmitting information about patients ("desk of patients") to a doctor's mobile phone during a visit and examination of a patient. These new approaches for the PocketNeuro project were analyzed in terms of medical information security and adaptation of the diagnostic images to the doctor's mobile phone. Images were extracted from a DICOM library. Matlab and its library were used as software to test our approaches and to validate our results. Experiments performed on a database of 30 256 x 256 pixel-sized neuronal medical images indicated that our new approaches for PocketNeuro project are valid and support plans for large-scale studies between French and Swiss hospitals using secured connections.

Detecting local ligand-binding site similarity in nonhomologous proteins by surface patch comparison.

PubMed

Sael, Lee; Kihara, Daisuke

2012-04-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. Copyright © 2011 Wiley Periodicals, Inc.

Investigation of dielectric pocket induced variations in tunnel field effect transistor

NASA Astrophysics Data System (ADS)

Upasana; Narang, Rakhi; Saxena, Manoj; Gupta, Mridula

2016-04-01

The performance of conventional Tunnel FETs struggling from ambipolar issues, insufficient on-current, lower transconductance value, higher delay and lower cut off frequency has been improved by introducing several material and device engineering concepts in past few years. Keeping this in view, another interesting and reliable option i.e. Dielectric Pocket TFET (featuring a dielectric pocket placement near tunneling junction) has been comprehensively and qualitatively demonstrated using ATLAS device simulator. The architecture has been explored in terms of various device electrostatic parameters such as potential, energy band profile, electron and hole concentration, electric field variation and band to band generation rate (GBTB) near the tunneling junction where the Dielectric Pocket (DP) has been introduced. Subsequently, a detailed investigation by changing the position and dielectric constant of pocket at respective junctions has been made where DP induced variations in drain current, transconductance and parasitic capacitance have been examined. The work highlights major improvements over conventional TFET in terms of lower subthreshold swing and threshold voltage, higher drain current and transconductance, improved on-to-off current ratio, suppressed ambipolar conduction and improved dynamic power dissipation issues for low voltage analog and digital applications.

Periodontal Pocket Depth, Hyperglycemia, and Progression of Chronic Kidney Disease: A Population-Based Longitudinal Study.

PubMed

Chang, Jia-Feng; Yeh, Jih-Chen; Chiu, Ya-Lin; Liou, Jian-Chiun; Hsiung, Jing-Ru; Tung, Tao-Hsin

2017-01-01

No large epidemiological study has been conducted to investigate the interaction and joint effects of periodontal pocket depth and hyperglycemia on progression of chronic kidney disease in patients with periodontal diseases. Periodontal pocket depth was utilized for the grading severity of periodontal disease in 2831 patients from January 2002 to June 2013. Progression of chronic kidney disease was defined as progression of color intensity in glomerular filtration rate and albuminuria grid of updated Kidney Disease-Improving Global Outcomes guidelines. Multivariable-adjusted hazard ratios (aHR) in various models were presented across different levels of periodontal pocket depth and hemoglobin A1c (HbA1c) in forest plots and 3-dimensional histograms. During 7621 person-years of follow-up, periodontal pocket depth and HbA1C levels were robustly associated with incremental risks for progression of chronic kidney disease (aHR 3.1; 95% confidence interval [CI], 2.0-4.6 for periodontal pocket depth >4.5 mm, and 2.5; 95% CI, 1.1-5.4 for HbA1C >6.5%, respectively). The interaction between periodontal pocket depth and HbA1C on progression of chronic kidney disease was strong (P <.01). Patients with higher periodontal pocket depth (>4.5 mm) and higher HbA1C (>6.5%) had the greatest risk (aHR 4.2; 95% CI, 1.7-6.8) compared with the lowest aHR group (periodontal pocket depth ≤3.8 mm and HbA1C ≤6%). Our study identified combined periodontal pocket depth and HbA1C as a valuable predictor of progression of chronic kidney disease in patients with periodontal diseases. While considering the interaction between periodontal diseases and hyperglycemia, periodontal survey and optimizing glycemic control are warranted to minimize the risk of worsening renal function. Copyright © 2016 Elsevier Inc. All rights reserved.

Unveiling a novel transient druggable pocket in BACE-1 through molecular simulations: Conformational analysis and binding mode of multisite inhibitors

PubMed Central

Di Pietro, Ornella; Laughton, Charles A.

2017-01-01

The critical role of BACE-1 in the formation of neurotoxic ß-amyloid peptides in the brain makes it an attractive target for an efficacious treatment of Alzheimer’s disease. However, the development of clinically useful BACE-1 inhibitors has proven to be extremely challenging. In this study we examine the binding mode of a novel potent inhibitor (compound 1, with IC50 80 nM) designed by synergistic combination of two fragments—huprine and rhein—that individually are endowed with very low activity against BACE-1. Examination of crystal structures reveals no appropriate binding site large enough to accommodate 1. Therefore we have examined the conformational flexibility of BACE-1 through extended molecular dynamics simulations, paying attention to the highly flexible region shaped by loops 8–14, 154–169 and 307–318. The analysis of the protein dynamics, together with studies of pocket druggability, has allowed us to detect the transient formation of a secondary binding site, which contains Arg307 as a key residue for the interaction with small molecules, at the edge of the catalytic cleft. The formation of this druggable “floppy” pocket would enable the binding of multisite inhibitors targeting both catalytic and secondary sites. Molecular dynamics simulations of BACE-1 bound to huprine-rhein hybrid compounds support the feasibility of this hypothesis. The results provide a basis to explain the high inhibitory potency of the two enantiomeric forms of 1, together with the large dependence on the length of the oligomethylenic linker. Furthermore, the multisite hypothesis has allowed us to rationalize the inhibitory potency of a series of tacrine-chromene hybrid compounds, specifically regarding the apparent lack of sensitivity of the inhibition constant to the chemical modifications introduced in the chromene unit. Overall, these findings pave the way for the exploration of novel functionalities in the design of optimized BACE-1 multisite inhibitors

Interplay between Magnetism, Superconductivity, and Orbital Order in 5-Pocket Model for Iron-Based Superconductors: Parquet Renormalization Group Study.

PubMed

Classen, Laura; Xing, Rui-Qi; Khodas, Maxim; Chubukov, Andrey V

2017-01-20

We report the results of the parquet renormalization group (RG) analysis of the phase diagram of the most general 5-pocket model for Fe-based superconductors. We use as an input the orbital structure of excitations near the five pockets made out of d_{xz}, d_{yz}, and d_{xy} orbitals and argue that there are 40 different interactions between low-energy fermions in the orbital basis. All interactions flow under the RG, as one progressively integrates out fermions with higher energies. We find that the low-energy behavior is amazingly simple, despite the large number of interactions. Namely, at low energies the full 5-pocket model effectively reduces either to a 3-pocket model made of one d_{xy} hole pocket and two electron pockets or a 4-pocket model made of two d_{xz}/d_{yz} hole pockets and two electron pockets. The leading instability in the effective 4-pocket model is a spontaneous orbital (nematic) order, followed by s^{+-} superconductivity. In the effective 3-pocket model, orbital fluctuations are weaker, and the system develops either s^{+-} superconductivity or a stripe spin-density wave. In the latter case, nematicity is induced by composite spin fluctuations.

Pocket money, eating behaviors, and weight status among Chinese children: The Childhood Obesity Study in China mega-cities.

PubMed

Li, Miao; Xue, Hong; Jia, Peng; Zhao, Yaling; Wang, Zhiyong; Xu, Fei; Wang, Youfa

2017-07-01

Both the obesity rate and pocket money are rising among children in China. This study examined family correlates of children's pocket money, associations of pocket money with eating behaviors and weight status, and how the associations may be modified by schools' unhealthy food restrictions in urban China. Data were collected in 2015 from 1648 students in 16 primary and middle schools in four mega-cities in China (4 schools/city): Beijing, Shanghai, Nanjing, and Xi'an. Cluster robust negative binomial regression models were fit to assess family correlates of pocket money, associations of pocket money with child eating behaviors and weight outcomes, and possible modifying effects of schools' unhealthy food restrictions. Sixty-nine percent of students received pocket money weekly. Students received more pocket money if mothers frequently ate out of home (IRR=2.28 [1.76, 2.94]) and/or family rarely had dinner together (IRR=1.42, 95%=[1.01, 1.99]). Students got less pocket money if parents were concerned about child's future health due to unhealthy eating (IRR=0.56 [0.32,0.98]). Students with more pocket money more frequently consumed (by 25-89%) sugary beverages, snacks, fast food, or at street food stalls, and were 45-90% more likely to be overweight/obese. Associations of pocket money with unhealthy eating and overweight/obesity were weaker in schools with unhealthy food restrictions. Pocket money is a risk factor for unhealthy eating and obesity in urban China. School policies may buffer pocket money's negative influence on students' eating and weight status. Copyright © 2017. Published by Elsevier Inc.

Out-of-pocket fertility patient expense: data from a multicenter prospective infertility cohort.

PubMed

Wu, Alex K; Odisho, Anobel Y; Washington, Samuel L; Katz, Patricia P; Smith, James F

2014-02-01

The high costs of fertility care may deter couples from seeking care. Urologists often are asked about the costs of these treatments. To our knowledge previous studies have not addressed the direct out-of-pocket costs to couples. We characterized these expenses in patients seeking fertility care. Couples were prospectively recruited from 8 community and academic reproductive endocrinology clinics. Each participating couple completed face-to-face or telephone interviews and cost diaries at study enrollment, and 4, 10 and 18 months of care. We determined overall out-of-pocket costs, in addition to relationships between out-of-pocket costs and treatment type, clinical outcomes and socioeconomic characteristics on multivariate linear regression analysis. A total of 332 couples completed cost diaries and had data available on treatment and outcomes. Average age was 36.8 and 35.6 years in men and women, respectively. Of this cohort 19% received noncycle based therapy, 4% used ovulation induction medication only, 22% underwent intrauterine insemination and 55% underwent in vitro fertilization. The median overall out-of-pocket expense was $5,338 (IQR 1,197-19,840). Couples using medication only had the lowest median out-of-pocket expenses at $912 while those using in vitro fertilization had the highest at $19,234. After multivariate adjustment the out-of-pocket expense was not significantly associated with successful pregnancy. On multivariate analysis couples treated with in vitro fertilization spent an average of $15,435 more than those treated with intrauterine insemination. Couples spent about $6,955 for each additional in vitro fertilization cycle. These data provide real-world estimates of out-of-pocket costs, which can be used to help couples plan for expenses that they may incur with treatment. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Research: Comparison of the Accuracy of a Pocket versus Standard Pulse Oximeter.

PubMed

da Costa, João Cordeiro; Faustino, Paula; Lima, Ricardo; Ladeira, Inês; Guimarães, Miguel

2016-01-01

Pulse oximetry has become an essential tool in clinical practice. With patient self-management becoming more prevalent, pulse oximetry self-monitoring has the potential to become common practice in the near future. This study sought to compare the accuracy of two pulse oximeters, a high-quality standard pulse oximeter and an inexpensive pocket pulse oximeter, and to compare both devices with arterial blood co-oximetry oxygen saturation. A total of 95 patients (35.8% women; mean [±SD] age 63.1 ± 13.9 years; mean arterial pressure was 92 ± 12.0 mmHg; mean axillar temperature 36.3 ± 0.4°C) presenting to our hospital for blood gas analysis was evaluated. The Bland-Altman technique was performed to calculate bias and precision, as well as agreement limits. Student's t test was performed. Standard oximeter presented 1.84% bias and a precision error of 1.80%. Pocket oximeter presented a bias of 1.85% and a precision error of 2.21%. Agreement limits were -1.69% to 5.37% (standard oximeter) and -2.48% to 6.18% (pocket oximeter). Both oximeters presented bias, which was expected given previous research. The pocket oximeter was less precise but had agreement limits that were comparable with current evidence. Pocket oximeters can be powerful allies in clinical monitoring of patients based on a self-monitoring/efficacy strategy.

F pocket flexibility influences the tapasin dependence of two differentially disease-associated MHC Class I proteins.

PubMed

Abualrous, Esam T; Fritzsche, Susanne; Hein, Zeynep; Al-Balushi, Mohammed S; Reinink, Peter; Boyle, Louise H; Wellbrock, Ursula; Antoniou, Antony N; Springer, Sebastian

2015-04-01

The human MHC class I protein HLA-B*27:05 is statistically associated with ankylosing spondylitis, unlike HLA-B*27:09, which differs in a single amino acid in the F pocket of the peptide-binding groove. To understand how this unique amino acid difference leads to a different behavior of the proteins in the cell, we have investigated the conformational stability of both proteins using a combination of in silico and experimental approaches. Here, we show that the binding site of B*27:05 is conformationally disordered in the absence of peptide due to a charge repulsion at the bottom of the F pocket. In agreement with this, B*27:05 requires the chaperone protein tapasin to a greater extent than the conformationally stable B*27:09 in order to remain structured and to bind peptide. Taken together, our data demonstrate a method to predict tapasin dependence and physiological behavior from the sequence and crystal structure of a particular class I allotype. Also watch the Video Abstract. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of Small Molecules against Botulinum Neurotoxin B Binding to Neuronal Cells at Ganglioside GT1b Binding Site with Low to Moderate Affinity

DTIC Science & Technology

2014-10-01

BoNT serotype B (BoNT/B) for the trisaccharide GT1b were identified from the x-ray crystal structure of the BoNT/B/trisaccharide (GT1b) complex ( PDB ...trisaccharide and all the water from the structure and identified four potential binding pockets (Pocket-1, Pocket-2, and Pocket-4) as shown in...four potential binding sites or pockets on BoNT serotype B (BoNT/B) for the trisaccharide GT1b were identified from the x-ray crystal structure of the

Searching for protein binding sites from Molecular Dynamics simulations and paramagnetic fragment-based NMR studies.

PubMed

Bernini, Andrea; Henrici De Angelis, Lucia; Morandi, Edoardo; Spiga, Ottavia; Santucci, Annalisa; Assfalg, Michael; Molinari, Henriette; Pillozzi, Serena; Arcangeli, Annarosa; Niccolai, Neri

2014-03-01

Hotspot delineation on protein surfaces represents a fundamental step for targeting protein-protein interfaces. Disruptors of protein-protein interactions can be designed provided that the sterical features of binding pockets, including the transient ones, can be defined. Molecular Dynamics, MD, simulations have been used as a reliable framework for identifying transient pocket openings on the protein surface. Accessible surface area and intramolecular H-bond involvement of protein backbone amides are proposed as descriptors for characterizing binding pocket occurrence and evolution along MD trajectories. TEMPOL induced paramagnetic perturbations on (1)H-(15)N HSQC signals of protein backbone amides have been analyzed as a fragment-based search for surface hotspots, in order to validate MD predicted pockets. This procedure has been applied to CXCL12, a small chemokine responsible for tumor progression and proliferation. From combined analysis of MD data and paramagnetic profiles, two CXCL12 sites suitable for the binding of small molecules were identified. One of these sites is the already well characterized CXCL12 region involved in the binding to CXCR4 receptor. The other one is a transient pocket predicted by Molecular Dynamics simulations, which could not be observed from static analysis of CXCL12 PDB structures. The present results indicate how TEMPOL, instrumental in identifying this transient pocket, can be a powerful tool to delineate minor conformations which can be highly relevant in dynamic discovery of antitumoral drugs. Copyright © 2013 Elsevier B.V. All rights reserved.

Detecting Local Ligand-Binding Site Similarity in Non-Homologous Proteins by Surface Patch Comparison

PubMed Central

Sael, Lee; Kihara, Daisuke

2012-01-01

Functional elucidation of proteins is one of the essential tasks in biology. Function of a protein, specifically, small ligand molecules that bind to a protein, can be predicted by finding similar local surface regions in binding sites of known proteins. Here, we developed an alignment free local surface comparison method for predicting a ligand molecule which binds to a query protein. The algorithm, named Patch-Surfer, represents a binding pocket as a combination of segmented surface patches, each of which is characterized by its geometrical shape, the electrostatic potential, the hydrophobicity, and the concaveness. Representing a pocket by a set of patches is effective to absorb difference of global pocket shape while capturing local similarity of pockets. The shape and the physicochemical properties of surface patches are represented using the 3D Zernike descriptor, which is a series expansion of mathematical 3D function. Two pockets are compared using a modified weighted bipartite matching algorithm, which matches similar patches from the two pockets. Patch-Surfer was benchmarked on three datasets, which consist in total of 390 proteins that bind to one of 21 ligands. Patch-Surfer showed superior performance to existing methods including a global pocket comparison method, Pocket-Surfer, which we have previously introduced. Particularly, as intended, the accuracy showed large improvement for flexible ligand molecules, which bind to pockets in different conformations. PMID:22275074

Pocket Money: Influence on Body Mass Index and Dental Caries among Urban Adolescents.

PubMed

Punitha, V C; Amudhan, A; Sivaprakasam, P; Rathnaprabhu, V

2014-12-01

To explore the influence of pocket money on Dental Caries and Body Mass Index. A cross-sectional study was conducted wherein urban adolescent schoolchildren of age 13-18(n=916) were selected by two stage random sampling technique. Dental caries was measured using the DMFT Index. The children's nutritional status was assessed by means of anthropometric measurements. Body Mass Index using weight and height of children was evaluated using the reference standard of the WHO 2007. RESULTS showed that 50% of children receive pocket money from parents. The average amount received was Rs. 360/month. There was a significant correlation between age and amount of money received (r=0.160, p=.001). The average amount received by male children was significantly higher (Rs. 400) when compared to female children (Rs. 303). It was observed that income of the family (>30,000 Rs./month) and socioeconomic status (Upper class) was significantly dependent on the amount of money received by children (p<0.05). There was no significant difference in the occurrence of caries among children receiving pocket money or not. When BMI categories and pocket money were considered, statistically significant difference was seen among overweight and obese and normal weight children (p<.05). Higher proportion (40.1%) of overweight and obese adolescent children frequented the fast food restaurants every week when compared to the underweight (31.7%) and normal weight children (29.9%). Adolescent children receiving pocket money from parents could influence their eating habits in turn affect general health. Parents and teachers should motivate children on healthy spending of their pocket money.

Do Out-of-Pocket Health Expenditures Rise with Age Among Older Americans?

ERIC Educational Resources Information Center

Stewart, Susan T.

2004-01-01

Purpose: Relationships are examined between age and out-of-pocket costs for different health goods and services among the older population. Design and Methods: Age patterns in health service use and out-of-pocket costs are examined by use of the 1990 Elderly Health Supplement to the Panel Study of Income Dynamics (N = 1,031, age 66+). Multivariate…

Dissecting the active site of a photoreceptor protein

NASA Astrophysics Data System (ADS)

Hoff, Wouter; Hara, Miwa; Ren, Jie; Moghadam, Farzaneh; Xie, Aihua; Kumauchi, Masato

While enzymes are quite large molecules, functionally important chemical events are often limited to a small region of the protein: the active site. The physical and chemical properties of residues at such active sites are often strongly altered compared to the same groups dissolved in water. Understanding such effects is important for unraveling the mechanisms underlying protein function and for protein engineering, but has proven challenging. Here we report on our ongoing efforts on using photoactive yellow protein (PYP), a bacterial photoreceptor, as a model system for such effects. We will report on the following questions: How many residues affect active site properties? Are these residues in direct physical contact with the active site? Can functionally important residues be recognized in the crystal structure of a protein? What structural resolution is needed to understand active sites? What spectroscopic techniques are most informative? Which weak interactions dominate active site properties?

Pocket-depths-related effectiveness of an intrapocket anaesthesia gel in periodontal maintenance patients.

PubMed

Derman, S H M; Lowden, C E; Kaus, P; Noack, M J

2014-05-01

The aim of this study was to determine the impact of the pocket depth on the effectiveness of an intrapocket anaesthesia gel during SRP in periodontal maintenance patients. Effectiveness was measured by pain levels during SRP via visual analogue scale (VAS) and verbal rating scale (VRS). Secondary endpoint was the evaluation of patients' preferred choice of anaesthesia for SRP. A total of 638 patients undergoing the periodontal maintenance programme and with the need for SRP participated in this observational study. After SRP, patients filled in questionnaires to record pain levels experienced and anaesthesia preference for future use. Mann-Whitney U-test was used to analyse intergroup difference in pain perception and anaesthesia choice. Overall, increasing pocket depths were accompanied by higher pain levels, irrespective of maximum or commonest pocket depths (P < 0.05). For SRP procedures, patients definitely prefer the anaesthesia gel (72.4%). In this study, an effectiveness of local anaesthesia gel (lidocaine/prilocaine) related to pocket depths was found in periodontal maintenance patients during SRP. Increasing pocket depths were accompanied by increasing procedural pain levels. Nevertheless, the anaesthesia gel is well accepted and in the majority of cases was found to be the preferred option for future SRP treatments. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mapping General Anesthetic Sites in Heteromeric γ-Aminobutyric Acid Type A Receptors Reveals a Potential For Targeting Receptor Subtypes.

PubMed

Forman, Stuart A; Miller, Keith W

2016-11-01

IV general anesthetics, including propofol, etomidate, alphaxalone, and barbiturates, produce important actions by enhancing γ-aminobutyric acid type A (GABAA) receptor activation. In this article, we review scientific studies that have located and mapped IV anesthetic sites using photoaffinity labeling and substituted cysteine modification protection. These anesthetics bind in transmembrane pockets between subunits of typical synaptic GABAA receptors, and drugs that display stereoselectivity also show remarkably selective interactions with distinct interfacial sites. These results suggest strategies for developing new drugs that selectively modulate distinct GABAA receptor subtypes.

Patterns and determinants of communal latrine usage in urban poverty pockets in Bhopal, India.

PubMed

Biran, A; Jenkins, M W; Dabrase, P; Bhagwat, I

2011-07-01

To explore and explain patterns of use of communal latrine facilities in urban poverty pockets. Six poverty pockets with communal latrine facilities representing two management models (Sulabh and municipal) were selected. Sampling was random and stratified by poverty pocket population size. A seventh, community-managed facility was also included. Data were collected by exit interviews with facility users and by interviews with residents from a randomly selected representative sample of poverty pocket households, on social, economic and demographic characteristics of households, latrine ownership, defecation practices, costs of using the facility and distance from the house to the facility. A tally of facility users was kept for 1 day at each facility. Data were analysed using logistic regression modelling to identify determinants of communal latrine usage. Communal latrines differed in their facilities, conditions, management and operating characteristics, and rates of usage. Reported usage rates among non-latrine-owning households ranged from 15% to 100%. There was significant variation in wealth, occupation and household structure across the poverty pockets as well as in household latrine ownership. Households in pockets with municipal communal latrine facilities appeared poorer. Households in pockets with Sulabh-managed communal facilities were significantly more likely to own a household latrine. Determinants of communal facility usage among households without a latrine were access and convenience (distance and opening hours), facility age, cleanliness/upkeep and cost. The ratio of male to female users was 2:1 across all facilities for both adults and children. Provision of communal facilities reduces but does not end the problem of open defecation in poverty pockets. Women appear to be relatively poorly served by communal facilities and, cost is a barrier to use by poorer households. Results suggest improving facility convenience and access and modifying fee

Active site dynamics of ribonuclease.

PubMed Central

Brünger, A T; Brooks, C L; Karplus, M

1985-01-01

The stochastic boundary molecular dynamics method is used to study the structure, dynamics, and energetics of the solvated active site of bovine pancreatic ribonuclease A. Simulations of the native enzyme and of the enzyme complexed with the dinucleotide substrate CpA and the transition-state analog uridine vanadate are compared. Structural features and dynamical couplings for ribonuclease residues found in the simulation are consistent with experimental data. Water molecules, most of which are not observed in crystallographic studies, are shown to play an important role in the active site. Hydrogen bonding of residues with water molecules in the free enzyme is found to mimic the substrate-enzyme interactions of residues involved in binding. Networks of water stabilize the cluster of positively charged active site residues. Correlated fluctuations between the uridine vanadate complex and the distant lysine residues are mediated through water and may indicate a possible role for these residues in stabilizing the transition state. Images PMID:3866234

Fermi arcs vs. fermi pockets in electron-doped perovskite iridates

DOE PAGES

He, Junfeng; Hafiz, H.; Mion, Thomas R.; ...

2015-02-23

We report on an angle resolved photoemission (ARPES) study of bulk electron-doped perovskite iridate, (Sr 1-xLa x)₃Ir₂O₇. Fermi surface pockets are observed with a total electron count in keeping with that expected from La substitution. Depending on the energy and polarization of the incident photons, these pockets show up in the form of disconnected “Fermi arcs”, reminiscent of those reported recently in surface electron-doped Sr₂IrO₄. Our observed spectral variation is consistent with the coexistence of an electronic supermodulation with structural distortion in the system.

Low dielectric response in enzyme active site

PubMed Central

Mertz, Edward L.; Krishtalik, Lev I.

2000-01-01

The kinetics of charge transfer depend crucially on the dielectric reorganization of the medium. In enzymatic reactions that involve charge transfer, atomic dielectric response of the active site and of its surroundings determines the efficiency of the protein as a catalyst. We report direct spectroscopic measurements of the reorganization energy associated with the dielectric response in the active site of α-chymotrypsin. A chromophoric inhibitor of the enzyme is used as a spectroscopic probe. We find that water strongly affects the dielectric reorganization in the active site of the enzyme in solution. The reorganization energy of the protein matrix in the vicinity of the active site is similar to that of low-polarity solvents. Surprisingly, water exhibits an anomalously high dielectric response that cannot be described in terms of the dielectric continuum theory. As a result, sequestering the active site from the aqueous environment inside low-dielectric enzyme body dramatically reduces the dielectric reorganization. This reduction is particularly important for controlling the rate of enzymatic reactions. PMID:10681440

Molecular Docking Studies to Explore Potential Binding Pockets and Inhibitors for Chikungunya Virus Envelope Glycoproteins.

PubMed

Nguyen, Phuong T V; Yu, Haibo; Keller, Paul A

2017-03-11

The chikungunya virus (CHIKV) envelope glycoproteins are considered important potential targets for anti-CHIKV drug discovery due to their crucial roles in virus attachment and virus entry. In this study, using two available crystal structures of the immature and mature forms of envelope glycoproteins, virtual screenings based on blind dockings and focused dockings were carried out to identify potential binding pockets and hit compounds for the virus. The chemical library database of compounds, NCI Diversity Set II, was used in these docking studies. In addition to reproducing previously reported examples, new binding pockets were identified, e.g., Pocket 2 in the 3N40, and Pocket 2 and Pocket 3 in the 3N42. Convergences in conformational sampling in docking using AutoDock Vina were evaluated. An analysis of docking results was carried out to understand interactions of the envelope glycoproteins complexes. Some key residues for interactions, for example Gly91 and His230, are identified as possessing important roles in the fusion process.

Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

PubMed Central

Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

2016-01-01

Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

Pocket Money: Influence on Body Mass Index and Dental Caries among Urban Adolescents

PubMed Central

Amudhan, A.; Sivaprakasam, P.; Rathnaprabhu, V.

2014-01-01

Objective: To explore the influence of pocket money on Dental Caries and Body Mass Index. Materials and Methods: A cross-sectional study was conducted wherein urban adolescent schoolchildren of age 13-18(n=916) were selected by two stage random sampling technique. Dental caries was measured using the DMFT Index. The children’s nutritional status was assessed by means of anthropometric measurements. Body Mass Index using weight and height of children was evaluated using the reference standard of the WHO 2007. Results: Results showed that 50% of children receive pocket money from parents. The average amount received was Rs. 360/month. There was a significant correlation between age and amount of money received (r=0.160, p=.001). The average amount received by male children was significantly higher (Rs. 400) when compared to female children (Rs. 303). It was observed that income of the family (>30,000 Rs./month) and socioeconomic status (Upper class) was significantly dependent on the amount of money received by children (p<0.05). There was no significant difference in the occurrence of caries among children receiving pocket money or not. When BMI categories and pocket money were considered, statistically significant difference was seen among overweight and obese and normal weight children (p<.05). Higher proportion (40.1%) of overweight and obese adolescent children frequented the fast food restaurants every week when compared to the underweight (31.7%) and normal weight children (29.9%). Conclusion: Adolescent children receiving pocket money from parents could influence their eating habits in turn affect general health. Parents and teachers should motivate children on healthy spending of their pocket money. PMID:25653973

Approach for targeting Ras with small molecules that activate SOS-mediated nucleotide exchange.

PubMed

Burns, Michael C; Sun, Qi; Daniels, R Nathan; Camper, DeMarco; Kennedy, J Phillip; Phan, Jason; Olejniczak, Edward T; Lee, Taekyu; Waterson, Alex G; Rossanese, Olivia W; Fesik, Stephen W

2014-03-04

Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active upstream receptor tyrosine kinases results in the deregulation of cellular signals governing growth and survival in ∼30% of all human cancers. However, the discovery of potent inhibitors of Ras has been difficult to achieve. Here, we report the identification of small molecules that bind to a unique pocket on the Ras:Son of Sevenless (SOS):Ras complex, increase the rate of SOS-catalyzed nucleotide exchange in vitro, and modulate Ras signaling pathways in cells. X-ray crystallography of Ras:SOS:Ras in complex with these molecules reveals that the compounds bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of multiple X-ray cocrystal structures. Mutational analyses confirmed the functional relevance of this binding site and showed it to be essential for compound activity. These molecules increase Ras-GTP levels and disrupt MAPK and PI3K signaling in cells at low micromolar concentrations. These small molecules represent tools to study the acute activation of Ras and highlight a pocket on SOS that may be exploited to modulate Ras signaling.

Single Walled Carbon Nanotube Based Air Pocket Encapsulated Ultraviolet Sensor.

PubMed

Kim, Sun Jin; Han, Jin-Woo; Kim, Beomseok; Meyyappan, M

2017-11-22

Carbon nanotube (CNT) is a promising candidate as a sensor material for the sensitive detection of gases/vapors, biomarkers, and even some radiation, as all these external variables affect the resistance and other properties of nanotubes, which forms the basis for sensing. Ultraviolet (UV) radiation does not impact the nanotube properties given the substantial mismatch of bandgaps and therefore, CNTs have never been considered for UV sensing, unlike the popular ZnO and other oxide nanwires. It is well-known that UV assists the adsorption/desorption characteristics of oxygen on carbon nanotubes, which changes the nanotube resistance. Here, we demonstrate a novel sensor structure encapsulated with an air pocket, where the confined air is responsible for the UV sensing mechanism and assures sensor stability and repeatability over time. In addition to the protection from any contamination, the air pocket encapsulated sensor offers negligible baseline drift and fast recovery compared to previously reported sensors. The air pocket isolated from the outside environment can act as a stationary oxygen reservoir, resulting in consistent sensor characteristics. Furthermore, this sensor can be used even in liquid environments.

Film cooling air pocket in a closed loop cooled airfoil

DOEpatents

Yu, Yufeng Phillip; Itzel, Gary Michael; Osgood, Sarah Jane; Bagepalli, Radhakrishna; Webbon, Waylon Willard; Burdgick, Steven Sebastian

2002-01-01

Turbine stator vane segments have radially inner and outer walls with vanes extending between them. The inner and outer walls are compartmentalized and have impingement plates. Steam flowing into the outer wall plenum passes through the impingement plate for impingement cooling of the outer wall upper surface. The spent impingement steam flows into cavities of the vane having inserts for impingement cooling the walls of the vane. The steam passes into the inner wall and through the impingement plate for impingement cooling of the inner wall surface and for return through return cavities having inserts for impingement cooling of the vane surfaces. To provide for air film cooing of select portions of the airfoil outer surface, at least one air pocket is defined on a wall of at least one of the cavities. Each air pocket is substantially closed with respect to the cooling medium in the cavity and cooling air pumped to the air pocket flows through outlet apertures in the wall of the airfoil to cool the same.

Damage reduction to ponderosa pine seedlings from northern pocket gophers by vegetation management through grass seeding and herbicide treatment

USGS Publications Warehouse

Engeman, Richard M.; Barnes, V.G.; Anthony, R.M.; Krupa, Heather W.

1998-01-01

2,4-D herbicide treatment was applied to 2 treatment units to remove the forbs that are the preferred food of pocket gophers. One of these units also was seeded with grasses prior to the 2,4-D treatment. The effect of 2,4-D and grass seeding plus 2,4-D treatments were compared to an untreated control unit. Long-term monitoring (7 yr) was conducted on the 3 units for vegetative cover (7 yr), pocket gopher activity, and individual survival times and time until gopher damage for 2 cohorts of seedlings (5 and 6 yrs). The 2,4-D treatments greatly reduced vegetative cover of the forbs and seeding increased grass cover on the unit receiving that treatment. Pocket gopher activity was reduced somewhat on the unit receiving only the 2,4-D treatment and more so on the unit receiving grass seeding and 2,4-D, although gophers remained active to some degree throughout the study. Both cohorts of seedlings for both treatments units showed greater average times until gopher damage over seedlings on the control unit. However, seedling survival from all sources of mortality was not positively affected by the treatments for the first cohort of seedlings. The 2,4-D treatment appeared to have killed some of the seedlings; however, seedlings that survived the treatment were in a situation where they were less likely to be damaged by gophers and seemed to have improved growth rates.

Retiree out-of-pocket healthcare spending: a study of consumer expectations and policy implications.

PubMed

Hoffman, Allison K; Jackson, Howell E

2013-01-01

Even though most American retirees benefit from Medicare coverage, a mounting body of research predicts that many will face large and increasing out-of-pocket expenditures for healthcare costs in retirement and that many already struggle to finance these costs. It is unclear, however, whether the general population understands the likely magnitude of these out-of-pocket expenditures well enough to plan for them effectively. This study is the first comprehensive examination of Americans' expectations regarding their out-of-pocket spending on healthcare in retirement. We surveyed over 1700 near retirees and retirees to assess their expectations regarding their own spending and then compared their responses to experts' estimates. Our main findings are twofold. First, overall expectations of out-of-pocket spending are mixed. While a significant proportion of respondents estimated out-of-pocket costs in retirement at or above expert estimates of what the typical retiree will spend, a disproportionate number estimated their future spending substantially below what experts view as likely. Estimates by members of some demographic subgroups, including women and younger respondents, deviated relatively further from the experts' estimates. Second, respondents consistently misjudged spending uncertainty. In particular, respondents significantly underestimated how much individual health experience and changes in government policy can affect individual out-of-pocket spending. We discuss possible policy responses, including efforts to improve financial planning and ways to reduce unanticipated financial risk through reform of health insurance regulation.

Iranian Households’ Payments on Food and Health Out-of-Pocket Expenditures: Evidence of Inequality

PubMed Central

GHIASVAND, Hesam; NAGHDI, Seyran; ABOLHASSANI, Nazanin; SHAARBAFCHIZADEH, Nasrin; MOGHRI, Javad

2015-01-01

Background: Inequality in households’ payments on food and health expenditures presents the accessibility and utilization patterns between them. This study investigated the Iranian rural and urban households’ inequality in payments on food and Out-of-Pocket health expenditures from 1998 to 2012. Methods: This descriptive study was conducted through the analysis of Iranian Statistics Centre data on Iranian households’ income and expenditures. The Gini Coefficients, Concentration and Kakwani indices have been calculated for Iranian rural and urban households’ Out-of-Pocket health and food expenditures. Results: The means of Iranian rural and urban total consumption expenditures inequality were 0.48 and 0.48, respectively. The means of concentration index of food expenditures for rural and urban regions were 0.35 and 0.34, respectively. The means of Out-of-Pocket payments for health services for rural and urban regions were 0.51 and 0.5, respectively. Finally the means of Kakwani index of Out-of-Pocket health payments in rural and urban households were −0.005 and −0.018, respectively. Conclusion: There are relative high levels of inequality in Iranian households’ payments on food and Out-of-Pocket health expenditures. PMID:26587474

Out-of-pocket health spending by poor and near-poor elderly Medicare beneficiaries.

PubMed

Gross, D J; Alecxih, L; Gibson, M J; Corea, J; Caplan, C; Brangan, N

1999-04-01

To estimate out-of-pocket health care spending by lower-income Medicare beneficiaries, and to examine spending variations between those who receive Medicaid assistance and those who do not receive such aid. DATA SOURCES AND COLLECTION: 1993 Medicare Current Beneficiary Survey (MCBS) Cost and Use files, supplemented with data from the Bureau of the Census (Current Population Survey); the Congressional Budget Office; the Health Care Financing Administration, Office of the Actuary (National Health Accounts); and the Social Security Administration. We analyzed out-of-pocket spending through a Medicare Benefits Simulation model, which projects out-of-pocket health care spending from the 1993 MCBS to 1997. Out-of-pocket health care spending is defined to include Medicare deductibles and coinsurance; premiums for private insurance, Medicare Part B, and Medicare HMOs; payments for non-covered goods and services; and balance billing by physicians. It excludes the costs of home care and nursing facility services, as well as indirect tax payments toward health care financing. Almost 60 percent of beneficiaries with incomes below the poverty level did not receive Medicaid assistance in 1997. We estimate that these beneficiaries spent, on average, about half their income out-of-pocket for health care, whether they were enrolled in a Medicare HMO or in the traditional fee-for-service program. The 75 percent of beneficiaries with incomes between 100 and 125 percent of the poverty level who were not enrolled in Medicaid spent an estimated 30 percent of their income out-of-pocket on health care if they were in the traditional program and about 23 percent of their income if they were enrolled in a Medicare HMO. Average out-of-pocket spending among fee-for-service beneficiaries varied depending on whether beneficiaries had Medigap policies, employer-provided supplemental insurance, or no supplemental coverage. Those without supplemental coverage spent more on health care goods and

Engineering the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by site saturation mutagenesis for D-phenylalanine synthesis.

PubMed

Gao, Xiuzhen; Huang, Fang; Feng, Jinhui; Chen, Xi; Zhang, Hailing; Wang, Zhixiang; Wu, Qiaqing; Zhu, Dunming

2013-08-01

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg(-1), which was 35.1-fold enhancement over the wild-type enzyme.

Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guo, Feng; Stanevich, Vitali; Wlodarchak, Nathan

Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A activemore » site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.« less

Out-of-pocket costs for paediatric admissions in district hospitals in Kenya

PubMed Central

Barasa, Edwine W; Ayieko, Philip; Cleary, Susan; English, Mike

2012-01-01

Objective To describe out-of-pocket costs of inpatient care for children under 5 years of age in district hospitals in Kenya. Methods A total of 256 caretakers of admitted children were interviewed in 2-week surveys conducted in eight hospitals in four provinces in Kenya. Caretakers were asked to report care seeking behaviour and expenditure related to accessing inpatient care. Family socio-economic status was assessed through reported expenditure in the previous month. Results Seventy eight percent of caretakers were required to pay user charges to access inpatient care for children. User charges (mean, US$ 8.1; 95% CI, 6.4–9.7) were 59% of total out-of-pocket costs, while transport costs (mean, US$ 4.9; 95% CI, 3.9–6.0) and medicine costs (mean, US$ 0.7; 95% CI, 0.5–1.0) were 36% and 5%, respectively. The mean total out-of-pocket cost per paediatric admission was US$ 14.1 (95% CI, 11.9–16.2). Out-of-pocket expenditures on health were catastrophic for 25.4% (95% CI, 18.4–33.3) of caretakers interviewed. Out-of-pocket expenditures were regressive, with a greater burden being experienced by households with lower socio-economic status. Conclusion Despite a policy of user fee exemption for children under 5 years of age in Kenya, our findings show that high unofficial user fees are still charged in district hospitals. Financing mechanisms that will offer financial risk protection to children seeking care need to be developed to remove barriers to child survival. PMID:22716184

Gating by tryptophan 73 exposes a cryptic pocket at the protein-binding interface of the oncogenic eIF4E protein.

PubMed

Lama, Dilraj; Brown, Christopher J; Lane, David P; Verma, Chandra S

2015-10-27

Targeting protein-protein interacting sites for potential therapeutic applications is a challenge in the development of inhibitors, and this becomes more difficult when these interfaces are relatively planar, as in the eukaryotic translation initiation factor 4E (eIF4E) protein. eIF4E is an oncogene that is overexpressed in numerous forms of cancer, making it a prime target as a therapeutic molecule. We report here the presence of a cryptic pocket at the protein-binding interface of eIF4E, which opens transiently during molecular dynamics simulations of the protein in solvent water and is observed to be stable when solvent water is mixed with benzene molecules. This pocket can also be seen in the ensemble of structures available from the solution-state conformations of eIF4E. The accessibility of the pocket is gated by the side-chain transitions of an evolutionarily conserved tryptophan residue. It is found to be feasible for accommodating clusters of benzene molecules, which signify the plasticity and ligandability of the pocket. We also observe that the newly formed cavity provides a favorable binding environment for interaction of a well-recognized small molecule inhibitor of eIF4E. The occurrence of this transiently accessible cavity highlights the existence of a more pronounced binding groove in a region that has traditionally been considered to be planar. Together, the data suggest that an alternate binding cavity exists on eIF4E and could be exploited for the rational design and development of a new class of lead compounds against the protein.

Dynamic control of droplets and pockets formation in homogeneous porous media immiscible displacements

NASA Astrophysics Data System (ADS)

Lins, T. F.; Azaiez, J.

2018-03-01

Interfacial instabilities of immiscible two-phase radial flow displacements in homogeneous porous media are analyzed for constant and time-dependent sinusoidal cyclic injection schemes. The analysis is carried out through numerical simulations based on the immersed interface and level set methods. The effects of the fluid properties and the injection flow parameters, namely, the period and the amplitude, on the formation of droplets and pockets are analyzed. It was found that larger capillary numbers or smaller viscosity ratios lead to more droplets/pockets that tend to appear earlier in time. Furthermore, the period and amplitude of the cyclic schemes were found to have a strong effect on droplets/pockets formations, and depending on their values, these can be enhanced or attenuated. In particular, the results revealed that there is a critical amplitude above which droplets and pockets formation is suppressed up to a specified time. This critical amplitude depends on the fluid properties, namely, the viscosity ratio and surface tension as well as on the period of the time-dependent scheme. The results of this study indicate that it is possible to use time-dependent cyclic schemes to control the formation and development of droplets/pockets in the flow and in particular to delay their appearance through an appropriate combination of the displacement scheme's amplitude and period.

RNA processing: pocket guides to ribosomal RNA.

PubMed

Peculis, B

1997-08-01

The functional role of a recently identified class of small nucleolar (sno) RNAs has been elucidated: the 'box H/ACA' snoRNAs act as guide RNAs, specifying the position of evolutionarily conserved pseudouridines in ribosomal (r)RNA via an rRNA-snoRNA base-pairing interaction that forms a 'pseudouridine pocket'.

Out-of-pocket costs associated with rotavirus gastroenteritis requiring hospitalization in Malaysia.

PubMed

Chai, P F; Lee, W S

2009-11-20

From August 2006 to July 2007 a prospective study of out-of-pocket costs incurred by care-givers of children hospitalized for rotavirus gastroenteritis was conducted in a hospital in Malaysia. Data on caretaker out-of-pocket costs were collected from 260 children hospitalized with diarrhoea. A stool sample was collected from 198 of these children of which 46 (23%) were positive for rotavirus by latex agglutination assay. The mean (median; interquartile range) out-of-pocket cost incurred by the care-givers was US$194 (US$169; US$47-738), constituting 26% of average monthly income of the households surveyed. Major components of the cost were hospital expenses (45%) and productivity loss (37%). These findings will allow further assessment of the cost-effectiveness of any future rotavirus immunization program in Malaysia.

Surgical management of retraction pockets of the pars tensa with cartilage and perichondrial grafts.

PubMed

Spielmann, P; Mills, R

2006-09-01

Stable, self-cleansing retraction pockets of the pars tensa are common incidental findings and require no treatment. In other cases, recurrent discharge occurs and there may also be associated conductive hearing loss. In a minority of cases, cholesteatoma may develop. This paper presents the results of surgery using a graft composed of cartilage and perichondrium for retraction pockets involving the posterior half of the tympanic membrane, as well as early results using a larger graft designed to manage retraction of the entire tympanic membrane. Data on 51 patients with posterior retraction pockets are presented. Forty-two (82 per cent) patients had no aural discharge one year following surgery and the tympanic membrane was not retracted in 43 (84 per cent). The larger 'Mercedes-Benz' graft was used in four patients and the results obtained suggested that it may prove a successful technique for extensive retraction pockets.

Advanced Geometric Optics on a Programmable Pocket Calculator.

ERIC Educational Resources Information Center

Nussbaum, Allen

1979-01-01

Presents a ray-tracing procedure based on some ideas of Herzberger and the matrix approach to geometrical optics. This method, which can be implemented on a programmable pocket calculator, applies to any conic surface, including paraboloids, spheres, and planes. (Author/GA)

Omega-oxidation impairs oxidizability of polyenoic fatty acids by 15-lipoxygenases: consequences for substrate orientation at the active site.

PubMed Central

Ivanov, I; Schwarz, K; Holzhütter, H G; Myagkova, G; Kühn, H

1998-01-01

During oxygenation by 15-lipoxygenases, polyenoic fatty acids are bound at the active site in such a way that the omega-terminus of the fatty acids penetrates into the substrate binding pocket. In contrast, for arachidonic acid 5-lipoxygenation, an inverse head to tail orientation has been suggested. However, an inverse orientation may be hindered by the large energy barrier associated with burying the charged carboxylate group in the hydrophobic environment of the substrate binding cleft. We studied the oxygenation kinetics of omega-modified fatty acids by 15-lipoxygenases and found that omega-hydroxylation strongly impaired substrate affinity (higher Km), but only moderately altered Vmax. In contrast, omega-carboxylation completely prevented the lipoxygenase reaction; however, methylation of the additional carboxylate group restored the activity. Arg403 of the human 15-lipoxygenase has been implicated in fatty acid binding by forming a salt bridge with the carboxylate group, and thus mutation of this amino acid to an uncharged residue was supposed to favour an inverse substrate orientation. The prepared Arg403-->Leu mutant of the rabbit 15-lipoxygenase was found to be a less effective catalyst of linoleic acid oxygenation. However, the oxygenation rate of omega-hydroxyarachidonic acid was similar when the wild-type and mutant enzyme were compared, and the patterns of oxygenation products were identical for both enzyme species. These data suggest that introduction of a polar, or even charged residue, at the omega-terminus of substrate fatty acids in connection with mutation of Arg403 may not alter substrate alignment at the active site of 15-lipoxygenases. PMID:9820810

Brine Pockets in the Icy Shell on Europa: Distribution, Chemistry, and Habitability

NASA Technical Reports Server (NTRS)

Zolotov, M. Yu; Shock, E. L.; Barr, A. C.; Pappalardo, R. T.

2004-01-01

On Earth, sea ice is rich in brine, salt, and gas inclusions that form through capturing of seawater during ice formation. Cooling of the ice over time leads to sequential freezing of captured sea-water, precipitation of salts, exsolution of gases, and formation of brine channels and pockets. Distribution and composition of brines in sea ice depend on the rate of ice formation, vertical temperature gradient, and the age of the ice. With aging, the abundance of brine pockets decreases through downward migration. De- spite low temperatures and elevated salinities, brines in sea ice provide a habitat for photosynthetic and chemosynthetic organisms. On Europa, brine pockets and channels could exist in the icy shell that may be from a few km to a few tens of km thick and is probably underlain by a water ocean. If the icy shell is relatively thick, convection could develop, affecting the temperature pattern in the ice. To predict the distribution and chemistry of brine pockets in the icy shell we have combined numerical models of the temperature distribution within a convecting shell, a model for oceanic chemistry, and a model for freezing of Europan oceanic water. Possible effects of brine and gas inclusions on ice rheology and tectonics are discussed.

Engineering the meso-Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum by Site Saturation Mutagenesis for d-Phenylalanine Synthesis

PubMed Central

Gao, Xiuzhen; Huang, Fang; Feng, Jinhui; Chen, Xi; Zhang, Hailing; Wang, Zhixiang; Wu, Qiaqing

2013-01-01

In order to enlarge the substrate binding pocket of the meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum to accommodate larger 2-keto acids, four amino acid residues (Phe146, Thr171, Arg181, and His227) were targeted for site saturation mutagenesis. Among all mutants, the single mutant H227V had a specific activity of 2.39 ± 0.06 U · mg−1, which was 35.1-fold enhancement over the wild-type enzyme. PMID:23728814

Comparative study to investigate the effect of meloxicam or minocycline HCl in situ gel system on local treatment of periodontal pockets.

PubMed

Kassem, Abeer Ahmed; Ismail, Fatma Ahmed; Naggar, Vivian Fahim; Aboulmagd, Elsayed

2014-08-01

In situ gelling formulations allow easy application to the target area. Gelation is induced by physiological stimuli at the site of application where the formula attains semisolid properties and exerts sustained drug release. In situ gelling formulations containing either 3% meloxicam (Mx) or 2% minocycline HCl (MH) were prepared for local application into the periodontal pockets. Gel formulations were based on the thermosensitive Pluronic(®) (Pl) and the pH-sensitive Carbopol(®) (C) polymers. C gels were prepared in combination with HPMC (H) to decrease its acidity. The total percent drug released from Pl formulae was 21.72% after 1 week for Mx and 85% after 3 days for MH. Their release kinetics data indicated anomalous non-Fickian behavior that could be controlled by both diffusion and chain relaxation. Addition of MH to C/H gels (1:2.5) resulted in liquefaction, followed by drug precipitation. Regarding C/H gel containing Mx, it showed a prolonged release rate up to 7 days with an initial burst effect; the kinetics data revealed Fickian-diffusion mechanism. The in vitro antibacterial activity studies for MH gel in Pl revealed that the drug released exceeded the minimum inhibitory concentration (MIC) of MH against Staphylococcus aureus ATCC 6538; placebo gel showed no effect on the microorganism. Clinical evaluation of Pl gels containing either Mx or MH showed significant improvement in chronic periodontitis patients, manifested by decrease in pocket depth and gingival index and increase in bone density.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

PubMed

Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

2014-12-16

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

Activation of Phenylalanine Hydroxylase by Phenylalanine Does Not Require Binding in the Active Site

PubMed Central

2015-01-01

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain. PMID:25453233

Out-of-pocket health care expenditures due to excess of body weight in Portugal.

PubMed

Veiga, Paula

2008-03-01

The prevalence for excessive weight has also been increasing dramatically in Portugal over the last decades. The consequences for families as well as for the publicly funded Portuguese health care system are a matter of policy interest. This paper uses an econometric model to compute the fraction of the national out-of-pocket health care expenditures attributable to overweight and obesity among Portuguese adults. Given that public health care system pays for a substantial share of the national health care expenditures, the estimated the out-of-pocket expenditures is only a share of the total expenditures. Per-capita expenditures and the burden that obesity and overweight impose on families are also estimated. Two waves of the Portuguese National Health Survey (NHS), namely; 1995/1996 and 1998/1999 are considered. The results suggest that out-of-pocket expenditures due to excess weight have increased sharply during these 3 years. The two-part model estimates suggest that the obese and overweight are more likely to incur out-of-pocket health care expenditures but, in the restricted sample of those that incur expenditures, there is weak or no evidence that the obese or overweight spend, on average, more than those of normal weight. Overall, it is estimated that in 1995/1996, more than 1.8% out-of-pocket health care expenditures were attributable to obesity and 2% to overweight (although not statistically significant). The estimated percentages are over 2.9% for obesity and 4% for overweight in 1998/1999. Combined, the estimated attributable percentage of national out-of-pocket expenditures due to excess weight was 3.8% in 1995/1996 and 6.9% in 1998/1999. Per-capita expenditures due to obesity or overweight are small, on average, in absolute terms, but they can be a significant cost for low income families. With respect to public policy concerns, the results suggest that measures which only slightly increase the out-of-pocket health care expenditures of being obese

Non-competitive inhibition by active site binders.

PubMed

Blat, Yuval

2010-06-01

Classical enzymology has been used for generations to understand the interactions of inhibitors with their enzyme targets. Enzymology tools enabled prediction of the biological impact of inhibitors as well as the development of novel, more potent, ones. Experiments designed to examine the competition between the tested inhibitor and the enzyme substrate(s) are the tool of choice to identify inhibitors that bind in the active site. Competition between an inhibitor and a substrate is considered a strong evidence for binding of the inhibitor in the active site, while the lack of competition suggests binding to an alternative site. Nevertheless, exceptions to this notion do exist. Active site-binding inhibitors can display non-competitive inhibition patterns. This unusual behavior has been observed with enzymes utilizing an exosite for substrate binding, isomechanism enzymes, enzymes with multiple substrates and/or products and two-step binding inhibitors. In many of these cases, the mechanisms underlying the lack of competition between the substrate and the inhibitor are well understood. Tools like alternative substrates, testing the enzyme reaction in the reverse direction and monitoring inhibition time dependence can be applied to enable distinction between 'badly behaving' active site binders and true exosite inhibitors.

The impact of health insurance programs on out-of-pocket expenditures in Indonesia: an increase or a decrease?

PubMed

Aji, Budi; De Allegri, Manuela; Souares, Aurelia; Sauerborn, Rainer

2013-07-18

We used panel data from the Indonesian Family Life Survey to investigate the impact of health insurance programs on reducing out-of-pocket expenditures. We employed three linear panel data models, two of which accounted for endogeneity: pooled ordinary least squares (OLS), pooled two-stage least squares (2SLS) for instrumental variable (IV), and fixed effects (FE). The study revealed that two health insurance programs had a significantly negative impact on out-of-pocket expenditures by using IV estimates. In the IV model, Askeskin decreased out-of-pocket expenditures by 34% and Askes by 55% compared with non-Askeskin and non-Askes, respectively, while Jamsostek was found to bear a nonsignificant effect on out-of-pocket expenditures. In the FE model, only Askeskin had a significant negative effect with an 11% reduction on out-of-pocket expenditures. This study showed that two large existing health insurance programs in Indonesia, Askeskin and Askes, effectively reduced household out-of-pocket expenditures. The ability of programs to offer financial protection by reducing out-of-pocket expenditures is likely to be a direct function of their benefits package and co-payment policies.

The Impact of Health Insurance Programs on Out-of-Pocket Expenditures in Indonesia: An Increase or a Decrease?

PubMed Central

Aji, Budi; De Allegri, Manuela; Souares, Aurelia; Sauerborn, Rainer

2013-01-01

We used panel data from the Indonesian Family Life Survey to investigate the impact of health insurance programs on reducing out-of-pocket expenditures. We employed three linear panel data models, two of which accounted for endogeneity: pooled ordinary least squares (OLS), pooled two-stage least squares (2SLS) for instrumental variable (IV), and fixed effects (FE). The study revealed that two health insurance programs had a significantly negative impact on out-of-pocket expenditures by using IV estimates. In the IV model, Askeskin decreased out-of-pocket expenditures by 34% and Askes by 55% compared with non-Askeskin and non-Askes, respectively, while Jamsostek was found to bear a nonsignificant effect on out-of-pocket expenditures. In the FE model, only Askeskin had a significant negative effect with an 11% reduction on out-of-pocket expenditures. This study showed that two large existing health insurance programs in Indonesia, Askeskin and Askes, effectively reduced household out-of-pocket expenditures. The ability of programs to offer financial protection by reducing out-of-pocket expenditures is likely to be a direct function of their benefits package and co-payment policies. PMID:23873263

Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zull, Lawrence M.; Yeniscavich, William

2008-01-15

The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive materialmore » contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.« less

Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

PubMed

Khashan, Raed S

2015-01-01

As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

Out-of-pocket medical costs and third-party healthcare costs for children with Down syndrome.

PubMed

Kageleiry, Andrew; Samuelson, David; Duh, Mei Sheng; Lefebvre, Patrick; Campbell, John; Skotko, Brian G

2017-03-01

Prior analyses have estimated the lifetime total societal costs of a person with Down syndrome (DS); however, no studies capture the expected medical costs that patients with DS can expect to incur during childhood. The study utilized the OptumHealth Reporting and Insights administrative claims database from 1999 to 2013. Children with a diagnosis of DS were identified, and their time was divided into clinically relevant age categories. Patients with DS in each age category were matched to controls without chromosomal conditions. Out-of-pocket medical costs and third-party expenditures were compared between the patient-age cohorts with DS and matched controls. Patients with DS had significantly higher mean annual out-of-pocket costs than their matched controls within each age and cost category. Total annual incremental out-of-pocket costs associated with DS were highest among individuals from birth to age 1 ($1,907, P < 0.001). The main drivers of the incremental out-of-pocket costs associated with DS were inpatient costs in the 1st year of life ($925, P < 0.001) and outpatient costs in later years (ranging $183-$623, all P < 0.001). Overall, patients with DS incurred incremental out-of-pocket medical costs of $18,248 between birth and age 18 years; third-party payers incurred incremental costs of $230,043 during the same period. Across all age categories, mean total out-of-pocket annual costs were greater for individuals with DS than those of matched controls. On average, parents of children with DS pay an additional $84 per month for out-of-pocket medical expenses when costs are amortized over 18 years. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study†

PubMed Central

Peng, Dungeng; Satterlee, James D.; Ma, Li-Hua; Dallas, Jerry L.; Smith, Kevin M.; Zhang, Xuhong; Sato, Michihiko; La Mar, Gerd N.

2011-01-01

Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which secures host iron, shares many properties with mammalian HOs, but also exhibits some key differences. The crystal structure appears more compact and the crystal-undetected C-terminus interacts with substrate in solution. The unique nature of substrate-protein, specifically pyrrole-I/II-helix-2, peripheral interactions in NmHO are probed by 2D 1H NMR to reveal unique structural features controlling substrate orientation. The thermodynamics of substrate orientational isomerism are mapped for substrates with individual vinyl → methyl → hydrogen substitutions and with enzyme C-terminal deletions. NmHO exhibits significantly stronger orientational preference, reflecting much stronger and selective pyrrole-I/II interactions with the protein matrix, than in mammalian HOs. Thus, replacing bulky vinyls with hydrogens results in a 180° rotation of substrate about the α,γ-meso axis in the active site. A "collapse" of the substrate pocket as substrate size decreases is reflected in movement of helix-2 toward the substrate as indicated by significant and selective increased NOESY cross peak intensity, increase in steric Fe-CN tilt reflected in the orientation of the major magnetic axis, and decrease in steric constraints controlling the rate of aromatic ring reorientation. The active site of NmHO appears "stressed" for native protohemin and its "collapse" upon replacing vinyls by hydrogen leads to a factor ~102 increase in substrate affinity. Interaction of the C-terminus with the active site destabilizes the crystallographic protohemin orientation by ~0.7 kcal/mol, which is consistent with optimizing the His207-Asp27 H-bond. Implications of the active site "stress" for product release are discussed. PMID:21870860

Medicare Advantage Members' Expected Out-Of-Pocket Spending For Inpatient And Skilled Nursing Facility Services.

PubMed

Keohane, Laura M; Grebla, Regina C; Mor, Vincent; Trivedi, Amal N

2015-06-01

Inpatient and skilled nursing facility (SNF) cost sharing in Medicare Advantage (MA) plans may reduce unnecessary use of these services. However, large out-of-pocket expenses potentially limit access to care and encourage beneficiaries at high risk of needing inpatient and postacute care to avoid or leave MA plans. In 2011 new federal regulations restricted inpatient and skilled nursing facility cost sharing and mandated limits on out-of-pocket spending in MA plans. After these regulations, MA members in plans with low premiums averaged $1,758 in expected out-of-pocket spending for an episode of seven hospital days and twenty skilled nursing facility days. Among members with the same low-premium plan in 2010 and 2011, 36 percent of members belonged to plans that added an out-of-pocket spending limit in 2011. However, these members also had a $293 increase in average cost sharing for an inpatient and skilled nursing facility episode, possibly to offset plans' expenses in financing out-of-pocket limits. Some MA beneficiaries may still have difficulty affording acute and postacute care despite greater regulation of cost sharing. Project HOPE—The People-to-People Health Foundation, Inc.

Medicare Advantage Members’ Expected Out-Of-Pocket Spending For Inpatient And Skilled Nursing Facility Services

PubMed Central

Keohane, Laura M.; Grebla, Regina C.; Mor, Vincent; Trivedi, Amal N.

2015-01-01

Inpatient and skilled nursing facility (SNF) cost sharing in Medicare Advantage (MA) plans may reduce unnecessary use of these services. However, large out-of-pocket expenses potentially limit access to care and encourage beneficiaries at high risk of needing inpatient and postacute care to avoid or leave MA plans. In 2011 new federal regulations restricted inpatient and skilled nursing facility cost sharing and mandated limits on out-of-pocket spending in MA plans. After these regulations, MA members in plans with low premiums averaged $1,758 in expected out-of-pocket spending for an episode of seven hospital days and twenty skilled nursing facility days. Among members with the same low-premium plan in 2010 and 2011, 36 percent of members belonged to plans that added an out-of-pocket spending limit in 2011. However, these members also had a $293 increase in average cost sharing for an inpatient and skilled nursing facility episode, possibly to offset plans’ expenses in financing out-of-pocket limits. Some MA beneficiaries may still have difficulty affording acute and postacute care despite greater regulation of cost sharing. PMID:26056208

What regulates the catalytic activities in AGE catalysis? An answer from quantum mechanics/molecular mechanics simulations.

PubMed

Zhang, Yulai; Zhang, Hongxing; Zheng, Qingchuan

2017-12-06

The AGE superfamily (AGEs) is made up of kinds of isomerase which are very important both physiologically and industrially. One of the most intriguing aspects of AGEs has to do with the mechanism that regulates their activities in single conserved active pocket. In order to clarify the relationship among single conserved active pocket and two activities in AGEs, results for the epimerization activity catalyzed by RaCE and the isomerization activity catalyzed by SeYihS were obtained by using QM/MM umbrella sampling simulations and 2D-FES calculations. Our results show that both of them have similar enzyme-substrate combination mode for inner pyranose ring in single conserved active pocket even though they have different substrate specificity. This means that the pathways of ring opening catalyzed by them are similar. However, one non-conserved residue (Leu183 in RaCE, Met175 in SeYihS) in the active site, which has different steric hindrance, causes a small but effective change in the direction of ring opening in stage 1. And then this change will induce a fundamentally different catalytic activity for RaCE and SeYihS in stage 2. Our results give a novel viewpoint about the regulatory mechanism between CE and YihS in AGEs, and may be helpful for further experiments of rational enzyme design based on the (α/α) 6 -barrel basic scaffold.

Alzheimer Disease and Related Disorders and Out-of-Pocket Health Care Spending and Burden Among Elderly Medicare Beneficiaries.

PubMed

Dwibedi, Nilanjana; Findley, Patricia A; Wiener R, Constance; Shen, Chan; Sambamoorthi, Usha

2018-03-01

To estimate the excess burden of out-of-pocket health care spending associated with Alzheimer disease and related disorders (ADRD) among older individuals (age 65 y and older). We adopted a retrospective, cross-sectional study design with data from 2012 Medicare Current Beneficiary Survey. The study sample comprised of elderly community-dwelling individuals who had positive total health care expenditures, and enrolled in Medicare throughout the calendar year (462 with ADRD, and 7160 without ADRD). We estimated the per-capita total annual out-of-pocket spending on health care and out-of-pocket spending by service type: inpatient, outpatient, home health, prescription drugs, and other services. We measured out-of-pocket spending burden by calculating the percentage of income spent on health care and defined high out-of-pocket spending burden as having this percentage above 10%. Multivariable analyses included ordinary least squares regressions and logistic regressions and these analyses adjusted for predisposing, enabling, need, personal health care practices and external environment characteristics. The average annual per-capita out-of-pocket health care spending was greater among individuals with ADRD compared with those without ADRD ($3285 vs. $1895); home health and prescription drugs accounted for 52% of total out-of-pocket spending among individuals with ADRD and 34% among individuals without ADRD. Elderly individuals with ADRD were more likely to have high out-of-pocket spending burden (adjusted odds ratio, 1.49; 95% confidence interval, 1.13-1.97) compared with those without ADRD. ADRD is associated with excess out-of-pocket health care spending, primarily driven by prescription drugs and home health care use.

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

PubMed Central

Spurny, Radovan; Debaveye, Sarah; Farinha, Ana; Veys, Ken; Vos, Ann M.; Gossas, Thomas; Atack, John; Bertrand, Sonia; Bertrand, Daniel; Danielson, U. Helena; Tresadern, Gary; Ulens, Chris

2015-01-01

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential. PMID:25918415

The Role of Electronic Pocket Dictionaries as an English Learning Tool among Chinese Students

ERIC Educational Resources Information Center

Jian, Hua-Li; Sandnes, Frode Eika; Law, Kris M. Y.; Huang, Yo-Ping; Huang, Yueh-Min

2009-01-01

This study addressed the role of electronic pocket dictionaries as a language learning tool among university students in Hong Kong and Taiwan. The target groups included engineering and humanities students at both undergraduate and graduate level. Speed of reference was found to be the main motivator for using an electronic pocket dictionary.…

Out-of-Pocket Household Expenditures on Medical Injections in Cambodia.

PubMed

Ozawa, Sachiko; Yemeke, Tatenda T; Tawah, Alie F; Kulkarni, Vivek; Villar Uribe, Manuela

2018-02-09

Cambodia has one of the highest rates of overall medical injection usage worldwide. Therapeutic injections, which are often unnecessary, contribute to the spread of blood-borne diseases. This study describes injection practices and associated household expenditures in rural northwest Cambodia. We assessed care-seeking patterns of surveyed adult family members who sought healthcare in the previous 30 days, including location of care, medical injection use, and out-of-pocket household expenditures for treatment. A regression model was used to explore the impact of injection use on out-of-pocket household expenditures. Among 480 households sampled, 298 included members who had been sick within the previous 30 days; a total of 342 episodes of care had been sought. Private providers accounted for over 66% (n = 226) of all episodes of care, with public and informal providers accounting for 20% (n = 69) and 14% (n = 47), respectively. Injections were administered in over 120 (35%) episodes of care, with 81% of injections administered by private providers. Patients who received injections incurred total out-of-pocket household expenditures that were, on average, 126,590 Cambodian Riel (KHR) (US$31.65) higher than those who did not receive injections (p < 0.01), equivalent to nearly half of the country's total annual health expenditure per capita. Receiving injections and perceived severity of illness were significantly associated with higher out-of-pocket household expenditures. This study found high levels of medical injection use, particularly among private healthcare providers, which was significantly associated with high healthcare expenditures. Reducing the number of medical injections would not only reduce disease transmission risk but also contribute to reduced healthcare costs and greater financial protection.

Occupation of the cytochrome P450 substrate pocket by diverse compounds at general anesthesia concentrations.

PubMed

LaBella, F S; Stein, D; Queen, G

1998-10-02

Each of a diverse array of compounds, at concentrations reported to effect general anesthesia, when added to liver microsomes, forms a complex with cytochromes P450 to generate, with reference to a cuvette containing microsomes only, a characteristic absorbance-difference spectrum. This spectrum results from a change in the electron-spin state of the heme iron atom induced upon entry by the anesthetic molecule into the enzyme catalytic pocket. The difference spectrum, representing the anesthetic-P450 complex, is characteristic of substances that are substrates for the enzyme. For the group of compounds as a whole, the magnitudes of the absorbance-difference spectra vary only about twofold, although the anesthetic potencies vary by several orders of magnitude. The dissociation constants (Ks), calculated from absorbance data and representing affinities of the anesthetics for P450, agree closely with the respective EC50 (concentration that effects anesthesia in 50% of individuals) values, and with the respective Ki (concentration that inhibits P450 catalytic activities half-maximally) values reported by us previously. The absorbance complex resulting from the occupation of the catalytic pocket by endogenous substrates, androstenedione and arachidonic acid, is inhibited, competitively, by anesthetics. Occupation of and perturbation of the heme catalytic pocket by anesthetic, as monitored by the absorbance-difference spectrum, is rapidly reversible. The presumed in vivo consequences of perturbation by general anesthetics of heme proteins is suppression of the generation of chemical signals that determine cell sensitivity and response.

Patients' annual income adequacy, insurance premiums and out-of-pocket expenses related to heart failure care.

PubMed

Piamjariyakul, Ubolrat; Yadrich, Donna Macan; Russell, Christy; Myer, Jane; Prinyarux, Chanawee; Vacek, James L; Ellerbeck, Edward F; Smith, Carol E

2014-01-01

To (1) identify the amount patients spend for insurance premiums, co-payments, deductibles, and other out-of-pocket costs related to HF and chronic health care services and estimate their annual non-reimbursed and out-of-pocket costs; and (2) identify patients' concerns about nonreimbursed and out-of-pocket expenses. HF is one of the most expensive illnesses for our society with multiple health services and financial burdens for families. Mixed methods with quantitative questionnaires and qualitative interviews. Patients (N = 149) reported annual averages for non-reimbursed health services co-payments and out-of-pocket costs ranging from $3913 to $5829 depending on insurance coverage. Thirty one patients (21%) reported inadequate health coverage related to their non-reimbursed costs. Non-reimbursed costs related to HF care are substantial and vary depending on their insurance, health services use, and out-of-pocket costs. Patient referral to social services to assist with expenses could provide some relief from the burden of high HF-related costs. Copyright © 2014 Elsevier Inc. All rights reserved.

Ionizable Side Chains at Catalytic Active Sites of Enzymes

PubMed Central

Jimenez-Morales, David; Liang, Jie

2012-01-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1072 Å3. The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes. PMID:22484856

ATP-Binding Pocket-Targeted Suppression of Src and Syk by Luteolin Contributes to Its Anti-Inflammatory Action

PubMed Central

Lee, Jeong-Oog; Kim, Mi-Yeon

2015-01-01

Luteolin is a flavonoid identified as a major anti-inflammatory component of Artemisia asiatica. Numerous reports have demonstrated the ability of luteolin to suppress inflammation in a variety of inflammatory conditions. However, its exact anti-inflammatory mechanism has not been fully elucidated. In the present study, the anti-inflammatory mode of action in activated macrophages of luteolin from Artemisia asiatica was examined by employing immunoblotting analysis, a luciferase reporter gene assay, enzyme assays, and an overexpression strategy. Luteolin dose-dependently inhibited the secretion of nitric oxide (NO) and prostaglandin E2 (PGE2) and diminished the levels of mRNA transcripts of inducible NO synthase (iNOS), tumor necrosis factor- (TNF-) α, and cyclooxygenase-2 (COX-2) in lipopolysaccharide- (LPS-) and pam3CSK-treated macrophage-like RAW264.7 cells without displaying cytotoxicity. Luteolin displayed potent NO-inhibitory activity and also suppressed the nuclear translocation of NF-κB (p65 and p50) via blockade of Src and Syk, but not other mitogen-activated kinases. Overexpression of wild type Src and point mutants thereof, and molecular modelling studies, suggest that the ATP-binding pocket may be the luteolin-binding site in Src. These results strongly suggest that luteolin may exert its anti-inflammatory action by suppressing the NF-κB signaling cascade via blockade of ATP binding in Src and Syk. PMID:26236111

Complex home care: Part 2- family annual income, insurance premium, and out-of-pocket expenses.

PubMed

Piamjariyakul, Ubolrat; Yadrich, Donna Macan; Ross, Vicki M; Smith, Carol E; Clements, Faye; Williams, Arthur R

2010-01-01

Annual costs paid by families for intravenous infusion of home parenteral nutrition (HPN) health insurance premiums, deductibles, co-payments for health services, and the wide range of out-of-pocket home health care expenses are significant. The costs of managing complex chronic care at home cannot be completely understood until all out-of-pocket costs have been defined, described, and tabulated. Non-reimbursed and out-of-pocket costs paid by families over years for complex chronic care negatively impact the financial stability of families. National health care reform must take into account the long-term financial burdens of families caring for those with complex home care. Any changes that may increase the out-of-pocket costs or health insurance costs to these families can also have a negative long-term impact on society when greater numbers of patients declare bankruptcy or qualify for medical disability.

Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding*

PubMed Central

Derbyshire, Emily R.; Deng, Sarah; Marletta, Michael A.

2010-01-01

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of α1β1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain β1(1–385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (β1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat β1 distal heme pocket (Ile-145 → Tyr and Ile-149 → Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin FeII-O2 complexes and is likely the first observation of a FeII-O2 complex in the full-length α1β1 protein. Additionally, these data suggest that atypical sGCs stabilize O2 binding by a hydrogen bonding network involving tyrosine and glutamine. PMID:20231286

Discriminative structural approaches for enzyme active-site prediction.

PubMed

Kato, Tsuyoshi; Nagano, Nozomi

2011-02-15

Predicting enzyme active-sites in proteins is an important issue not only for protein sciences but also for a variety of practical applications such as drug design. Because enzyme reaction mechanisms are based on the local structures of enzyme active-sites, various template-based methods that compare local structures in proteins have been developed to date. In comparing such local sites, a simple measurement, RMSD, has been used so far. This paper introduces new machine learning algorithms that refine the similarity/deviation for comparison of local structures. The similarity/deviation is applied to two types of applications, single template analysis and multiple template analysis. In the single template analysis, a single template is used as a query to search proteins for active sites, whereas a protein structure is examined as a query to discover the possible active-sites using a set of templates in the multiple template analysis. This paper experimentally illustrates that the machine learning algorithms effectively improve the similarity/deviation measurements for both the analyses.

Effect of azithromycin on acid reflux, hiatus hernia and proximal acid pocket in the postprandial period.

PubMed

Rohof, W O; Bennink, R J; de Ruigh, A A; Hirsch, D P; Zwinderman, A H; Boeckxstaens, G E

2012-12-01

The risk for acidic reflux is mainly determined by the position of the gastric acid pocket. It was hypothesised that compounds affecting proximal stomach tone might reduce gastro-oesophageal reflux by changing the acid pocket position. To study the effect of azithromycin (Azi) on acid pocket position and acid exposure in patients with gastro-oesophageal reflux disease (GORD). Nineteen patients with GORD were included, of whom seven had a large hiatal hernia (≥3 cm) (L-HH) and 12 had a small or no hiatal hernia (S-HH). Patients were randomised to Azi 250 mg/day or placebo during 3 days in a crossover manner. On each study day, reflux episodes were detected using concurrent high-resolution manometry and pH-impedance monitoring after a standardised meal. The acid pocket was visualised using scintigraphy, and its position was determined relative to the diaphragm. Azi reduced the number of acid reflux events (placebo 8.0±2.2 vs Azi 5.6±1.8, p<0.01) and postprandial acid exposure (placebo 10.5±3.8% vs Azi 5.9±2.5%, p<0.05) in all patients without affecting the total number of reflux episodes. Acid reflux occurred mainly when the acid pocket was located above, or at the level of, the diaphragm, rather than below the diaphragm. Treatment with Azi reduced hiatal hernia size and resulted in a more distal position of the acid pocket compared with placebo (below the diaphragm 39% vs 29%, p=0.03). Azi reduced the rate of acid reflux episodes in patients with S-HH (38% to 17%) to a greater extent than in patients with L-HH (69% to 62%, p=0.04). Azi reduces acid reflux episodes and oesophageal acid exposure. This effect was associated with a smaller hiatal hernia size and a more distal position of the acid pocket, further indicating the importance of the acid pocket in the pathogenesis of GORD. http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=1970 NTR1970.

Study Of Physician And Patient Communication Identifies Missed Opportunities To Help Reduce Patients’ Out-Of-Pocket Spending

PubMed Central

Ubel, Peter A.; Zhang, Cecilia J.; Hesson, Ashley; Davis, J. Kelly; Kirby, Christine; Barnett, Jamison; Hunter, Wynn G.

2018-01-01

Some experts contend that requiring patients to pay out of pocket for a portion of their care will bring consumer discipline to health care markets. But are physicians prepared to help patients factor out-of-pocket expenses into medical decisions? In this qualitative study of audiorecorded clinical encounters, we identified physician behaviors that stand in the way of helping patients navigate out-of-pocket spending. Some behaviors reflected a failure to fully engage with patients’ financial concerns, from never acknowledging such concerns to dismissing them too quickly. Other behaviors reflected a failure to resolve uncertainty about out-of-pocket expenses or reliance on temporary solutions without making long-term plans to reduce spending. Many of these failures resulted from systemic barriers to health care spending conversations, such as a lack of price transparency. For consumer health care markets to work as intended, physicians need to be prepared to help patients navigate out-of-pocket expenses when financial concerns arise during clinical encounters. PMID:27044966

Evidence for a small hole pocket in the Fermi surface of underdoped YBa2Cu3Oy

PubMed Central

Doiron-Leyraud, N.; Badoux, S.; René de Cotret, S.; Lepault, S.; LeBoeuf, D.; Laliberté, F.; Hassinger, E.; Ramshaw, B. J.; Bonn, D. A.; Hardy, W. N.; Liang, R.; Park, J.-H..; Vignolles, D.; Vignolle, B.; Taillefer, L.; Proust, C.

2015-01-01

In underdoped cuprate superconductors, the Fermi surface undergoes a reconstruction that produces a small electron pocket, but whether there is another, as yet, undetected portion to the Fermi surface is unknown. Establishing the complete topology of the Fermi surface is key to identifying the mechanism responsible for its reconstruction. Here we report evidence for a second Fermi pocket in underdoped YBa2Cu3Oy, detected as a small quantum oscillation frequency in the thermoelectric response and in the c-axis resistance. The field-angle dependence of the frequency shows that it is a distinct Fermi surface, and the normal-state thermopower requires it to be a hole pocket. A Fermi surface consisting of one electron pocket and two hole pockets with the measured areas and masses is consistent with a Fermi-surface reconstruction by the charge–density–wave order observed in YBa2Cu3Oy, provided other parts of the reconstructed Fermi surface are removed by a separate mechanism, possibly the pseudogap. PMID:25616011

Multiple Fermi pockets revealed by Shubnikov-de Haas oscillations in WTe2

NASA Astrophysics Data System (ADS)

Xiang, Fei-Xiang; Veldhorst, Menno; Dou, Shi-Xue; Wang, Xiao-Lin

2015-11-01

The recently discovered non-saturating and parabolic magnetoresistance and the pressure-induced superconductivity at low temperature in WTe2 imply its rich electronic structure and possible practical applications. Here we use magnetotransport measurements to investigate the electronic structure of WTe2 single crystals. A non-saturating and parabolic magnetoresistance is observed from low temperature to high temperature up to 200 K with magnetic fields up to 8 T. Shubnikov-de Haas (SdH) oscillations with beating patterns are observed, the fast Fourier transform of which reveals three oscillation frequencies, corresponding to three pairs of Fermi pockets with comparable effective masses, m* ∼ 0.31~me . By fitting the Hall resistivity, we infer that they can be attributed to one pair of electron pockets and two pairs of hole pockets, together with nearly perfect compensation of the electron-hole carrier concentration. These magnetotransport measurements reveal the complex electronic structure in WTe2, explaining the non-saturating magnetoresistance.

Understanding the physical and chemical nature of the warfarin drug binding site in human serum albumin: experimental and theoretical studies.

PubMed

Abou-Zied, Osama K

2015-01-01

Human serum albumin (HSA) is one of the major carrier proteins in the body and constitutes approximately half of the protein found in blood plasma. It plays an important role in lipid metabolism, and its ability to reversibly bind a large variety of pharmaceutical compounds makes it a crucial determinant of drug pharmacokinetics and pharmacodynamics. This review deals with one of the protein's major binding sites "Sudlow I" which includes a binding pocket for the drug warfarin (WAR). The binding nature of this important site can be characterized by measuring the spectroscopic changes when a ligand is bound. Using several drugs, including WAR, and other drug-like molecules as ligands, the results emphasize the nature of Sudlow I as a flexible binding site, capable of binding a variety of ligands by adapting its binding pockets. The high affinity of the WAR pocket for binding versatile molecular structures stems from the flexibility of the amino acids forming the pocket. The binding site is shown to have an ionization ability which is important to consider when using drugs that are known to bind in Sudlow I. Several studies point to the important role of water molecules trapped inside the binding site in molecular recognition and ligand binding. Water inside the protein's cavity is crucial in maintaining the balance between the hydrophobic and hydrophilic nature of the binding site. Upon the unfolding and refolding of HSA, more water molecules are trapped inside the binding site which cause some swelling that prevents a full recovery from the denatured state. Better understanding of the mechanism of binding in macromolecules such as HSA and other proteins can be achieved by combining experimental and theoretical studies which produce significant synergies in studying complex biochemical phenomena.

Ionizable side chains at catalytic active sites of enzymes.

PubMed

Jimenez-Morales, David; Liang, Jie; Eisenberg, Bob

2012-05-01

Catalytic active sites of enzymes of known structure can be well defined by a modern program of computational geometry. The CASTp program was used to define and measure the volume of the catalytic active sites of 573 enzymes in the Catalytic Site Atlas database. The active sites are identified as catalytic because the amino acids they contain are known to participate in the chemical reaction catalyzed by the enzyme. Acid and base side chains are reliable markers of catalytic active sites. The catalytic active sites have 4 acid and 5 base side chains, in an average volume of 1,072 Å(3). The number density of acid side chains is 8.3 M (in chemical units); the number density of basic side chains is 10.6 M. The catalytic active site of these enzymes is an unusual electrostatic and steric environment in which side chains and reactants are crowded together in a mixture more like an ionic liquid than an ideal infinitely dilute solution. The electrostatics and crowding of reactants and side chains seems likely to be important for catalytic function. In three types of analogous ion channels, simulation of crowded charges accounts for the main properties of selectivity measured in a wide range of solutions and concentrations. It seems wise to use mathematics designed to study interacting complex fluids when making models of the catalytic active sites of enzymes.

Out-of-pocket expenses for maternity care in rural Bangladesh: a public-private comparison.

PubMed

Rahman, Moshiur; Rob, Ubaidur; Noor, Forhana Rahman; Bellows, Benjamin

Out-of-pocket expenses incurred by women for availing maternal healthcare services at public and private health facilities in Bangladesh were examined using a baseline household survey evaluating the impact of demand side financing vouchers on utilization and service delivery for maternal healthcare. The survey was conducted in 2010 among 3,300 women who gave birth in the previous 12 months from the start of data collection. Information on costs incurred to receive antenatal, delivery, and postnatal care services was collected. Findings reveal that the majority of women reported paying out-of-pocket expenses for availing maternal healthcare services both at public and private health facilities. Out-of-pocket expenses include registration, consultation, laboratory examination, medicine, transportation, and other associated costs incurred for receiving maternal healthcare services. On average, women paid US$3.60 out-of-pocket expenses for receiving antenatal care at public health facilities and US$12.40 at private health facilities. Similarly, women paid one and half times more for normal (US$42.30) and cesarean deliveries (US$136.20) at private health facilities compared to public health facilities. On the other hand, costs for postnatal care services did not vary significantly between public and private health facilities. Utilization of maternal healthcare services can be improved if out-of-pocket expenses can be minimized. At the same time, effective demand generation strategies are necessary to encourage women to utilize health facilities.

Pocket radiation dosimeter: dosimeter charger assembly

DOEpatents

Manning, F.W.

1982-03-17

This invention is a novel pocket-type radiation dosimeter comprising an electrometric radiation dosimeter and a charging circuit therefor. The instrument is especially designed to be amenable to mass production, to have a long shelf life, and to be compact, lightweight, and usable by the layman. The dosimeter proper may be of conventional design. The charging circuit includes a shake-type electrostatic generator, a voltage doubler for integrating generator output voltages of one polarity, and a switch operated by an external permanent magnet.

Pocket radiation dosimeter--dosimeter charger assembly

DOEpatents

Manning, Frank W.

1984-01-01

This invention is a novel pocket-type radiation dosimeter comprising an electrometric radiation dosimeter and a charging circuit therefor. The instrument is especially designed to be amenable to mass production, to have a long shelf life, and to be compact, lightweight, and usable by the layman. The dosimeter proper may be of conventional design. The charging circuit includes a shake-type electrostatic generator, a voltage doubler for integrating generator output voltages of one polarity, and a switch operated by an external permanent magnet.

Tertiary structure prediction and identification of druggable pocket in the cancer biomarker – Osteopontin-c

PubMed Central

2014-01-01

Background Osteopontin (Eta, secreted sialoprotein 1, opn) is secreted from different cell types including cancer cells. Three splice variant forms namely osteopontin-a, osteopontin-b and osteopontin-c have been identified. The main astonishing feature is that osteopontin-c is found to be elevated in almost all types of cancer cells. This was the vital point to consider it for sequence analysis and structure predictions which provide ample chances for prognostic, therapeutic and preventive cancer research. Methods Osteopontin-c gene sequence was determined from Breast Cancer sample and was translated to protein sequence. It was then analyzed using various software and web tools for binding pockets, docking and druggability analysis. Due to the lack of homological templates, tertiary structure was predicted using ab-initio method server – I-TASSER and was evaluated after refinement using web tools. Refined structure was compared with known bone sialoprotein electron microscopic structure and docked with CD44 for binding analysis and binding pockets were identified for drug designing. Results Signal sequence of about sixteen amino acid residues was identified using signal sequence prediction servers. Due to the absence of known structures of similar proteins, three dimensional structure of osteopontin-c was predicted using I-TASSER server. The predicted structure was refined with the help of SUMMA server and was validated using SAVES server. Molecular dynamic analysis was carried out using GROMACS software. The final model was built and was used for docking with CD44. Druggable pockets were identified using pocket energies. Conclusions The tertiary structure of osteopontin-c was predicted successfully using the ab-initio method and the predictions showed that osteopontin-c is of fibrous nature comparable to firbronectin. Docking studies showed the significant similarities of QSAET motif in the interaction of CD44 and osteopontins between the normal and splice

Development of radiopaque, biocompatible, antimicrobial, micro-particle fillers for micro-CT imaging of simulated periodontal pockets.

PubMed

Elashiry, M; Meghil, M M; Kalathingal, S; Buchanan, A; Rajendran, M; Elrefai, R; Ochieng, M; Elawady, A; Arce, R M; Sandhage, K H; Cutler, C W

2018-04-01

Approximately 10 9 bacteria can be harbored within periodontal pockets (PP) along with inflammatory byproducts implicated in the pathophysiology of systemic diseases linked to periodontitis (PD). Calculation of this inflammatory burden has involved estimation of total pocket surface area using analog data from conventional periodontal probing which is unable to determine the three-dimensional (3-D) nature of PP. The goals of this study are to determine the radiopacity, biocompatibility, and antimicrobial activity of transient micro-particle fillers in vitro and demonstrate their capability for 3-D imaging of artificial PP (U.S. Patent publication number: 9814791 B2). Relative radiopacity values of various metal oxide fillers were obtained from conventional radiography and micro-computed tomography (μCT) using in vitro models. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were used to measure the biocompatibility of calcium tungstate (CaWO 4 ) particles by determination of viable keratinocytes percentage (%) after exposure. After introducing an antibacterial compound (K21) to the radiopaque agent, antimicrobial tests were conducted using Porphyromonas gingivalis (P. gingivalis) and Streptococcus gordonii (S. gordonii) strains and blood agar plates. CaWO 4 micro-particle-bearing fillers exhibited an X-ray radiopacity distinct from tooth structures that enabled 3-D visualization of an artificial periodontal pocket created around a human tooth. MTT assays indicated that CaWO 4 micro-particles are highly biocompatible (increasing the viability of exposed keratinocytes). Radiopaque micro-particle fillers combined with K21 showed significant antimicrobial activity for P. gingivalis and S. gordonii. The plausibility of visualizing PP with 3-D radiographic imaging using new radiopaque, biocompatible, transient fillers was demonstrated in vitro. Antibacterial (or other) agents added to this formula could provide beneficial therapeutic features

An integrated strategy for improving contrast, durability, and portability of a Pocket Colposcope for cervical cancer screening and diagnosis

PubMed Central

Mueller, Jenna; Asma, Betsy; Asiedu, Mercy; Krieger, Marlee S.; Chitalia, Rhea; Dahl, Denali; Taylor, Peyton; Schmitt, John W.; Ramanujam, Nimmi

2018-01-01

Introduction We have previously developed a portable Pocket Colposcope for cervical cancer screening in resource-limited settings. In this manuscript we report two different strategies (cross-polarization and an integrated reflector) to improve image contrast levels achieved with the Pocket Colposcope and evaluate the merits of each strategy compared to a standard-of-care digital colposcope. The desired outcomes included reduced specular reflection (glare), increased illumination beam pattern uniformity, and reduced electrical power budget. In addition, anti-fogging and waterproofing features were incorporated to prevent the Pocket Colposcope from fogging in the vaginal canal and to enable rapid disinfection by submersion in chemical agents. Methods Cross-polarization (Generation 3 Pocket Colposcope) and a new reflector design (Generation 4 Pocket Colposcope) were used to reduce glare and improve contrast. The reflector design (including the angle and height of the reflector sidewalls) was optimized through ray-tracing simulations. Both systems were characterized with a series of bench tests to assess specular reflection, beam pattern uniformity, and image contrast. A pilot clinical study was conducted to compare the Generation 3 and 4 Pocket Colposcopes to a standard-of-care colposcope (Leisegang Optik 2). Specifically, paired images of cervices were collected from the standard-of-care colposcope and either the Generation 3 (n = 24 patients) or the Generation 4 (n = 32 patients) Pocket Colposcopes. The paired images were blinded by device, randomized, and sent to an expert physician who provided a diagnosis for each image. Corresponding pathology was obtained for all image pairs. The primary outcome measures were the level of agreement (%) and κ (kappa) statistic between the standard-of-care colposcope and each Pocket Colposcope (Generation 3 and Generation 4). Results Both generations of Pocket Colposcope had significantly higher image contrast when compared to

Impact of out-of-pocket spending caps on financial burden of those with group health insurance.

PubMed

Riggs, Kevin R; Buttorff, Christine; Alexander, G Caleb

2015-05-01

The Affordable Care Act (ACA) mandates that all private health insurance include out-of-pocket spending caps. Insurance purchased through the ACA's Health Insurance Marketplace may qualify for income-based caps, whereas group insurance will not have income-based caps. Little is known about how out-of-pocket caps impact individuals' health care financial burden. We aimed to estimate what proportion of non-elderly individuals with group insurance will benefit from out-of-pocket caps, and the effect that various cap levels would have on their financial burden. We applied the expected uniform spending caps, hypothetical reduced uniform spending caps (reduced by one-third), and hypothetical income-based spending caps (similar to the caps on Health Insurance Marketplace plans) to nationally representative data from the Medical Expenditure Panel Survey (MEPS). Participants were non-elderly individuals (aged < 65 years) with private group health insurance in the 2011 and 2012 MEPS surveys (n =26,666). (1) The percentage of individuals with reduced family out-of-pocket spending as a result of the various caps; and (2) the percentage of individuals experiencing health care services financial burden (family out-of-pocket spending on health care, not including premiums, greater than 10% of total family income) under each scenario. With the uniform caps, 1.2% of individuals had lower out-of-pocket spending, compared with 3.8% with reduced uniform caps and 2.1% with income-based caps. Uniform caps led to a small reduction in percentage of individuals experiencing financial burden (from 3.3% to 3.1%), with a modestly larger reduction as a result of reduced uniform caps (2.9%) and income-based caps (2.8%). Mandated uniform out-of-pocket caps for those with group insurance will benefit very few individuals, and will not result in substantial reductions in financial burden.

Pocket facts 2008 : Swedish Road Administration, roads and traffic

DOT National Transportation Integrated Search

2008-05-01

Pocket Facts offers an overall picture of the road : transport system. Here you will find a selection of : brief facts about roads, road transports, vehicles and : people in traffic. In addition, it presents the functions : and organisation at the SR...

Portable Anthrax Testing with Lab-in-a-Pocket

ScienceCinema

Finley, Melissa; Koskelo, Markku; Edwards, Thayne

2018-05-30

BaDx (Bacillus anthracis Diagnostics) is a lab-in-a-pocket device to sample, sense, and diagnose bacteria that cause anthrax. It accomplishes these tasks in environments with no power, refrigerated storage, or laboratory equipment. BaDx was designed to be used with minimal or no training, and to keep handlers safe.

Portable Anthrax Testing with Lab-in-a-Pocket

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finley, Melissa; Koskelo, Markku; Edwards, Thayne

2014-10-24

BaDx (Bacillus anthracis Diagnostics) is a lab-in-a-pocket device to sample, sense, and diagnose bacteria that cause anthrax. It accomplishes these tasks in environments with no power, refrigerated storage, or laboratory equipment. BaDx was designed to be used with minimal or no training, and to keep handlers safe.

Out-of-pocket costs for childhood stroke: the impact of chronic illness on parents' pocketbooks.

PubMed

Plumb, Patricia; Seiber, Eric; Dowling, Michael M; Lee, JoEllen; Bernard, Timothy J; deVeber, Gabrielle; Ichord, Rebecca N; Bastian, Rachel; Lo, Warren D

2015-01-01

Direct costs for children who had stroke are similar to those for adults. There is no information regarding the out-of-pocket costs families encounter. We described the out-of-pocket costs families encountered in the first year after a child's ischemic stroke. Twenty-two subjects were prospectively recruited at four centers in the United States and Canada in 2008 and 2009 as part of the "Validation of the Pediatric NIH Stroke Scale" study; families' indirect costs were tracked for 1 year. Every 3 months, parents reported hours they did not work, nonreimbursed costs for medical visits or other health care, and mileage. They provided estimates of annual income. We calculated total out-of-pocket costs in US dollars and reported costs as a proportion of annual income. Total median out-of-pocket cost for the year after an ischemic stroke was $4354 (range, $0-$28,666; interquartile range, $1008-$8245). Out-of-pocket costs were greatest in the first 3 months after the incident stroke, with the largest proportion because of lost wages, followed by transportation, and nonreimbursed health care. For the entire year, median costs represented 6.8% (range, 0%-81.9%; interquartile range, 2.7%-17.2%) of annual income. Out-of-pocket expenses are significant after a child's ischemic stroke. The median costs are noteworthy provided that the median American household had cash savings of $3650 at the time of the study. These results with previous reports of direct costs provide a more complete view of the overall costs to families and society. Childhood stroke creates an under-recognized cost to society because of decreased parental productivity. Copyright © 2015 Elsevier Inc. All rights reserved.

Metal active site elasticity linked to activation of homocysteine in methionine synthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.

2008-04-02

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry uponmore » binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.« less

Three Dimensional Flow and Pressure Patterns in a Single Pocket of a Hydrostatic Journal Bearing

NASA Technical Reports Server (NTRS)

Braun, M. Jack; Dzodzo, Milorad B.

1996-01-01

The flow in a hydrostatic pocket is described by a mathematical model that uses the three dimensional Navier-Stokes equations written in terms of the primary variables, u, v, w, and p. Using a conservative formulation, a finite volume multi-block method is applied through a collocated, body fitted grid. The flow is simulated in a shallow pocket with a depth/length ratio of 0.02. The flow structures obtained and described by the authors in their previous two dimensional models are made visible in their three dimensional aspect for the Couette flow. It has been found that the flow regimes formed central and secondary vortical cells with three dimensional corkscrew-like structures that lead the fluid on an outward bound path in the axial direction of the pocket. The position of the central vortical cell center is at the exit region of the capillary restrictor feedline. It has also been determined that a fluid turn around zone occupies all the upstream space between the floor of the pocket and the runner, thus preventing any flow exit through the upstream port. The corresponding pressure distribution under the shaft presented as well. It was clearly established that for the Couette dominated case the pressure varies significantly in the pocket in the circumferential direction, while its variation is less pronounced axially.

Mental health care and out-of-pocket expenditures in Europe: results from the ESEMeD project.

PubMed

Sevilla-Dedieu, Christine; Kovess-Masfety, Viviane; Gilbert, Fabien; Vilagut, Gemma; Koenig, Hans-Helmut; Bruffaerts, Ronny; Haro, Josep Maria; Alonso, Jordi

2011-06-01

Most published data on out-of-pocket spending on mental health originate from the United States, where insurance payments for mental health have traditionally been much less generous than benefits for other health care services. Given the difference in the structure of health care funding in Europe, it is clearly important to obtain similar information on out-of-pocket expenditure in different European countries. To estimate out-of-pocket costs paid by people who receive mental health care in six European countries (Belgium, France, Germany, Italy, the Netherlands, and Spain). Of the 8,796 participants in a cross-sectional survey conducted in these six European countries, 1,128 reported having consulted a professional for a mental health problem in the year preceding the interview and provided information on how many times in the past year they consulted each type of provider, and the money they and their family members had paid out-of-pocket for their mental health care. In addition to sociodemographic characteristics, information on mental health status was collected using the CIDI 3.0. Descriptive statistics on out-of-pocket expenditure and share of income across countries were generated. Two-part models were employed to identify the relationship between the different covariates, notably the types of providers consulted, and out-of-pocket expenditure. Overall, 41.0% of those who used services for a mental health problem paid something for the care they received. This represented a minority of respondents in all countries except Belgium (87.9%) and Italy (61.7%). The financial burden of these costs relative to income was found to be low (1.2%), ranging from 0.4% in Germany to 2.3% in France. Out-of-pocket expenditure differed according to the type of providers consulted, with non-physician health professionals and medical specialists being more often associated with significant expenditure. Although the study is limited principally by data collection from self

Red Pine Pocket Mortality - Unknown Cause (Pest Alert)

Treesearch

USDA Forest Service

1985-01-01

Continuing mortality of red pine from an unknown cause has been observed in 30 to 40 year old plantations in southern and west central Wisconsin. A single tree or small group of trees die, followed by mortality of adjacent trees. These circular pockets of dead trees expand up to 0.3 acre per year.

A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansine site of tubulin.

PubMed

Menchon, Grégory; Prota, Andrea E; Lucena-Agell, Daniel; Bucher, Pascal; Jansen, Rolf; Irschik, Herbert; Müller, Rolf; Paterson, Ian; Díaz, J Fernando; Altmann, Karl-Heinz; Steinmetz, Michel O

2018-05-29

Microtubule-targeting agents (MTAs) like taxol and vinblastine are among the most successful chemotherapeutic drugs against cancer. Here, we describe a fluorescence anisotropy-based assay that specifically probes for ligands targeting the recently discovered maytansine site of tubulin. Using this assay, we have determined the dissociation constants of known maytansine site ligands, including the pharmacologically active degradation product of the clinical antibody-drug conjugate trastuzumab emtansine. In addition, we discovered that the two natural products spongistatin-1 and disorazole Z with established cellular potency bind to the maytansine site on β-tubulin. The high-resolution crystal structures of spongistatin-1 and disorazole Z in complex with tubulin allowed the definition of an additional sub-site adjacent to the pocket shared by all maytansine-site ligands, which could be exploitable as a distinct, separate target site for small molecules. Our study provides a basis for the discovery and development of next-generation MTAs for the treatment of cancer.

Quantum mechanical design of enzyme active sites.

PubMed

Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

2008-02-01

The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

Out-of-pocket healthcare payments on chronic conditions impoverish urban poor in Bangalore, India.

PubMed

Bhojani, Upendra; Thriveni, Bs; Devadasan, Roopa; Munegowda, Cm; Devadasan, Narayanan; Kolsteren, Patrick; Criel, Bart

2012-11-16

The burden of chronic conditions is on the rise in India, necessitating long-term support from healthcare services. Healthcare, in India, is primarily financed through out-of-pocket payments by households. Considering scarce evidence available from India, our study investigates whether and how out-of-pocket payments for outpatient care affect individuals with chronic conditions. A large census covering 9299 households was conducted in Bangalore, India. Of these, 3202 households that reported presence of chronic condition were further analysed. Data was collected using a structured household-level questionnaire. Out-of-pocket payments, catastrophic healthcare expenditure, and the resultant impoverishment were measured using a standard technique. The response rate for the census was 98.5%. Overall, 69.6% (95%CI=68.0-71.2) of households made out-of-pocket payments for outpatient care spending a median of 3.2% (95%CI=3.0-3.4) of their total income. Overall, 16% (95%CI=14.8-17.3) of households suffered financial catastrophe by spending more than 10% of household income on outpatient care. Occurrence and intensity of financial catastrophe were inequitably high among poor. Low household income, use of referral hospitals as place for consultation, and small household size were associated with a greater likelihood of incurring financial catastrophe.The out-of-pocket spending on chronic conditions doubled the number of people living below the poverty line in one month, with further deepening of their poverty. In order to cope, households borrowed money (4.2% instances), and sold or mortgaged their assets (0.4% instances). This study provides evidence from India that the out-of-pocket payment for chronic conditions, even for outpatient care, pushes people into poverty. Our findings suggest that improving availability of affordable medications and diagnostics for chronic conditions, as well as strengthening the gate keeping function of the primary care services are important

Out-of-pocket healthcare payments on chronic conditions impoverish urban poor in Bangalore, India

PubMed Central

2012-01-01

Background The burden of chronic conditions is on the rise in India, necessitating long-term support from healthcare services. Healthcare, in India, is primarily financed through out-of-pocket payments by households. Considering scarce evidence available from India, our study investigates whether and how out-of-pocket payments for outpatient care affect individuals with chronic conditions. Methods A large census covering 9299 households was conducted in Bangalore, India. Of these, 3202 households that reported presence of chronic condition were further analysed. Data was collected using a structured household-level questionnaire. Out-of-pocket payments, catastrophic healthcare expenditure, and the resultant impoverishment were measured using a standard technique. Results The response rate for the census was 98.5%. Overall, 69.6% (95%CI=68.0-71.2) of households made out-of-pocket payments for outpatient care spending a median of 3.2% (95%CI=3.0-3.4) of their total income. Overall, 16% (95%CI=14.8-17.3) of households suffered financial catastrophe by spending more than 10% of household income on outpatient care. Occurrence and intensity of financial catastrophe were inequitably high among poor. Low household income, use of referral hospitals as place for consultation, and small household size were associated with a greater likelihood of incurring financial catastrophe. The out-of-pocket spending on chronic conditions doubled the number of people living below the poverty line in one month, with further deepening of their poverty. In order to cope, households borrowed money (4.2% instances), and sold or mortgaged their assets (0.4% instances). Conclusions This study provides evidence from India that the out-of-pocket payment for chronic conditions, even for outpatient care, pushes people into poverty. Our findings suggest that improving availability of affordable medications and diagnostics for chronic conditions, as well as strengthening the gate keeping function of

Effect of soy addition on microwavable pocket-type flat doughs.

PubMed

Serventi, Luca; Sachleben, Joseph; Vodovotz, Yael

2011-01-01

Microwavable frozen baked goods are widely used by the food industry. However, the altered heat and mass transfer patterns associated with microwave radiation result in tough and rubbery baked products due to reduced plasticization of the polymers. Ingredients with high water-holding capacity and high content of polar lipids have been shown to enhance gluten plasticization and to improve water retention. Therefore, this study explored the physicochemical changes imparted by microwave baking of pocket-type flat doughs with and without soy added at 10%, 20%, and 26% and compared these to their conventionally baked counterparts. Microwave baking resulted in a soft, rubbery, and tough wheat product with increased "freezable" water. Soy was added to the formulation as a means to improve polymer plasticization. Conventional baking of soy doughs resulted in rubbery and tough products due to changes in water state and mobility (freezable water approximately 15 compared with 7.09 of the control). However, soy reduced the cohesiveness of the microwave baked products reaching the lowest value at 20% soy addition (cohesiveness 0.33 ± 1, comparable to that of the conventionally baked control). These data suggest that reduction of water mobility induced by soy proteins and polar lipids (confirmed by thermogravimetric analysis [TGA] and ¹H nuclear magnetic resonance [¹H NMR]) possibly plasticized the starch-gluten network of microwave baked soy doughs. Thus, soy was shown to improve the texture of microwave baked pocket-type flat doughs although further formula optimization is warranted. Microwavable pocket-type flat doughs are used frequently by the food industry to enrobe meat, vegetable, and sweet items for convenient meal delivery. Microwave heating of such doughs induces the development of crustless products compared to conventionally baked products, resulting in a tough and rubbery texture. Partial substitution of wheat flour with soy, in the form of soy flour and soy

Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor

PubMed Central

Maillet, Emeline L.; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Osman, Roman; Max, Marianna

2015-01-01

The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2’s VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. PMID:26377607

Pseudogap-generated a coexistence of Fermi arcs and Fermi pockets in cuprate superconductors

NASA Astrophysics Data System (ADS)

Zhao, Huaisong; Gao, Deheng; Feng, Shiping

2017-03-01

One of the most intriguing puzzle is why there is a coexistence of Fermi arcs and Fermi pockets in the pseudogap phase of cuprate superconductors? This puzzle is calling for an explanation. Based on the t - J model in the fermion-spin representation, the coexistence of the Fermi arcs and Fermi pockets in cuprate superconductors is studied by taking into account the pseudogap effect. It is shown that the pseudogap induces an energy band splitting, and then the poles of the electron Green's function at zero energy form two contours in momentum space, however, the electron spectral weight on these two contours around the antinodal region is gapped out by the pseudogap, leaving behind the low-energy electron spectral weight only located at the disconnected segments around the nodal region. In particular, the tips of these disconnected segments converge on the hot spots to form the closed Fermi pockets, generating a coexistence of the Fermi arcs and Fermi pockets. Moreover, the single-particle coherent weight is directly related to the pseudogap, and grows linearly with doping. The calculated result of the overall dispersion of the electron excitations is in qualitative agreement with the experimental data. The theory also predicts that the pseudogap-induced peak-dip-hump structure in the electron spectrum is absent from the hot-spot directions.

20. Living room, view to east wall and pocket doors ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

20. Living room, view to east wall and pocket doors to front parlor; view to east; 65mm lens with electronic flash illumination. - Warner Hutton House, 13495 Sousa Lane, Saratoga, Santa Clara County, CA

Comparison of stromal hydration techniques for clear corneal cataract incisions: conventional hydration versus anterior stromal pocket hydration.

PubMed

Mifflin, Mark D; Kinard, Krista; Neuffer, Marcus C

2012-06-01

Anterior stromal pocket hydration was compared with conventional hydration for preventing wound leak after 2.8 mm uniplanar clear corneal incisions (CCIs) in patients having routine cataract surgery. Conventional hydration involves hydration of the lateral walls of the main incision with visible whitening of the stroma. The anterior stromal pocket hydration technique involves creation of an additional supraincisional stromal pocket overlying the main incision, which is then hydrated instead of the main incision. Sixty-six eyes of 48 patients were included in the data analysis with 33 assigned to each study group. The anterior stromal pocket hydration technique was significantly better than conventional hydration in preventing wound leak due to direct pressure on the posterior lip of the incision. Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Changes in out-of-pocket charges associated with obstetric care provided under Medicare in Australia.

PubMed

Callander, Emily; Fox, Haylee

2018-06-01

Recent health reforms alongside unregulated provider fees have led to increased attention being given to out-of-pocket healthcare costs. This study utilised annual statistics published by the Department of Health for services provided under the Medicare Benefits Schedule (MBS) from 1992/3 to 2016/17 to identify changes in out-of-pocket charges for obstetric items over time, and estimate the change in demand for obstetric items in response to price increases. Since 1992/3 out-of-pocket charges increased by 1035% for out-of-hospital items and 77% for in-hospital items. Demand for obstetric items has reduced with increasing charges. © 2018 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists.

Chemistry and Microtextures of Melt Pockets in the Los Angeles Basaltic Shergottite

NASA Astrophysics Data System (ADS)

Walton, E. L.; Spray, J. G.

2002-01-01

Los Angeles is a non-brecciated, highly shocked basaltic shergottite. The shock effects in Los Angeles can be classified into two basic types: (1) mechanical deformations and transformations that take place essentially in the solid state, and (2) localized regions of shock melting forming glassy to microcrystal line enclaves. A brief description of the former type, based largely on optical observations, is given. The complete transformation of plagioclase to maskelynite, mosaicism and planar fracturing of clinopyroxenes, pyrrhotite veins within clinopyroxene, polycrystallinity of some pyrrhotite grains, and a 350 pinlong fault with approximately 15-micrometer displacement is described. These results are in agreement with this study. This work presents a detailed description of the shock-related microtexture and mineralogy of the localized melt pockets which have formed by in situ melting of local mineral phases. The melt pockets range from 3 mm x 3 min 0.07 mm x 1.25 mm, in direct contact with the host matrix. Two polished thin sections of stone 1 (1.3 cm x 1 cm, 2.1 cm x 1.2 cm) have been investigated to determine the mineralogy and microtextures of the shock-induced melt pockets using a JEOL 6400 digital scanning electron microscope equipped with a Link Analytical eXL energy dispersive spectrometer (EDS) fitted with a Si (Li) LZ-4 Pentafet detector, and a FEG-SEM equipped with the In-Lens Thermal FEG which produces high probe current sufficient for EBSP, WDS, and EDS. These instruments are capable of characterizing compositional variations and microtextures of the melt pockets that are unresolvable using optical techniques.

Exploration of the effect of sequence variations located inside the binding pocket of HIV-1 and HIV-2 proteases.

PubMed

Triki, Dhoha; Billot, Telli; Visseaux, Benoit; Descamps, Diane; Flatters, Delphine; Camproux, Anne-Claude; Regad, Leslie

2018-04-10

HIV-2 protease (PR2) is naturally resistant to most FDA (Food and Drug Administration)-approved HIV-1 protease inhibitors (PIs), a major antiretroviral class. In this study, we compared the PR1 and PR2 binding pockets extracted from structures complexed with 12 ligands. The comparison of PR1 and PR2 pocket properties showed that bound PR2 pockets were more hydrophobic with more oxygen atoms and fewer nitrogen atoms than PR1 pockets. The structural comparison of PR1 and PR2 pockets highlighted structural changes induced by their sequence variations and that were consistent with these property changes. Specifically, substitutions at residues 31, 46, and 82 induced structural changes in their main-chain atoms that could affect PI binding in PR2. In addition, the modelling of PR1 mutant structures containing V32I and L76M substitutions revealed a cooperative mechanism leading to structural deformation of flap-residue 45 that could modify PR2 flexibility. Our results suggest that substitutions in the PR1 and PR2 pockets can modify PI binding and flap flexibility, which could underlie PR2 resistance against PIs. These results provide new insights concerning the structural changes induced by PR1 and PR2 pocket variation changes, improving the understanding of the atomic mechanism of PR2 resistance to PIs.

Case of pacemaker pocket infection caused by Finegoldia magna.

PubMed

Hosseini Dehkordi, Seyed Hamed; Osorio, Georgina

2017-10-01

Finegoldia magna (formerly called Peptostreptococcus magnus) is a Gram-positive anaerobic coccus which is increasingly recognized as an opportunistic pathogen. We present a case of F. magna associated non-valvular cardiovascular device-related infection in an 83 year-old male who received a permanent pacemaker for sick sinus syndrome seven weeks prior to his presentation. Five weeks after the implantation, the pacemaker and leads were explanted because of clinical evidence of pacemaker pocket infection. He was initially treated with sulfamethoxazole-trimethoprim based on the Gram stain results from the removed pacemaker. However, two weeks later, he was readmitted with sepsis and was successfully treated with ampicillin-sulbactam. Culture results from the pacemaker and pocket as well as blood cultures grew F. magna. Clinicians should be aware of the possibility of F. magna infection when initial gram stain results show "gram positive cocci". Copyright © 2017 Elsevier Ltd. All rights reserved.

53. Retail Pockets, Looking West, date unknown Historic Photograph, Photogapher ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

53. Retail Pockets, Looking West, date unknown Historic Photograph, Photogapher Unknown; Collection of William Everett, Jr. (Wilkes-Barre, PA), photocopy by Joseph E.B. Elliot - Huber Coal Breaker, 101 South Main Street, Ashley, Luzerne County, PA

7. DETAIL VIEW OF ROCKER ARM, SHOWING POCKETS, LUGS, INCLINED ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

7. DETAIL VIEW OF ROCKER ARM, SHOWING POCKETS, LUGS, INCLINED STOPPING BLOCK AT SHOREWARD END OF TRACK GIRDER - Seddon Island Scherzer Rolling Lift Bridge, Spanning Garrison Channel from Tampa to Seddon Island, Tampa, Hillsborough County, FL

Correlates of out-of-pocket and catastrophic health expenditures in Tanzania: results from a national household survey.

PubMed

Brinda, Ethel Mary; Andrés, Antonio Rodríguez; Andrés, Rodriguez Antonio; Enemark, Ulrika

2014-03-05

Inequality in health services access and utilization are influenced by out-of-pocket health expenditures in many low and middle-income countries (LMICs). Various antecedents such as social factors, poor health and economic factors are proposed to direct the choice of health care service use and incurring out-of-pocket payments. We investigated the association of these factors with out-of-pocket health expenditures among the adult and older population in the United Republic of Tanzania. We also investigated the prevalence and associated determinants contributing to household catastrophic health expenditures. We accessed the data of a multistage stratified random sample of 7279 adult participants, aged between 18 and 59 years, as well as 1018 participants aged above 60 years, from the first round of the Tanzania National Panel survey. We employed multiple generalized linear and logistic regression models to evaluate the correlates of out-of-pocket as well as catastrophic health expenditures, accounting for the complex sample design effects. Increasing age, female gender, obesity and functional disability increased the adults' out-of-pocket health expenditures significantly, while functional disability and visits to traditional healers increased the out-of-pocket health expenditures in older participants. Adult participants, who lacked formal education or worked as manual laborers earned significantly less (p < 0.001) and spent less on health (p < 0.001), despite having higher levels of disability. Large household size, household head's occupation as a manual laborer, household member with chronic illness, domestic violence against women and traditional healer's visits were significantly associated with high catastrophic health expenditures. We observed that the prevalence of inequalities in socioeconomic factors played a significant role in determining the nature of both out-of-pocket and catastrophic health expenditures. We propose that investment in social welfare

Skin lesions over the pocket area that may mimic cardiac implantable electronic device infection: A case series.

PubMed

Korantzopoulos, Panagiotis; Plakoutsi, Sofia; Florou, Elizabeth; Bechlioulis, Aris

2018-05-21

The early and correct diagnosis of cardiac implantable electronic device (CIED) infections is critical given that early aggressive treatment with complete removal of the system along with antimicrobial therapy dramatically improves outcomes. Pocket infection manifested by local signs of inflammation is the most common form of CIED infections. Conditions mimicking pocket infection have been described in the literature. These include various types of malignancy and rarely allergic reactions/contact dermatitis to pacemaker compounds. We aimed to describe skin lesions and disorders over the pocket area that mimic CIED infection. We present a series of 5 cases with skin lesions that mimic pocket infection. We document these cases with corresponding photographs. Most of them have not been described in this setting. We report the following cases of conditions that proved not to be CIED infection: One case of superficial cellulitis, one case of herpes zoster over the pocket area, one case of spontaneous bruising over the pocket a long time after implantation in a patient taking oral anticoagulation, and 2 cases of contact dermatitis due to prolonged postoperative application of povidone iodine. All cases had favorable outcome after conservative treatment and no CIED infection was developed during follow-up. Clinicians should be aware of rare conditions that mimic CIED infection. Incorrect diagnosis of these disorders may falsely lead to CIED extraction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Women Saw Large Decrease In Out-Of-Pocket Spending For Contraceptives After ACA Mandate Removed Cost Sharing.

PubMed

Becker, Nora V; Polsky, Daniel

2015-07-01

The Affordable Care Act mandates that private health insurance plans cover prescription contraceptives with no consumer cost sharing. The positive financial impact of this new provision on consumers who purchase contraceptives could be substantial, but it has not yet been estimated. Using a large administrative claims data set from a national insurer, we estimated out-of-pocket spending before and after the mandate. We found that mean and median per prescription out-of-pocket expenses have decreased for almost all reversible contraceptive methods on the market. The average percentages of out-of-pocket spending for oral contraceptive pill prescriptions and intrauterine device insertions by women using those methods both dropped by 20 percentage points after implementation of the ACA mandate. We estimated average out-of-pocket savings per contraceptive user to be $248 for the intrauterine device and $255 annually for the oral contraceptive pill. Our results suggest that the mandate has led to large reductions in total out-of-pocket spending on contraceptives and that these price changes are likely to be salient for women with private health insurance. Project HOPE—The People-to-People Health Foundation, Inc.

Novobiocin and additional inhibitors of the Hsp90 C-terminal nucleotide-binding pocket.

PubMed

Donnelly, Alison; Blagg, Brian S J

2008-01-01

The 90 kDa heat shock proteins (Hsp90), which are integrally involved in cell signaling, proliferation, and survival, are ubiquitously expressed in cells. Many proteins in tumor cells are dependent upon the Hsp90 protein folding machinery for their stability, refolding, and maturation. Inhibition of Hsp90 uniquely targets client proteins associated with all six hallmarks of cancer. Thus, Hsp90 has emerged as a promising target for the treatment of cancer. Hsp90 exists as a homodimer, which contains three domains. The N-terminal domain contains an ATP-binding site that binds the natural products geldanamycin and radicicol. The middle domain is highly charged and has high affinity for co-chaperones and client proteins. Initial studies by Csermely and co-workers suggested a second ATP-binding site in the C-terminus of Hsp90. This C-terminal nucleotide binding pocket has been shown to not only bind ATP, but cisplatin, novobiocin, epilgallocatechin-3-gallate (EGCG) and taxol. The coumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 were isolated from several streptomyces strains and exhibit potent activity against Gram-positive bacteria. These compounds bind type II topoisomerases, including DNA gyrase, and inhibit the enzyme-catalyzed hydrolysis of ATP. As a result, novobiocin analogues have garnered the attention of numerous researchers as an attractive agent for the treatment of bacterial infection. Novobiocin was reported to bind weakly to the newly discovered Hsp90 C-terminal ATP binding site ( approximately 700 M in SkBr3 cells) and induce degradation of Hsp90 client proteins. Structural modification of this compound has led to an increase of 1000-fold in activity in anti-proliferative assays. Recent studies of structure-activity relationship (SAR) by Renoir and co-workers highlighted the crucial role of the C-4 and/or C-7 positions of the coumarin and removal of the noviose moiety, which appeared to be essential for degradation of Hsp90 client

How Medicaid Expansion Affected Out-of-Pocket Health Care Spending for Low-Income Families.

PubMed

Glied, Sherry; Chakraborty, Ougni; Russo, Therese

2017-08-01

ISSUE. Prior research shows that low-income residents of states that expanded Medicaid under the Affordable Care Act are less likely to experience financial barriers to health care access, but the impact on out-of-pocket spending has not yet been measured. GOAL. Assess how the Medicaid expansion affected out-of-pocket health care spending for low-income families compared to those in states that did not expand and consider whether effects differed in states that expanded under conventional Medicaid rules vs. waiver programs. METHODS. Analysis of the Consumer Expenditure Survey 2010–2015. KEY FINDINGS AND CONCLUSIONS. Compared to families in nonexpansion states, low-income families in states that did expand Medicaid saved an average of $382 in annual spending on health care. In these states, low-income families were less like to report any out-of-pocket spending on insurance premiums or medical care than were similar families in nonexpansion states. For families that did have some out-of-pocket spending, spending levels were lower in states that expanded Medicaid. Low-income families in Medicaid expansion states were also much less likely to have catastrophically high spending levels. The form of coverage expansion — conventional Medicaid or waiver rules — did not have a statistically significant effect on these outcomes.

O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7.

PubMed

Shadrina, Maria S; Peslherbe, Gilles H; English, Ann M

2015-09-01

Hemoglobin transports O2 by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O2 significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O2 to the solvent via a transient E7 channel. To reveal the O2 escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O2 diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7's conformation, we observe the same four or five escape routes leading directly from the α- or β-distal heme pockets to the solvent. Only 21-53% of O2 escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O2 from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O2 escapes to the solvent directly from the α- or β-distal pockets of the mutant increases to 70-88%. In contrast to O2, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes.

Household out-of-pocket medical expenditures and national health insurance in Taiwan: income and regional inequality

PubMed Central

Chu, Tu-Bin; Liu, Tsai-Ching; Chen, Chin-Shyan; Tsai, Yi-Wen; Chiu, Wen-Ta

2005-01-01

Background Unequal geographical distribution of medical care resources and insufficient healthcare coverage have been two long-standing problems with Taiwan's public health system. The implementation of National Health Insurance (NHI) attempted to mitigate the inequality in health care use. This study examines the degree to which Taiwan's National Health Insurance (NHI) has reduced out-of-pocket medical expenditures in households in different regions and varying levels of income. Methods Data used in this study were drawn from the 1994 and 1996 Surveys of Family Income and Expenditure. We pooled the data from 1994 and 1996 and included a year dummy variable (NHI), equal to 1 if the household data came from 1996 in order to assess the impact of NHI on household out-of-pocket medical care expenditures shortly after its implementation in 1995. Results An individual who was older, female, married, unemployed, better educated, richer, head of a larger family household, or living in the central and eastern areas was more likely to have greater household out-of-pocket medical expenditures. NHI was found to have effectively reduced household out-of-pocket medical expenditures by 23.08%, particularly for more affluent households. With the implementation of NHI, lower and middle income quintiles had smaller decreases in out-of-pocket medical expenditure. NHI was also found to have reduced household out-of-pocket medical expenditures more for households in eastern Taiwan. Conclusion Although NHI was established to create free medical care for all, further effort is needed to reduce the medical costs for certain disadvantaged groups, particularly the poor and aborigines, if equality is to be achieved. PMID:16137336

Out-of-pocket payments for health care services in Bulgaria: financial burden and barrier to access.

PubMed

Atanasova, Elka; Pavlova, Milena; Moutafova, Emanuela; Rechel, Bernd; Groot, Wim

2013-12-01

In recent years, Bulgaria has increasingly relied on out-of-pocket payments as one of the main sources of health care financing. However, it is largely unknown whether the official patient charges, combined with informal payments, are affordable for the population. Our study aimed to explore the scale of out-of-pocket payments for health care services and their affordability. Data were collected in two nationally representative surveys, conducted in Bulgaria in 2010 and 2011, using face-to-face interviews based on a standardized questionnaire. To select respondents, a multi-stage random probability method was used. The questionnaire included questions on the out-of-pocket payments for health care services used by the respondent during the preceding 12 months. In total, 75.7% (2010) and 84.0% (2011) of outpatient service users reported to have paid out-of-pocket, with 12.6% (2010) and 9.7% (2011) of users reporting informal payments. Of those who had used inpatient services, 66.5% (2010) and 63.1% (2011) reported to have made out-of-pocket payments, with 31.8% (2010) and 18.3% (2011) reporting to have paid informally. We found large inability to pay indicated by the need to borrow money and/or forego services. Regression analysis showed that the inability to pay is especially pronounced among those with poor health status and chronic diseases and those on low household incomes. The high level of both formal and informal out-of-pocket payments for health care services in Bulgaria poses a considerable burden for households and undermines access to health services for poorer parts of the population.

Active and regulatory sites of cytosolic 5'-nucleotidase.

PubMed

Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

2010-12-01

Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation. © 2010 The Authors Journal compilation © 2010 FEBS.

Water in the Active Site of Ketosteroid Isomerase

PubMed Central

Hanoian, Philip; Hammes-Schiffer, Sharon

2011-01-01

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two waters in the Y16S mutant, one water in the Y16F and FFF mutants, and intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of 1H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less

Water in the active site of ketosteroid isomerase.

PubMed

Hanoian, Philip; Hammes-Schiffer, Sharon

2011-08-09

Classical molecular dynamics simulations were utilized to investigate the structural and dynamical properties of water in the active site of ketosteroid isomerase (KSI) to provide insight into the role of these water molecules in the enzyme-catalyzed reaction. This reaction is thought to proceed via a dienolate intermediate that is stabilized by hydrogen bonding with residues Tyr16 and Asp103. A comparative study was performed for the wild-type (WT) KSI and the Y16F, Y16S, and Y16F/Y32F/Y57F (FFF) mutants. These systems were studied with three different bound ligands: equilenin, which is an intermediate analog, and the intermediate states of two steroid substrates. Several distinct water occupation sites were identified in the active site of KSI for the WT and mutant systems. Three additional sites were identified in the Y16S mutant that were not occupied in WT KSI or the other mutants studied. The number of water molecules directly hydrogen bonded to the ligand oxygen was approximately two in the Y16S mutant and one in the Y16F and FFF mutants, with intermittent hydrogen bonding of one water molecule in WT KSI. The molecular dynamics trajectories of the Y16F and FFF mutants reproduced the small conformational changes of residue 16 observed in the crystal structures of these two mutants. Quantum mechanical/molecular mechanical calculations of (1)H NMR chemical shifts of the protons in the active site hydrogen-bonding network suggest that the presence of water in the active site does not prevent the formation of short hydrogen bonds with far-downfield chemical shifts. The molecular dynamics simulations indicate that the active site water molecules exchange much more frequently for WT KSI and the FFF mutant than for the Y16F and Y16S mutants. This difference is most likely due to the hydrogen-bonding interaction between Tyr57 and an active site water molecule that is persistent in the Y16F and Y16S mutants but absent in the FFF mutant and significantly less probable

A Novel AS1411 Aptamer-Based Three-Way Junction Pocket DNA Nanostructure Loaded with Doxorubicin for Targeting Cancer Cells in Vitro and in Vivo.

PubMed

Taghdisi, Seyed Mohammad; Danesh, Noor Mohammad; Ramezani, Mohammad; Yazdian-Robati, Rezvan; Abnous, Khalil

2018-05-07

Active targeting of nanostructures containing chemotherapeutic agents can improve cancer treatment. Here, a three-way junction pocket DNA nanostructure was developed for efficient doxorubicin (Dox) delivery into cancer cells. The three-way junction pocket DNA nanostructure is composed of three strands of AS1411 aptamer as both a therapeutic aptamer and nucleolin target, the potential biomarker of prostate (PC-3 cells) and breast (4T1 cells) cancers. The properties of the Dox-loaded three-way junction pocket DNA nanostructure were characterized and verified to have several advantages, including high serum stability and a pH-responsive property. Cellular uptake studies showed that the Dox-loaded DNA nanostructure was preferably internalized into target cancer cells (PC-3 and 4T1 cells). MTT cell viability assay demonstrated that the Dox-loaded DNA nanostructure had significantly higher cytotoxicity for PC-3 and 4T1 cells compared to that of nontarget cells (CHO cells, Chinese hamster ovary cell). The in vivo antitumor effect showed that the Dox-loaded DNA nanostructure was more effective in prohibition of the tumor growth compared to free Dox. These findings showed that the Dox-loaded three-way junction pocket DNA nanostructure could significantly reduce the cytotoxic effects of Dox against nontarget cells.

Apolar Distal Pocket Mutants of Yeast Cytochrome c Peroxidase: Hydrogen Peroxide Reactivity and Cyanide Binding of the TriAla, TriVal, and TriLeu Variants

PubMed Central

Bidwai, Anil K.; Meyen, Cassandra; Kilheeney, Heather; Wroblewski, Damian; Vitello, Lidia B.; Erman, James E.

2012-01-01

Three yeast cytochrome c peroxidase (CcP) variants with apolar distal heme pockets have been constructed. The CcP variants have Arg48, Trp51, and His52 mutated to either all alanines, CcP(triAla), all valines, CcP(triVal), or all leucines, CcP(triLeu). The triple mutants have detectable enzymatic activity at pH 6 but the activity is less than 0.02% that of wild-type CcP. The activity loss is primarily due to the decreased rate of reaction between the triple mutants and H2O2 compared to wild-type CcP. Spectroscopic properties and cyanide binding characteristics of the triple mutants have been investigated over the pH stability region of CcP, pH 4 to 8. The absorption spectra indicate that the CcP triple mutants have hemes that are predominantly five-coordinate, high-spin at pH 5 and six-coordinate, low-spin at pH 8. Cyanide binding to the triple mutants is biphasic indicating that the triple mutants have two slowly-exchanging conformational states with different cyanide affinities. The binding affinity for cyanide is reduced at least two orders of magnitude in the triple mutants compared to wild-type CcP and the rate of cyanide binding is reduced by four to five orders of magnitude. Correlation of the reaction rates of CcP and 12 distal pocket mutants with H2O2 and HCN suggests that both reactions require ionization of the reactants within the distal heme pocket allowing the anion to bind the heme iron. Distal pocket features that promote substrate ionization (basic residues involved in base-catalyzed substrate ionization or polar residues that can stabilize substrate anions) increase the overall rate of reaction with H2O2 and HCN while features that inhibit substrate ionization slow the reactions. PMID:23022490

Modeling of ultrasound transmission through a solid-liquid interface comprising a network of gas pockets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paumel, K.; Baque, F.; Moysan, J.

Ultrasonic inspection of sodium-cooled fast reactor requires a good acoustic coupling between the transducer and the liquid sodium. Ultrasonic transmission through a solid surface in contact with liquid sodium can be complex due to the presence of microscopic gas pockets entrapped by the surface roughness. Experiments are run using substrates with controlled roughness consisting of a network of holes and a modeling approach is then developed. In this model, a gas pocket stiffness at a partially solid-liquid interface is defined. This stiffness is then used to calculate the transmission coefficient of ultrasound at the entire interface. The gas pocket stiffnessmore » has a static, as well as an inertial component, which depends on the ultrasonic frequency and the radiative mass.« less

Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

PubMed

Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

2016-09-01

A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

Half-of-the-sites reactivity of outer-membrane phospholipase A against an active-site-directed inhibitor.

PubMed

Ubarretxena-Belandia, I; Cox, R C; Dijkman, R; Egmond, M R; Verheij, H M; Dekker, N

1999-03-01

The reaction of a novel active-site-directed phospholipase A1 inhibitor with the outer-membrane phospholipase A (OMPLA) was investigated. The inhibitor 1-p-nitrophenyl-octylphosphonate-2-tridecylcarbamoyl-3-et hanesulfonyl -amino-3-deoxy-sn-glycerol irreversibly inactivated OMPLA. The inhibition reaction did not require the cofactor calcium or an unprotonated active-site His142. The inhibition of the enzyme solubilized in hexadecylphosphocholine micelles was characterized by a rapid (t1/2 = 20 min) and complete loss of enzymatic activity, concurrent with the covalent modification of 50% of the active-site serines, as judged from the amount of p-nitrophenolate (PNP) released. Modification of the remaining 50% occurred at a much lower rate, indicative of half-of-the-sites reactivity against the inhibitor of this dimeric enzyme. Inhibition of monomeric OMPLA solubilized in hexadecyl-N,N-dimethyl-1-ammonio-3-propanesulfonate resulted in an equimolar monophasic release of PNP, concurrent with the loss of enzymatic activity (t1/2 = 14 min). The half-of-the-sites reactivity is discussed in view of the dimeric nature of this enzyme.

Physical activity and cancer risk: dose-response and cancer, all sites and site-specific.

PubMed

Thune, I; Furberg, A S

2001-06-01

The association between physical activity and overall and site-specific cancer risk is elaborated in relation to whether any observed dose-response association between physical activity and cancer can be interpreted in terms of how much physical activity (type, intensity, duration, frequency) is needed to influence site- and gender-specific cancer risk. Observational studies were reviewed that have examined the independent effect of the volume of occupational physical activity (OPA) and/or leisure time physical activity (LPA) on overall and site-specific cancer risk. The evidence of cohort and case-control studies suggests that both leisure time and occupational physical activity protect against overall cancer risk, with a graded dose-response association suggested in both sexes. Confounding effects such as diet, body weight, and parity are often included as a covariate in the analyses, with little influence on the observed associations. A crude graded inverse dose-response association was observed between physical activity and colon cancer in 48 studies including 40,674 colon/colorectal cancer cases for both sexes. A dose-response effect of physical activity on colon cancer risk was especially observed, when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed as MET-hours per week. An observed inverse association with a dose-response relationship between physical activity and breast cancer was also identified in the majority of the 41 studies including 108,031 breast cancer cases. The dose-response relationship was in particular observed in case-control studies and supported by observations in cohort studies when participation in activities of at least moderate activity (>4.5 MET) and demonstrated by activities expressed by MET-hours per week. This association between physical activity and breast cancer risk is possibly dependent on age at exposure, age at diagnosis, menopausal status and other effect

An alginate-antacid formulation localizes to the acid pocket to reduce acid reflux in patients with gastroesophageal reflux disease.

PubMed

Rohof, Wout O; Bennink, Roel J; Smout, Andre J P M; Thomas, Edward; Boeckxstaens, Guy E

2013-12-01

Alginate rafts (polysaccharide polymers that precipitate into a low-density viscous gel when they contact gastric acid) have been reported to form at the acid pocket, an unbuffered pool of acid that floats on top of ingested food and causes postprandial acid reflux. We studied the location of an alginate formulation in relation to the acid pocket and the corresponding effects on reflux parameters and acid pocket positioning in patients with gastroesophageal reflux disease (GERD). We randomly assigned patients with symptomatic GERD and large hiatal hernias to groups who were given either (111)In-labeled alginate-antacid (n = 8, Gaviscon Double Action Liquid) or antacid (n = 8, Antagel) after a standard meal. The relative positions of labeled alginate and acid pocket were analyzed for 2 hours by using scintigraphy; reflux episodes were detected by using high-resolution manometry and pH-impedance monitoring. The alginate-antacid label localized to the acid pocket. The number of acid reflux episodes was significantly reduced in patients receiving alginate-antacid (3.5; range, 0-6.5; P = .03) compared with those receiving antacid (15; range, 5-20), whereas time to acid reflux was significantly increased in patients receiving alginate-antacid (63 minutes; range, 23-92) vs those receiving antacid (14 minutes; range, 9-23; P = .01). The acid pocket was located below the diaphragm in 71% of patients given alginate-antacid vs 21% of those given antacid (P = .08). There was an inverse correlation between a subdiaphragm position of the acid pocket and acid reflux (r = -0.76, P < .001). In a study of 16 patients with GERD, we observed that the alginate-antacid raft localizes to the postprandial acid pocket and displaces it below the diaphragm to reduce postprandial acid reflux. These findings indicate the importance of the acid pocket in GERD pathogenesis and establish alginate-antacid as an appropriate therapy for postprandial acid reflux. Copyright © 2013 AGA Institute

Active Site Metal Occupancy and Cyclic Di-GMP Phosphodiesterase Activity of Thermotoga maritima HD-GYP.

PubMed

Miner, Kyle D; Kurtz, Donald M

2016-02-16

HD-GYPs make up a subclass of the metal-dependent HD phosphohydrolase superfamily and catalyze conversion of cyclic di(3',5')-guanosine monophosphate (c-di-GMP) to 5'-phosphoguanylyl-(3'→5')-guanosine (pGpG) and GMP. Until now, the only reported crystal structure of an HD-GYP that also exhibits c-di-GMP phosphodiesterase activity contains a His/carboxylate ligated triiron active site. However, other structural and phylogenetic correlations indicate that some HD-GYPs contain dimetal active sites. Here we provide evidence that an HD-GYP c-di-GMP phosphodiesterase, TM0186, from Thermotoga maritima can accommodate both di- and trimetal active sites. We show that an as-isolated iron-containing TM0186 has an oxo/carboxylato-bridged diferric site, and that the reduced (diferrous) form is necessary and sufficient to catalyze conversion of c-di-GMP to pGpG, but that conversion of pGpG to GMP requires more than two metals per active site. Similar c-di-GMP phosphodiesterase activities were obtained with divalent iron or manganese. On the basis of activity correlations with several putative metal ligand residue variants and molecular dynamics simulations, we propose that TM0186 can accommodate both di- and trimetal active sites. Our results also suggest that a Glu residue conserved in a subset of HD-GYPs is required for formation of the trimetal site and can also serve as a labile ligand to the dimetal site. Given the anaerobic growth requirement of T. maritima, we suggest that this HD-GYP can function in vivo with either divalent iron or manganese occupying di- and trimetal sites.

Deciphering Cryptic Binding Sites on Proteins by Mixed-Solvent Molecular Dynamics.

PubMed

Kimura, S Roy; Hu, Hai Peng; Ruvinsky, Anatoly M; Sherman, Woody; Favia, Angelo D

2017-06-26

In recent years, molecular dynamics simulations of proteins in explicit mixed solvents have been applied to various problems in protein biophysics and drug discovery, including protein folding, protein surface characterization, fragment screening, allostery, and druggability assessment. In this study, we perform a systematic study on how mixtures of organic solvent probes in water can reveal cryptic ligand binding pockets that are not evident in crystal structures of apo proteins. We examine a diverse set of eight PDB proteins that show pocket opening induced by ligand binding and investigate whether solvent MD simulations on the apo structures can induce the binding site observed in the holo structures. The cosolvent simulations were found to induce conformational changes on the protein surface, which were characterized and compared with the holo structures. Analyses of the biological systems, choice of probes and concentrations, druggability of the resulting induced pockets, and application to drug discovery are discussed here.

Out of Pocket Payment for Obstetrical Complications: A Cost Analysis Study in Iran

PubMed Central

Yavangi, Mahnaz; Sohrabi, Mohammad Reza; Riazi, Sahand

2013-01-01

Background: This study was conducted to determine the total expenditure and out of pocket payment on pregnancy complications in Tehran, the capital of Iran. Methods: A cross-sectional study conducted on 1172 patients who admitted in two general teaching referral Hospitals in Tehran. In this study, we calculated total and out of pocket inpatient costs for seven pregnancy complications including preeclampsia, intrauterine growth restriction (IUGR), abortion, ante-partum hemorrhage, preterm delivery, premature rupture of membranes and post-dated pregnancy. We used descriptive analysis and analysis of variance test to compare these pregnancy complications. Results: The average duration of hospitalization was 3.28 days and the number of visits by physicians for a patient was 9.79 on average. The average total cost for these pregnancy complications was 735.22 Unites States Dollars (USD) (standard deviation [SD] = 650.53). The average out of packet share was 277.08 USD (SD = 350.74), which was 37.69% of total expenditure. IUGR with payment of 398.76 USD (SD = 418.54) (52.06% of total expenditure) had the greatest amount of out of pocket expenditure in all complications. While, abortion had the minimum out of pocket amount that was 148.77 USD (SD = 244.05). Conclusions: Obstetrics complications had no catastrophic effect on families, but IUGR cost was about 30% of monthly household non-food costs in Tehran so more financial protection plans and insurances are recommended for these patients. PMID:24404365

An experimental study of geyser-like flows induced by a pressurized air pocket

NASA Astrophysics Data System (ADS)

Elayeb, I. S.; Leon, A.; Choi, Y.; Alnahit, A. O.

2015-12-01

Previous studies argues that the entrapment of pressurized air pockets within combined sewer systems can produce geyser flows, which is an oscillating jetting of a mixture of gas-liquid flows. To verify that pressurized air pockets can effectively produce geysers, laboratory experiments were conducted. However, past experiments were conducted in relatively small-scale apparatus (i.e. maximum φ2" vertical shaft). This study conducted a set of experiments in a larger apparatus. The experimental setup consists of an upstream head tank, a downstream head tank, a horizontal pipe (46.5ft long, φ6") and a vertical pipe (10ft long, φ6"). The initial condition for the experiments is constant flow discharge through the horizontal pipe. The experiments are initiated by injecting an air pocket with pre-determined volume and pressure at the upstream end of the horizontal pipe. The air pocket propagates through the horizontal pipe until it arrives to the vertical shaft, where it is released producing a geyser-like flow. Three flow rates in the horizontal pipe and three injected air pressures were tested. The variables measured were pressure at two locations in the horizontal pipe and two locations in the vertical pipe. High resolution videos at two regions in the vertical shaft were also recorded. To gain further insights in the physics of air-water interaction, the laboratory experiments were complemented with numerical simulations conducted using a commercial 3D CFD model, previously validated with experiments.

Simulation of Population Processes with a Programmable Pocket Calculator.

ERIC Educational Resources Information Center

Kidd, N. A. C.

1979-01-01

Presents a set of simulation models for use in teaching population dynamics. These models are designed specifically for use with a programmable pocket calculator, and can be used to demonstrate growth of populations with discrete or overlapping generations and also to explore effects of density-dependent and -independent mortality. (Author/CS)

Nearshore hydrodynamics at pocket beaches with contrasting wave exposure in southern Portugal

NASA Astrophysics Data System (ADS)

Horta, João; Oliveira, Sónia; Moura, Delminda; Ferreira, Óscar

2018-05-01

Pocket beaches on rocky coasts with headlands that control hydro-sedimentary processes are considered to be constrained sedimentary systems, generally with limited sediment inputs. Pocket beaches face severe changes over time. Under worst-case scenarios, these changes can result in the loss of the beach, causing waves to directly attack adjacent cliffs. Studies of nearshore hydrodynamics can help to understand such changes and optimise sediment nourishment procedures. The present work contributes to the knowledge of hydrodynamic forcing mechanisms at pocket beaches by providing a comprehensive description of the nearshore circulation at two beaches with contrasting wave exposures. Two pocket beaches in southern Portugal were studied by combining field measurements of waves and currents with numerical models (STWAVE and BOUSS-2D). The aim of this analysis was to evaluate nearshore hydrodynamics under different wave exposure forcing conditions (e.g. variable wave heights/directions and different tidal levels). The results show that the beach circulation can rapidly shift from longshore-to rip-dominated depending on changes in both the offshore wave direction and tidal levels. Waves with higher obliquity (for both low and moderate wave energy conditions) tend to generate longshore circulation in all considered tidal stages, while waves with lower obliquity tend to produce rip flow with higher-velocity rip currents during low to intermediate tidal stages. The results indicate that the location and intensity of rip currents strongly depend on geomorphological constraints, that is, the control exerted by shore platforms. A larger morphological control is observed at mean sea level because most platforms are submerged/exposed during high/low tide and therefore exert less control on nearshore circulation.

78 FR 54214 - Endangered and Threatened Wildlife and Plants; Removing Five Subspecies of Mazama Pocket Gopher...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-09-03

... section 3 of the Act, an endangered species is any species which is in danger of extinction throughout all... populations, do not occur elsewhere. Our Response: The presumption of extinction for the Tacoma pocket gopher... contrast to our presumption of extinction for the Tacoma pocket gopher, which is based on evidence from...

External protons destabilize the activated voltage sensor in hERG channels.

PubMed

Shi, Yu Patrick; Cheng, Yen May; Van Slyke, Aaron C; Claydon, Tom W

2014-03-01

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

Diffusional correlations among multiple active sites in a single enzyme.

PubMed

Echeverria, Carlos; Kapral, Raymond

2014-04-07

Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

Discovery of Novel Nonactive Site Inhibitors of the Prothrombinase Enzyme Complex.

PubMed

Kapoor, Karan; McGill, Nicole; Peterson, Cynthia B; Meyers, Harold V; Blackburn, Michael N; Baudry, Jerome

2016-03-28

The risk of serious bleeding is a major liability of anticoagulant drugs that are active-site competitive inhibitors targeting the Factor Xa (FXa) prothrombin (PT) binding site. The present work identifies several new classes of small molecule anticoagulants that can act as nonactive site inhibitors of the prothrombinase (PTase) complex composed of FXa and Factor Va (FVa). These new classes of anticoagulants were identified, using a novel agnostic computational approach to identify previously unrecognized binding pockets at the FXa-FVa interface. From about three million docking calculations of 281,128 compounds in a conformational ensemble of FXa heavy chains identified by molecular dynamics (MD) simulations, 97 compounds and their structural analogues were selected for experimental validation, through a series of inhibition assays. The compound selection was based on their predicted binding affinities to FXa and their ability to successfully bind to multiple protein conformations while showing selectivity for particular binding sites at the FXa/FVa interface. From these, thirty-one (31) compounds were experimentally identified as nonactive site inhibitors. Concentration-based assays further identified 10 compounds represented by four small-molecule families of inhibitors that achieve dose-independent partial inhibition of PTase activity in a nonactive site-dependent and self-limiting mechanism. Several compounds were identified for their ability to bind to protein conformations only seen during MD, highlighting the importance of accounting for protein flexibility in structure-based drug discovery approaches.

Pathological periodontal pockets are associated with raised diastolic blood pressure in obese adolescents.

PubMed

Zeigler, Cecilia C; Wondimu, Biniyam; Marcus, Claude; Modéer, Thomas

2015-03-24

Obesity, a well-known risk factor for developing cardiovascular disease (CVD), is associated with chronic periodontitis in adults. This cross-sectional pilot study on obese adolescents was designed to investigate whether periodontal disease in terms of pathological periodontal pockets is associated with raised blood pressure and other risk markers for CVD. The study included 75 obese subjects between 12 to 18 years of age, mean 14.5. Subjects answered a questionnaire regarding health, oral hygiene habits and sociodemographic factors. A clinical examination included Visible Plaque Index (VPI %), Gingival inflammation (BOP %) and the occurrence of pathological pockets exceeding 4 mm (PD ≥ 4 mm). Blood serum were collected and analyzed. The systolic and diastolic blood pressures were registered. Adolescents with pathological periodontal pockets (PD ≥ 4 mm; n = 14) had significantly higher BOP >25% (P = 0.002), higher diastolic blood pressure (P = 0.008), higher levels of Interleukin (IL)-6 (P < 0.001), Leptin (P = 0.018), Macrophage Chemoattractant Protein-1 (MCP-1) (P = 0.049) and thyroid stimulating hormone (TSH) (P = 0.004) in blood serum compared with subjects without pathological periodontal pockets (PD ≥ 4 mm; n = 61). The bivariate linear regression analysis demonstrated that PD ≥ 4 mm (P = 0.008) and systolic blood pressure (P < 0.001) were significantly associated with the dependent variable "diastolic blood pressure". The association between PD ≥ 4 mm and diastolic blood pressure remained significant (P = 0.006) even after adjusting for potential confounders BMI-sds, age, gender, mother's country of birth, BOP >25%, IL-6, IL-8, Leptin, MCP-1, TSH and total cholesterol in the multiple regression analysis. In conclusion, this study indicates an association between pathological periodontal pockets and diastolic blood pressure in obese adolescents. The association was unaffected by other risk markers for cardiovascular events or periodontal disease

Out-of-pocket health expenditures and antimicrobial resistance in low- and middle-income countries

PubMed Central

Alsan, Marcella; Schoemaker, Lena; Eggleston, Karen; Kammili, Nagamani; Kolli, Prasanthi; Bhattacharya, Jay

2015-01-01

Background The decreasing effectiveness of antimicrobial agents is a growing global public health concern. Low- and middle-income countries (LMIC) are vulnerable to the loss of antimicrobial efficacy given their high burden of infectious disease and the cost of treating resistant organisms. Methods We analyzed data from the World Health Organization’s Antibacterial Resistance Global Surveillance Report. We investigated the importance of out-of-pocket spending and copayment requirements for public sector medications on the level of bacterial resistance among LMIC, adjusting for environmental factors purported to be predictors of resistance, such as sanitation, animal husbandry and poverty as well as other structural components of the health sector. Findings Out-of-pocket health expenditures were the only factor demonstrating a statistically significant relationship with antimicrobial resistance. A ten point increase in the percentage of health expenditures that were out-of-pocket was associated with a 3·2 percentage point increase in resistant isolates [95% CI, 1·17 to 5·15, p-value=0·002]. This relationship was driven by countries requiring copayments for medications in the public health sector. Among these countries, moving from the 20th to 80th percentile of out-of-pocket health expenditures was associated with an increase in resistant bacterial isolates from 17·76 [95%CI 12·54 to 22·97] to 36·27 percentage points [95% CI 31·16 to 41·38]. Interpretation Out-of-pocket health expenditures were strongly correlated with antimicrobial resistance among LMIC. This relationship was driven by countries that require copayments on medications in the public sector. Our findings suggest cost-sharing of antimicrobials in the public sector may drive demand to the private sector where supply-side incentives to overprescribe are likely heightened and quality assurance less standardized. PMID:26164481

Structural basis of enzymatic activity for the ferulic acid decarboxylase (FADase) from Enterobacter sp. Px6-4.

PubMed

Gu, Wen; Yang, Jinkui; Lou, Zhiyong; Liang, Lianming; Sun, Yuna; Huang, Jingwen; Li, Xuemei; Cao, Yi; Meng, Zhaohui; Zhang, Ke-Qin

2011-01-21

Microbial ferulic acid decarboxylase (FADase) catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol) via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD) superfamily. Structural analysis revealed that FADase catalyzed reactions by an "open-closed" mechanism involving a pocket of 8 × 8 × 15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer.

Formation of a repressive complex in the mammalian circadian clock is mediated by the secondary pocket of CRY1

DOE PAGES

Michael, Alicia K.; Fribourgh, Jennifer L.; Chelliah, Yogarany; ...

2017-01-31

The basic helix-loop-helix PAS domain (bHLH-PAS) transcription factor CLOCK:BMAL1 (brain and muscle Arnt-like protein 1) sits at the core of the mammalian circadian transcription/translation feedback loop. Precise control of CLOCK:BMAL1 activity by coactivators and repressors establishes the ~24-h periodicity of gene expression. Formation of a repressive complex, defined by the core clock proteins cryptochrome 1 (CRY1):CLOCK:BMAL1, plays an important role controlling the switch from repression to activation each day. Here in this paper, we show that CRY1 binds directly to the PAS domain core of CLOCK: BMAL1, driven primarily by interaction with the CLOCK PAS-B domain. Integrative modeling and solutionmore » X-ray scattering studies unambiguously position a key loop of the CLOCK PAS-B domain in the secondary pocket of CRY1, analogous to the antenna chromophore-binding pocket of photolyase. CRY1 docks onto the transcription factor alongside the PAS domains, extending above the DNA-binding bHLH domain. Single point mutations at the interface on either CRY1 or CLOCK disrupt formation of the ternary complex, highlighting the importance of this interface for direct regulation of CLOCK:BMAL1 activity by CRY1.« less

Fabrication and Testing of a Modular Micro-Pocket Fission Detector Instrumentation System for Test Nuclear Reactors

NASA Astrophysics Data System (ADS)

Reichenberger, Michael A.; Nichols, Daniel M.; Stevenson, Sarah R.; Swope, Tanner M.; Hilger, Caden W.; Roberts, Jeremy A.; Unruh, Troy C.; McGregor, Douglas S.

2018-01-01

Advancements in nuclear reactor core modeling and computational capability have encouraged further development of in-core neutron sensors. Measurement of the neutron-flux distribution within the reactor core provides a more complete understanding of the operating conditions in the reactor than typical ex-core sensors. Micro-Pocket Fission Detectors have been developed and tested previously but have been limited to single-node operation and have utilized highly specialized designs. The development of a widely deployable, multi-node Micro-Pocket Fission Detector assembly will enhance nuclear research capabilities. A modular, four-node Micro-Pocket Fission Detector array was designed, fabricated, and tested at Kansas State University. The array was constructed from materials that do not significantly perturb the neutron flux in the reactor core. All four sensor nodes were equally spaced axially in the array to span the fuel-region of the reactor core. The array was filled with neon gas, serving as an ionization medium in the small cavities of the Micro-Pocket Fission Detectors. The modular design of the instrument facilitates the testing and deployment of numerous sensor arrays. The unified design drastically improved device ruggedness and simplified construction from previous designs. Five 8-mm penetrations in the upper grid plate of the Kansas State University TRIGA Mk. II research nuclear reactor were utilized to deploy the array between fuel elements in the core. The Micro-Pocket Fission Detector array was coupled to an electronic support system which has been specially developed to support pulse-mode operation. The Micro-Pocket Fission Detector array composed of four sensors was used to monitor local neutron flux at a constant reactor power of 100 kWth at different axial locations simultaneously. The array was positioned at five different radial locations within the core to emulate the deployment of multiple arrays and develop a 2-dimensional measurement of

Evaluation of Cation Hydrolysis Schemes with a Pocket Calculator.

ERIC Educational Resources Information Center

Clare, Brian W.

1979-01-01

Described is the use of two models of pocket calculators. The Hewlett-Packard HP67 and the Texas Instruments TI59, to solve problems arising in connection with ionic equilibria in solution. A three-parameter regression program is described and listed as a specific example, the hydrolysis of hexavalent uranium, is provided. (BT)

How the ACA's Health Insurance Expansions Have Affected Out-of-Pocket Cost-Sharing and Spending on Premiums.

PubMed

Glied, Sherry; Solís-Román, Claudia; Parikh, Shivani

2016-09-01

One important benefit gained by the millions of Americans with health insurance through the Affordable Care Act (ACA) is protection from high out-of-pocket health spending. While Medicaid unambiguously reduces out-of-pocket premium and medical costs for low-income people, it is less certain that marketplace coverage and other types of insurance purchased to comply with the law's individual mandate also protect from high health spending. Goal: To compare out-of-pocket spending in 2014 to spending in 2013; assess how this spending changed in states where many people enrolled in the marketplaces relative to states where few people enrolled; and project the decline in the percentage of people paying high amounts out-of-pocket. Methods: Linear regression models were used to estimate whether people under age 65 spent above certain thresholds. Key findings and conclusions: The probability of incurring high out-of-pocket costs and premium expenses declined as marketplace enrollment increased. The percentage reductions were greatest among those with incomes between 250 percent and 399 percent of poverty, those who were eligible for premium subsidies, and those who previously were uninsured or had very limited nongroup coverage. These effects appear largely attributable to marketplace enrollment rather than to other ACA provisions or to economic trends.

Impacts of chronic non-communicable diseases on households' out-of-pocket healthcare expenditures in Sri Lanka.

PubMed

Pallegedara, Asankha

2018-01-10

This article examines the effects of chronic non-communicable diseases (NCDs) on households' out-of-pocket health expenditures in Sri Lanka. We explore the disease specific impacts on out-of-pocket health care expenses from chronic NCDs such as heart diseases, hypertension, cancer, diabetics and asthma. We use nationwide cross-sectional household income and expenditure survey 2012/2013 data compiled by the department of census and statistics of Sri Lanka. Employing propensity score matching method to account for selectivity bias, we find that chronic NCD affected households appear to spend significantly higher out-of-pocket health care expenditures and encounter grater economic burden than matched control group despite having universal public health care policy in Sri Lanka. The results also suggest that out-of-pocket expenses on medicines and other pharmaceutical products as well as expenses on medical laboratory tests and other ancillary services are particularly higher for households with chronic NCD patients. The findings underline the importance of protecting households against the financial burden due to NCDs.

POOL server: machine learning application for functional site prediction in proteins.

PubMed

Somarowthu, Srinivas; Ondrechen, Mary Jo

2012-08-01

We present an automated web server for partial order optimum likelihood (POOL), a machine learning application that combines computed electrostatic and geometric information for high-performance prediction of catalytic residues from 3D structures. Input features consist of THEMATICS electrostatics data and pocket information from ConCavity. THEMATICS measures deviation from typical, sigmoidal titration behavior to identify functionally important residues and ConCavity identifies binding pockets by analyzing the surface geometry of protein structures. Both THEMATICS and ConCavity (structure only) do not require the query protein to have any sequence or structure similarity to other proteins. Hence, POOL is applicable to proteins with novel folds and engineered proteins. As an additional option for cases where sequence homologues are available, users can include evolutionary information from INTREPID for enhanced accuracy in site prediction. The web site is free and open to all users with no login requirements at http://www.pool.neu.edu. m.ondrechen@neu.edu Supplementary data are available at Bioinformatics online.

Pocket formula for nuclear deformations of actinides

NASA Astrophysics Data System (ADS)

Manjunatha, H. C.; Sridhar, K. N.

2018-06-01

We have formulated a pocket formula for quadrupole (β2), octupole (β3), hexadecapole (β4) and hexacontatetrapole (β6) deformation of the nuclear ground state of all isotopes of actinide nuclei (89 < Z < 103). This formula is first of its kind and produces a nuclear deformation of all isotopes actinide nuclei 89 < Z < 103 with simple inputs of Z and A. Hence, this formula is useful in the fields of nuclear physics to study the structure and interaction of nuclei.

Discovery of the ammonium substrate site on glutamine synthetase, a third cation binding site.

PubMed Central

Liaw, S. H.; Kuo, I.; Eisenberg, D.

1995-01-01

because ADP binding induces movement of Asp 50' toward this monovalent cation site, essentially forming the site. This observation supports a two-step mechanism with ordered substrate binding: ATP first binds to GS, then Glu binds and attacks ATP to form gamma-glutamyl phosphate and ADP, which complete the ammonium binding site. The third substrate, an ammonium ion, then binds to GS, and then loses a proton to form the more active species ammonia, which attacks the gamma-glutamyl phosphate to yield Gln. (5) Because the products (Glu or Gln) of the reactions catalyzed by GS are determined by the molecule (water or ammonium) attacking the intermediate gamma-glutamyl phosphate, this negatively charged ammonium binding pocket has been designed naturally for high affinity of ammonium to GS, permitting glutamine synthesis to proceed in aqueous solution. PMID:8563633

How Accurate Is Your Activity Tracker? A Comparative Study of Step Counts in Low-Intensity Physical Activities

PubMed Central

2017-01-01

Background As commercially available activity trackers are being utilized in clinical trials, the research community remains uncertain about reliability of the trackers, particularly in studies that involve walking aids and low-intensity activities. While these trackers have been tested for reliability during walking and running activities, there has been limited research on validating them during low-intensity activities and walking with assistive tools. Objective The aim of this study was to (1) determine the accuracy of 3 Fitbit devices (ie, Zip, One, and Flex) at different wearing positions (ie, pants pocket, chest, and wrist) during walking at 3 different speeds, 2.5, 5, and 8 km/h, performed by healthy adults on a treadmill; (2) determine the accuracy of the mentioned trackers worn at different sites during activities of daily living; and (3) examine whether intensity of physical activity (PA) impacts the choice of optimal wearing site of the tracker. Methods We recruited 15 healthy young adults to perform 6 PAs while wearing 3 Fitbit devices (ie, Zip, One, and Flex) on their chest, pants pocket, and wrist. The activities include walking at 2.5, 5, and 8 km/h, pushing a shopping cart, walking with aid of a walker, and eating while sitting. We compared the number of steps counted by each tracker with gold standard numbers. We performed multiple statistical analyses to compute descriptive statistics (ie, ANOVA test), intraclass correlation coefficient (ICC), mean absolute error rate, and correlation by comparing the tracker-recorded data with that of the gold standard. Results All the 3 trackers demonstrated good-to-excellent (ICC>0.75) correlation with the gold standard step counts during treadmill experiments. The correlation was poor (ICC<0.60), and the error rate was significantly higher in walker experiment compared to other activities. There was no significant difference between the trackers and the gold standard in the shopping cart experiment. The wrist

A comprehensive cold gas performance study of the Pocket Rocket radiofrequency electrothermal microthruster

NASA Astrophysics Data System (ADS)

Ho, Teck Seng; Charles, Christine; Boswell, Roderick W.

2016-12-01

This paper presents computational fluid dynamics simulations of the cold gas operation of Pocket Rocket and Mini Pocket Rocket radiofrequency electrothermal microthrusters, replicating experiments performed in both sub-Torr and vacuum environments. This work takes advantage of flow velocity choking to circumvent the invalidity of modelling vacuum regions within a CFD simulation, while still preserving the accuracy of the desired results in the internal regions of the microthrusters. Simulated results of the plenum stagnation pressure is in precise agreement with experimental measurements when slip boundary conditions with the correct tangential momentum accommodation coefficients for each gas are used. Thrust and specific impulse is calculated by integrating the flow profiles at the exit of the microthrusters, and are in good agreement with experimental pendulum thrust balance measurements and theoretical expectations. For low thrust conditions where experimental instruments are not sufficiently sensitive, these cold gas simulations provide additional data points against which experimental results can be verified and extrapolated. The cold gas simulations presented in this paper will be used as a benchmark to compare with future plasma simulations of the Pocket Rocket microthruster.

Direct instrumental identification of catalytically active surface sites

NASA Astrophysics Data System (ADS)

Pfisterer, Jonas H. K.; Liang, Yunchang; Schneider, Oliver; Bandarenka, Aliaksandr S.

2017-09-01

The activity of heterogeneous catalysts—which are involved in some 80 per cent of processes in the chemical and energy industries—is determined by the electronic structure of specific surface sites that offer optimal binding of reaction intermediates. Directly identifying and monitoring these sites during a reaction should therefore provide insight that might aid the targeted development of heterogeneous catalysts and electrocatalysts (those that participate in electrochemical reactions) for practical applications. The invention of the scanning tunnelling microscope (STM) and the electrochemical STM promised to deliver such imaging capabilities, and both have indeed contributed greatly to our atomistic understanding of heterogeneous catalysis. But although the STM has been used to probe and initiate surface reactions, and has even enabled local measurements of reactivity in some systems, it is not generally thought to be suited to the direct identification of catalytically active surface sites under reaction conditions. Here we demonstrate, however, that common STMs can readily map the catalytic activity of surfaces with high spatial resolution: we show that by monitoring relative changes in the tunnelling current noise, active sites can be distinguished in an almost quantitative fashion according to their ability to catalyse the hydrogen-evolution reaction or the oxygen-reduction reaction. These data allow us to evaluate directly the importance and relative contribution to overall catalyst activity of different defects and sites at the boundaries between two materials. With its ability to deliver such information and its ready applicability to different systems, we anticipate that our method will aid the rational design of heterogeneous catalysts.

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations1

PubMed Central

Just, Victoria J.; Burrell, Matthew R.; Bowater, Laura; McRobbie, Iain; Stevenson, Clare E. M.; Lawson, David M.; Bornemann, Stephen

2007-01-01

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Δ162–163 or Δ162–164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Δ162–163 and S161A) strongly support the hypothesis that site 2 is purely structural. PMID:17680775

Role of active site rigidity in activity: MD simulation and fluorescence study on a lipase mutant.

PubMed

Kamal, Md Zahid; Mohammad, Tabrez Anwar Shamim; Krishnamoorthy, G; Rao, Nalam Madhusudhana

2012-01-01

Relationship between stability and activity of enzymes is maintained by underlying conformational flexibility. In thermophilic enzymes, a decrease in flexibility causes low enzyme activity while in less stable proteins such as mesophiles and psychrophiles, an increase in flexibility is associated with enhanced enzyme activity. Recently, we identified a mutant of a lipase whose stability and activity were enhanced simultaneously. In this work, we probed the conformational dynamics of the mutant and the wild type lipase, particularly flexibility of their active site using molecular dynamic simulations and time-resolved fluorescence techniques. In contrast to the earlier observations, our data show that active site of the mutant is more rigid than wild type enzyme. Further investigation suggests that this lipase needs minimal reorganization/flexibility of active site residues during its catalytic cycle. Molecular dynamic simulations suggest that catalytically competent active site geometry of the mutant is relatively more preserved than wild type lipase, which might have led to its higher enzyme activity. Our study implies that widely accepted positive correlation between conformation flexibility and enzyme activity need not be stringent and draws attention to the possibility that high enzyme activity can still be accomplished in a rigid active site and stable protein structures. This finding has a significant implication towards better understanding of involvement of dynamic motions in enzyme catalysis and enzyme engineering through mutations in active site.

Out-of-pocket costs of HAART limit HIV treatment responses in Botswana's private sector.

PubMed

Bisson, Gregory P; Frank, Ian; Gross, Robert; Lo Re, Vincent; Strom, Jordan B; Wang, Xingmei; Mogorosi, Mpho; Gaolathe, Tendani; Ndwapi, Ndwapi; Friedman, Harvey; Strom, Brian L; Dickinson, Diana

2006-06-12

A large number of HIV-infected patients in sub-Saharan Africa pay out-of-pocket for HAART. This analysis from Botswana indicates that higher median out-of-pocket regimen costs to patients for the initial 30 days of HAART are associated with failure to achieve a viral load< 400 copies/ml [US$32; interquartile range (IQR), 20-84 compared with US$22; (IQR, 17-36), P = 0.001]. HAART costs should be minimized as scale-up efforts in sub-Saharan Africa progress.

Correlates of out-of-pocket and catastrophic health expenditures in Tanzania: results from a national household survey

PubMed Central

2014-01-01

Background Inequality in health services access and utilization are influenced by out-of-pocket health expenditures in many low and middle-income countries (LMICs). Various antecedents such as social factors, poor health and economic factors are proposed to direct the choice of health care service use and incurring out-of-pocket payments. We investigated the association of these factors with out-of-pocket health expenditures among the adult and older population in the United Republic of Tanzania. We also investigated the prevalence and associated determinants contributing to household catastrophic health expenditures. Methods We accessed the data of a multistage stratified random sample of 7279 adult participants, aged between 18 and 59 years, as well as 1018 participants aged above 60 years, from the first round of the Tanzania National Panel survey. We employed multiple generalized linear and logistic regression models to evaluate the correlates of out-of-pocket as well as catastrophic health expenditures, accounting for the complex sample design effects. Results Increasing age, female gender, obesity and functional disability increased the adults’ out-of-pocket health expenditures significantly, while functional disability and visits to traditional healers increased the out-of-pocket health expenditures in older participants. Adult participants, who lacked formal education or worked as manual laborers earned significantly less (p < 0.001) and spent less on health (p < 0.001), despite having higher levels of disability. Large household size, household head’s occupation as a manual laborer, household member with chronic illness, domestic violence against women and traditional healer’s visits were significantly associated with high catastrophic health expenditures. Conclusion We observed that the prevalence of inequalities in socioeconomic factors played a significant role in determining the nature of both out-of-pocket and catastrophic health

A pocket-sized metabolic analyzer for assessment of resting energy expenditure.

PubMed

Zhao, Di; Xian, Xiaojun; Terrera, Mirna; Krishnan, Ranganath; Miller, Dylan; Bridgeman, Devon; Tao, Kevin; Zhang, Lihua; Tsow, Francis; Forzani, Erica S; Tao, Nongjian

2014-04-01

The assessment of metabolic parameters related to energy expenditure has a proven value for weight management; however these measurements remain too difficult and costly for monitoring individuals at home. The objective of this study is to evaluate the accuracy of a new pocket-sized metabolic analyzer device for assessing energy expenditure at rest (REE) and during sedentary activities (EE). The new device performs indirect calorimetry by measuring an individual's oxygen consumption (VO2) and carbon dioxide production (VCO2) rates, which allows the determination of resting- and sedentary activity-related energy expenditure. VO2 and VCO2 values of 17 volunteer adult subjects were measured during resting and sedentary activities in order to compare the metabolic analyzer with the Douglas bag method. The Douglas bag method is considered the Gold Standard method for indirect calorimetry. Metabolic parameters of VO2, VCO2, and energy expenditure were compared using linear regression analysis, paired t-tests, and Bland-Altman plots. Linear regression analysis of measured VO2 and VCO2 values, as well as calculated energy expenditure assessed with the new analyzer and Douglas bag method, had the following linear regression parameters (linear regression slope LRS0, and R-squared coefficient, r(2)) with p = 0: LRS0 (SD) = 1.00 (0.01), r(2) = 0.9933 for VO2; LRS0 (SD) = 1.00 (0.01), r(2) = 0.9929 for VCO2; and LRS0 (SD) = 1.00 (0.01), r(2) = 0.9942 for energy expenditure. In addition, results from paired t-tests did not show statistical significant difference between the methods with a significance level of α = 0.05 for VO2, VCO2, REE, and EE. Furthermore, the Bland-Altman plot for REE showed good agreement between methods with 100% of the results within ±2SD, which was equivalent to ≤10% error. The findings demonstrate that the new pocket-sized metabolic analyzer device is accurate for determining VO2, VCO2, and energy expenditure. Copyright © 2013 Elsevier Ltd and

Impact of the federal contraceptive coverage guarantee on out-of-pocket payments for contraceptives: 2014 update.

PubMed

Sonfield, Adam; Tapales, Athena; Jones, Rachel K; Finer, Lawrence B

2015-01-01

The Affordable Care Act requires most private health plans to cover contraceptive methods, services and counseling, without any out-of-pocket costs to patients; that requirement took effect for millions of Americans in January 2013. Data for this study come from a subset of the 1842 women aged 18-39 years who responded to all four waves of a national longitudinal survey. This analysis focuses on the 892 women who had private health insurance and who used a prescription contraceptive method during any of the four study periods. Women were asked about the amount they paid out of pocket in an average month for their method of choice. Between fall 2012 and spring 2014, the proportion of privately insured women paying zero dollars out of pocket for oral contraceptives increased substantially, from 15% to 67%. Similar changes occurred among privately insured women using injectable contraception, the vaginal ring and the intrauterine device. The implementation of the federal contraceptive coverage requirement appears to have had a notable impact on the out-of-pocket costs paid by privately insured women, and that impact has increased over time. This study measures the out-of-pocket costs for women with private insurance prior to the federal contraceptive coverage requirement and after it took effect; in doing so, it highlights areas of progress in eliminating these costs. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

52. View Looking East from Power Plant Silos, Retail Pockets ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

52. View Looking East from Power Plant Silos, Retail Pockets under Construction, dated 2 October 1956 Historic Photograph, Photographer Unknown; Collection of William Everett, Jr. (Wilkes-Barre, PA), photocopy by Joseph E.B. Elliot - Huber Coal Breaker, 101 South Main Street, Ashley, Luzerne County, PA

Characterization of molecular determinants of the conformational stability of macrophage migration inhibitory factor: leucine 46 hydrophobic pocket.

PubMed

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

PubMed Central

El-Turk, Farah; Fauvet, Bruno; Ashrafi, Amer; Ouertatani-Sakouhi, Hajer; Cho, Min-Kyu; Neri, Marilisa; Cascella, Michele; Rothlisberger, Ursula; Pojer, Florence; Zweckstetter, Markus; Lashuel, Hilal

2012-01-01

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state. PMID:23028743

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 8 2011-07-01 2011-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 8 2010-07-01 2010-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

40 CFR 61.154 - Standard for active waste disposal sites.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing waste...

Solvation of excess electrons trapped in charge pockets on molecular surfaces

NASA Astrophysics Data System (ADS)

Jalbout, Abraham F.

This work considers the ability of hydrogen fluoride (HF) to solvate excess electrons located on cyclic hydrocarbon surfaces. The principle applied involves the formation of systems in which excess electrons can be stabilized not only on concentrated molecular surface charge pockets but also by HF. Recent studies have shown that OH groups can form stable hydrogen-bonded networks on one side of a hydrocarbon surface (i.e. cyclohexane sheets), at the same time, the hydrogen atoms on the opposite side of this surface form a pocket of positive charge can attract the excess electron. This density can be further stabilized by the addition of an HF molecule that can form an 'anion with an internally solvated electron' (AISE) state. These systems are shown to be stable with respect to vertical electron detachment (VDE).

Glycosyltransfer in mutants of putative catalytic residue Glu303 of the human ABO(H) A and B blood group glycosyltransferases GTA and GTB proceeds through a labile active site.

PubMed

Blackler, Ryan J; Gagnon, Susannah M L; Polakowski, Robert; Rose, Natisha L; Zheng, Ruixiang B; Letts, James A; Johal, Asha R; Schuman, Brock; Borisova, Svetlana N; Palcic, Monica M; Evans, Stephen V

2017-04-01

The homologous glycosyltransferases α-1,3-N-acetylgalactosaminyltransferase (GTA) and α-1,3-galactosyltransferase (GTB) carry out the final synthetic step of the closely related human ABO(H) blood group A and B antigens. The catalytic mechanism of these model retaining enzymes remains under debate, where Glu303 has been suggested to act as a putative nucleophile in a double displacement mechanism, a local dipole stabilizing the intermediate in an orthogonal associative mechanism or a general base to stabilize the reactive oxocarbenium ion-like intermediate in an SNi-like mechanism. Kinetic analysis of GTA and GTB point mutants E303C, E303D, E303Q and E303A shows that despite the enzymes having nearly identical sequences, the corresponding mutants of GTA/GTB have up to a 13-fold difference in their residual activities relative to wild type. High-resolution single crystal X-ray diffraction studies reveal, surprisingly, that the mutated Cys, Asp and Gln functional groups are no more than 0.8 Å further from the anomeric carbon of donor substrate compared to wild type. However, complicating the analysis is the observation that Glu303 itself plays a critical role in maintaining the stability of a strained "double-turn" in the active site through several hydrogen bonds, and any mutation other than E303Q leads to significantly higher thermal motion or even disorder in the substrate recognition pockets. Thus, there is a remarkable juxtaposition of the mutants E303C and E303D, which retain significant activity despite disrupted active site architecture, with GTB/E303Q, which maintains active site architecture but exhibits zero activity. These findings indicate that nucleophilicity at position 303 is more catalytically valuable than active site stability and highlight the mechanistic elasticity of these enzymes. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

MCNP6 simulated performance of Micro-Pocket Fission Detectors (MPFDs) in the Transient REActor Test (TREAT) Facility

DOE PAGES

Reichenberger, Michael A.; Patel, Vishal K.; Roberts, Jeremy A.; ...

2017-03-03

Here, Micro-Pocket Fission Detectors (MPFDs) are under development for in-core neutron flux measurements at the Transient REActor Test facility (TREAT) and in other experiments at Idaho National Laboratory (INL). The sensitivity of MPFDs to the energy dependent neutron flux at TREAT has been determined for 0.0300-μm thick active material coatings of 242Pu, 232Th, natural uranium, and 93% enriched 235U. Self-shielding effects in the active material of the MPFD was also confirmed to be negligible. Finally, fission fragment energy deposition was found to be in conformance with previously reported results.

Effects of Common Pesticides on Prostaglandin D2 (PGD2) Inhibition in SC5 Mouse Sertoli Cells, Evidence of Binding at the COX-2 Active Site, and Implications for Endocrine Disruption.

PubMed

Kugathas, Subramaniam; Audouze, Karine; Ermler, Sibylle; Orton, Frances; Rosivatz, Erika; Scholze, Martin; Kortenkamp, Andreas

2016-04-01

There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects. We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis. Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket. The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos-methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances-o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)-showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor. Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrine-disrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action. Kugathas S, Audouze K, Ermler S, Orton F, Rosivatz E

Effects of Common Pesticides on Prostaglandin D2 (PGD2) Inhibition in SC5 Mouse Sertoli Cells, Evidence of Binding at the COX-2 Active Site, and Implications for Endocrine Disruption

PubMed Central

Kugathas, Subramaniam; Audouze, Karine; Ermler, Sibylle; Orton, Frances; Rosivatz, Erika; Scholze, Martin; Kortenkamp, Andreas

2015-01-01

Background: There are concerns that diminished prostaglandin action in fetal life could increase the risk of congenital malformations. Many endocrine-disrupting chemicals have been found to suppress prostaglandin synthesis, but to our knowledge, pesticides have never been tested for these effects. Objectives: We assessed the ability of pesticides that are commonly used in the European Union to suppress prostaglandin D2 (PGD2) synthesis. Methods: Changes in PGD2 secretion in juvenile mouse Sertoli cells (SC5 cells) were measured using an ELISA. Coincubation with arachidonic acid (AA) was conducted to determine the site of action in the PGD2 synthetic pathway. Molecular modeling studies were performed to assess whether pesticides identified as PGD2-active could serve as ligands of the cyclooxygenase-2 (COX-2) binding pocket. Results: The pesticides boscalid, chlorpropham, cypermethrin, cyprodinil, fenhexamid, fludioxonil, imazalil (enilconazole), imidacloprid, iprodione, linuron, methiocarb, o-phenylphenol, pirimiphos-methyl, pyrimethanil, and tebuconazole suppressed PGD2 production. Strikingly, some of these substances—o-phenylphenol, cypermethrin, cyprodinil, linuron, and imazalil (enilconazole)—showed potencies (IC50) in the range between 175 and 1,500 nM, similar to those of analgesics intended to block COX enzymes. Supplementation with AA failed to reverse this effect, suggesting that the sites of action of these pesticides are COX enzymes. The molecular modeling studies revealed that the COX-2 binding pocket can accommodate most of the pesticides shown to suppress PGD2 synthesis. Some of these pesticides are also capable of antagonizing the androgen receptor. Conclusions: Chemicals with structural features more varied than previously thought can suppress PGD2 synthesis. Our findings signal a need for in vivo studies to establish the extent of endocrine-disrupting effects that might arise from simultaneous interference with PGD2 signaling and androgen action

Characteristics and tolerances of the pocket mouse and incidence of disease. [CNS lesions during space flights

NASA Technical Reports Server (NTRS)

Lindberg, R. G.; Kraft, L. M.; Simmonds, R. C.; Bailey, O. T.; Dunlap, W. A.; Haymaker, W.

1975-01-01

Studies carried out on the pocket mouse colony on Apollo XVII are reported. They revealed no serological evidence of viral disease, no pathogenic enterobacteria or respiratory Mycoplasma on culture, a 25% incidence of sarcosporidiosis, and a 2% incidence of chronic meningitis or meningoencephalitis. It is concluded that the pocket mouse is a highly adaptive animal and very well-suited to space flight.

An Exploratory Examination of "Pockets of Success" in Creating Urban High Schools of Opportunity for LSES Students

ERIC Educational Resources Information Center

Gaines, Frank

2013-01-01

The purpose of this exploratory study was to examine "pockets of success" through the voices of participant stakeholders in low socio-economic status urban high schools and communities to identify opportunities and structures that can improve post-secondary outcomes for students. Examining those pockets of success to rise above the…

Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

PubMed Central

Smith, Catherine E.; Mendillo, Marc L.; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S.; Desai, Arshad; Putnam, Christopher D.; Kolodner, Richard D.

2013-01-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway. PMID:24204293

Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

PubMed

Smith, Catherine E; Mendillo, Marc L; Bowen, Nikki; Hombauer, Hans; Campbell, Christopher S; Desai, Arshad; Putnam, Christopher D; Kolodner, Richard D

2013-10-01

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

Out-of-pocket medical expenses for inpatient care among beneficiaries of the National Health Insurance Program in the Philippines.

PubMed

Tobe, Makoto; Stickley, Andrew; del Rosario, Rodolfo B; Shibuya, Kenji

2013-08-01

OBJECTIVE The National Health Insurance Program (NHIP) in the Philippines is a social health insurance system partially subsidized by tax-based financing which offers benefits on a fee-for-service basis up to a fixed ceiling. This paper quantifies the extent to which beneficiaries of the NHIP incur out-of-pocket expenses for inpatient care, and examines the characteristics of beneficiaries making these payments and the hospitals in which these payments are typically made. METHODS Probit and ordinary least squares regression analyses were carried out on 94 531 insurance claims from Benguet province and Baguio city during the period 2007 to 2009. RESULTS Eighty-six per cent of claims involved an out-of-pocket payment. The median figure for out-of-pocket payments was Philippine Pesos (PHP) 3016 (US$67), with this figure varying widely [inter-quartile range (IQR): PHP 9393 (US$209)]. Thirteen per cent of claims involved very large out-of-pocket payments exceeding PHP 19 213 (US$428)-the equivalent of 10% of the average annual household income in the region. Membership type, disease severity, age and residential location of the patient, length of hospitalization, and ownership and level of the hospital were all significantly associated with making out-of-pocket payments and/or the size of these payments. CONCLUSION Although the current NHIP reduces the size of out-of-pocket payments, NHIP beneficiaries are not completely free from the risk of large out-of-pocket payments (as the size of these payments varies widely and can be extremely large), despite NHIP's attempts to mitigate this by setting different benefit ceilings based on the level of the hospital and the severity of the disease. To reduce these large out-of-pocket payments and to increase financial risk protection further, it is essential to ensure more investment for health from social health insurance and/or tax-based government funding as well as shifting the provider payment mechanism from a fee

Pocket rocket: An electrothermal plasma micro-thruster

NASA Astrophysics Data System (ADS)

Greig, Amelia Diane

Recently, an increase in use of micro-satellites constructed from commercial off the shelf (COTS) components has developed, to address the large costs associated with designing, testing and launching satellites. One particular type of micro-satellite of interest are CubeSats, which are modular 10 cm cubic satellites with total weight less than 1.33 kg. To assist with orbit boosting and attitude control of CubeSats, micro-propulsion systems are required, but are currently limited. A potential electrothermal plasma micro-thruster for use with CubeSats or other micro-satellites is under development at The Australian National University and forms the basis for this work. The thruster, known as ‘Pocket Rocket’, utilises neutral gas heating from ion-neutral collisions within a weakly ionised asymmetric plasma discharge, increasing the exhaust thermal velocity of the propellant gas, thereby producing higher thrust than if the propellant was emitted cold. In this work, neutral gas temperature of the Pocket Rocket discharge is studied in depth using rovibrational spectroscopy of the nitrogen (N2) second positive system (C3Πu → B3Πg), using both pure N2 and argon/N2 mixtures as the operating gas. Volume averaged steady state gas temperatures are measured for a range of operating conditions, with an analytical collisional model developed to verify experimental results. Results show that neutral gas heating is occurring with volume averaged steady state temperatures reaching 430 K in N2 and 1060 K for argon with 1% N2 at standard operating conditions of 1.5 Torr pressure and 10 W power input, demonstrating proof of concept for the Pocket Rocket thruster. Spatiotemporal profiles of gas temperature identify that the dominant heating mechanisms are ion-neutral collisions within the discharge and wall heating from ion bombardment of the thruster walls. To complement the experimental results, computational fluid dynamics (CFD) simulations using the commercial CFD

Species-Specific Activation of TLR4 by Hypoacylated Endotoxins Governed by Residues 82 and 122 of MD-2

PubMed Central

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation. PMID:25203747

Species-specific activation of TLR4 by hypoacylated endotoxins governed by residues 82 and 122 of MD-2.

PubMed

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Parallel pocket incision: Less invasive surgical intervention for the treatment of intractable pressure ulcer with wound edge undermining.

PubMed

Yamamoto, Takumi; Yoshimatsu, Hidehiko; Hayashi, Akitatsu; Koshima, Isao

2015-10-01

The treatment of deep pressure ulcer with a wide wound edge undermining (pocket) is challenging, especially when conservative treatments are ineffective. As most patients with a pressure ulcer suffer from systemic comorbidities, invasive surgery cannot be performed on all patients, and less invasive treatment is required. Less invasive surgical intervention to a deep pressure ulcer, parallel pocket incision (PPI), was performed on 10 patients with intractable pressure ulcers with a pocket formation. In PPI procedures, two parallel skin incisions were made to open up the deepest fold of the pocket and to preserve the skin overlying the pocket lesion; through the created incisions, the necrotic tissues around the deepest fold of the undermining could be easily removed, which facilitated spontaneous wound healing. Postoperative results and complications were evaluated. All PPI procedures were safely performed under local infiltration anesthesia without major postoperative complication; minor bleeding was seen intraoperatively in three patients, which could be easily controlled with electric cautery coagulation. Nine of 10 ulcers were cured after PPI, and one could not be followed up due to the patient's death non-related to the pressure ulcer. For the nine cured patients, the average time for cure was 14.9 weeks, and no recurrence was observed at postoperative 6 months. PPI is a simple, technically easy, and less invasive surgical intervention to an intractable pressure ulcer with a pocket, which can be safely performed under local infiltration anesthesia even on a patient with severe systemic comorbidities. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

New insight into the architecture of oxy-anion pocket in unliganded conformation of GAT domains: A MD-simulation study.

PubMed

Bairagya, Hridoy R; Bansal, Manju

2016-03-01

Human Guanine Monophosphate Synthetase (hGMPS) converts XMP to GMP, and acts as a bifunctional enzyme with N-terminal "glutaminase" (GAT) and C-terminal "synthetase" domain. The enzyme is identified as a potential target for anti-cancer and immunosuppressive therapies. GAT domain of enzyme plays central role in metabolism, and contains conserved catalytic residues Cys104, His190, and Glu192. MD simulation studies on GAT domain suggest that position of oxyanion in unliganded conformation is occupied by one conserved water molecule (W1), which also stabilizes that pocket. This position is occupied by a negatively charged atom of the substrate or ligand in ligand bound crystal structures. In fact, MD simulation study of Ser75 to Val indicates that W1 conserved water molecule is stabilized by Ser75, while Thr152, and His190 also act as anchor residues to maintain appropriate architecture of oxyanion pocket through water mediated H-bond interactions. Possibly, four conserved water molecules stabilize oxyanion hole in unliganded state, but they vacate these positions when the enzyme (hGMPS)-substrate complex is formed. Thus this study not only reveals functionally important role of conserved water molecules in GAT domain, but also highlights essential role of other non-catalytic residues such as Ser75 and Thr152 in this enzymatic domain. The results from this computational study could be of interest to experimental community and provide a testable hypothesis for experimental validation. Conserved sites of water molecules near and at oxyanion hole highlight structural importance of water molecules and suggest a rethink of the conventional definition of chemical geometry of inhibitor binding site. © 2016 Wiley Periodicals, Inc.

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2012 CFR

2012-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2011 CFR

2011-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2013 CFR

2013-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2010 CFR

2010-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

10 CFR 63.16 - Review of site characterization activities. 2

Code of Federal Regulations, 2014 CFR

2014-01-01

... which such activities are carried out and to observe excavations, borings, and in situ tests, as they... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of site characterization activities. 2 2 In addition to the review of site characterization activities...

A combination of 19F NMR and surface plasmon resonance for site-specific hit selection and validation of fragment molecules that bind to the ATP-binding site of a kinase.

PubMed

Nagatoishi, Satoru; Yamaguchi, Sou; Katoh, Etsuko; Kajita, Keita; Yokotagawa, Takane; Kanai, Satoru; Furuya, Toshio; Tsumoto, Kouhei

2018-05-01

19 F NMR has recently emerged as an efficient, sensitive tool for analyzing protein binding to small molecules, and surface plasmon resonance (SPR) is also a popular tool for this purpose. Herein a combination of 19 F NMR and SPR was used to find novel binders to the ATP-binding pocket of MAP kinase extracellular regulated kinase 2 (ERK2) by fragment screening with an original fluorinated-fragment library. The 19 F NMR screening yielded a high primary hit rate of binders to the ERK2 ATP-binding pocket compared with the rate for the SPR screening. Hit compounds were evaluated and categorized according to their ability to bind to different binding sites in the ATP-binding pocket. The binding manner was characterized by using isothermal titration calorimetry and docking simulation. Combining 19 F NMR with other biophysical methods allows the identification of multiple types of hit compounds, thereby increasing opportunities for drug design using preferred fragments. Copyright © 2018 Elsevier Ltd. All rights reserved.

The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase.

PubMed

Campomanes, Pablo; Rothlisberger, Ursula; Alfonso-Prieto, Mercedes; Rovira, Carme

2015-09-02

Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H2O2). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H2O2 at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, ∼ 6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H2O2, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the "wet" active site of HRP have a substantial impact on the reaction barrier, compared to the "dry" active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H2O2 disproportionation.

Determinants Of Impoverishment Due To Out Of Pocket Payments In Nigeria.

PubMed

Aregbeshola, Bolaji Samson; Khan, Samina Mohsin

2017-01-01

Poverty is an extreme consequence of out of pocket payments in countries with health systems that do not provide financial risk protection through mandatory health insurance coverage for people in both the formal and informal sectors. The study assessed the determinants of impoverishment due to out of pocket payments in Nigeria. Secondary data from the Harmonized Nigeria Living Standard Survey (HNLSS) of 2009/10 was utilized to assess factors associated with impoverishment in Nigeria. Household and individual characteristics associated with impoverishment were determined using binary logistic regression. A significance level of p<0.05 was used. Results show that lack of health insurance, having a member above 65 years, large household size, household socio-economic status, type of illness suffered, type of health facility visited, geo-political zones, education of household heads and location were major determinants of impoverishment due to out of pocket health expenditure. Findings from the study show that most households and individuals are vulnerable to financial risk due to this regressive source of payments for health care services. This explains why the level of poverty keeps increasing in spite of the numerous poverty alleviation programs across the country. Policy makers and political actors need to design a new health system financing policy that will increase financial risk protection for people in both the formal and informal sectors. Governments and decision makers have to focus on health as a determinant of economic well-being.

[The imperfect equalizing device: Physician care discount cards and physician care out-of-pocket costs in Iceland.].

PubMed

Vilhjálmsson, Rúnar; Sigurðardóttir, Guðrún V

2003-05-01

Research shows that out-of-pocket health care costs in Iceland and other Western European countries have increased in recent years, and unequal access to health services has been documented. In an attempt to contain out-of-pocket-costs and avoid service inequities, Icelandic health authorities have for a number of years issued health care discount cards. The purpose ot the study was to investigate the distribution of out-of-pocket physician costs and discount cards, and the extent to which the cards reach those who are entitled to them. The study is based on a national panel survey titled Health and Living Conditions in Iceland. A random sample of 18-75 year olds was drawn from the National Register. 1924 respondents participated in the first wave (69% response rate) and 1592 of them (83%) in the second wave. Cross-tabular analysis was used to investigate variations in out-of-pocket physician costs and discount card status across sociodemographic groups. 19.9% of the respondents had accumulated out-of-pocket costs that made them eligible for a discount card. Furthermore, there was considerable variation in the percentage of eligible individuals across population groups. The discount card was poorly distributed, as only 45.7% of eligible individuals had actually obtained a card. This lack of coverage was greatest among younger individuals, parents of young children, individuals in larger households, the full-time employed, and those who had more education and income. The purpose of the discount card is to even out and contain out-of-pocket physician care costs, and sustain equal access to physician services. The purpose is no more than partially reached, as only a minority of eligible individuals are actual cardholders. This can be largely explained by the fact that health authorities have done little to promote the card, and make it cumbersome for patients to obtain it.

Biomarker-based diagnosis of pacemaker and implantable cardioverter defibrillator pocket infections: A prospective, multicentre, case-control evaluation

PubMed Central

Vrazic, Hrvoje; Haller, Bernhard; Braun, Siegmund; Petzold, Tobias; Ott, Ilka; Lennerz, Agnes; Michel, Jonathan; Blažek, Patrick; Deisenhofer, Isabel; Whittaker, Peter; Kolb, Christof

2017-01-01

Background The use of cardiac implantable electronic devices (CIED) has risen steadily, yet the rate of cardiac device infections (CDI) has disproportionately increased. Amongst all cardiac device infections, the pocket infection is the most challenging diagnosis. Therefore, we aimed to improve diagnosis of such pocket infection by identifying relevant biomarkers. Methods We enrolled 25 consecutive patients with invasively and microbiologically confirmed pocket infection. None of the patients had any confounding conditions. Pre-operative levels of 14 biomarkers were compared in infected and control (n = 50) patients. Our selected biomarkers included white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), lipopolysaccharide binding protein, high-sensitivity C-reactive protein (HS-CRP), polymorphonuclear-elastase, presepsin, various interleukins, tumor necrosis factor α (TNF-α), and granulocyte macrophage colony-stimulating factor (GM-CSF). Results Of the 25 patients with isolated pocket infection (70±13years, 76% male, 40% ICDs), none presented with leukocytosis. In contrast, they had higher serum levels of HS-CRP (p = 0.019) and PCT (p = 0.010) than control patients. Median PCT-level was 0.06 ng/mL (IQR 0.03–0.07 ng/mL) in the study group versus 0.03 ng/mL (IQR 0.02–0.04 ng/mL) in controls. An optimized PCT cut-off value of 0.05 ng/mL suggests pocket infection with a sensitivity of 60% and specificity of 82%. In addition TNF-α- and GM-CSF-levels were lower in the study group. Other biomarkers did not differ between groups. Conclusion Diagnosis of isolated pocket infections requires clinical awareness, physical examination, evaluation of blood cultures and echocardiography assessment. Nevertheless, measurement of PCT- and HS-CRP-levels can aid diagnosis. However, no conclusion can be drawn from normal WBC-values. Clinical trial registration clinicaltrials.gov identifier: NCT01619267 PMID:28264059

51. South side of Breaker, Retail Pockets under Construction Historic ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

51. South side of Breaker, Retail Pockets under Construction Historic Photograph, Photographer Unknown Originally taken by Glen Alden Safety Department, 18 August 1954; Collection of William Everett, Jr. (Wilkes-Barre, PA), photocopy by Joseph E.B. Elliot - Huber Coal Breaker, 101 South Main Street, Ashley, Luzerne County, PA

Estimating annual medical and out-of-pocket expenditures associated with traumatic injuries in the United States.

PubMed

Alghnam, Suliman; Vanness, David J; Gaskin, Darrell J; Thorpe, Roland J; Castillo, Renan

2016-02-01

Every year, as many as 31 million Americans sustain traumatic injuries, leaving survivors with risks of disabilities and health settings with staggering medical costs. Little is known on the societal burden of injuries in terms of medical and out-of-pocket expenditures. Therefore, we used a nationally representative sample to evaluate the association between injuries and health expenditures among a nationally representative US sample. This study used years 2006 to 2010 (Panels 11-14; n = 53,065) of the Medical Expenditure Panel Survey. Each panel was followed up for 2 years. Total expenditures included insurance payments and out-of-pocket costs. Two-part models were constructed to examine differences in annual medical expenditures between injured and noninjured populations controlling for confounding effects. A total of 4,210 individuals (7.9%) reported injuries representing 21.5 million individuals. Injured individuals were more likely to be males, to be white, and to report higher medical expenditures in the second year than the reference population (p < 0.01). Adjusted analyses showed that reporting any injury was associated with $2,577 (95% confidence interval [CI], $2,049-$3,103) and $186 (95% CI, $142-$230) increase in total and out-of-pocket costs, respectively. While a moderate or severe injury was associated with $4,779 (95% CI, $3,947-$5,610) increase in the average of medical expenditures and $256 (95% CI, $190-$322) increase in out-of-pocket costs adjusting for covariates. Our adjusted national medical cost of injuries was estimated at $56 billion and out-of-pocket cost to be approximately $4 billion. Injuries pose a substantial burden on medical expenditures in the United States. Investment in injury prevention can facilitate reducing medical expenditures and save resources. Prevention programs may use the out-of-pocket findings to highlight injury burden on individual's prosperity and thus facilitate engagement of the public in prevention. Economic

Low-Level Laser Therapy (LLLT) for periodontal pockets: a review

NASA Astrophysics Data System (ADS)

Pribac, Valentin; Todea, Carmen; Duma, Virgil-Florin

2016-03-01

The applications of lasers in medicine, both in the field of diagnosis and treatment are gaining momentum. In dentistry in particular, numerous types of lasers with a wide range of characteristics are being utilized in all fields. In consequence, a lot of experience and knowledge has been gained in the last two decades in this domain; this resulted in the development of novel technologies and devices. A brief overview is made in the first part of this article on these topics. The treatment of periodontal disease with laser therapy is pointed out, as well as the photodynamic therapy which is using LLLT for the activation of the sensitizing gel that is introduced in the periodontal pockets. This paper reviews also the application of photodynamic therapy in clinical trials which have different results; a standardization of the protocol utilized for this procedure is concluded to be necessary.

Identification of distant drug off-targets by direct superposition of binding pocket surfaces.

PubMed

Schumann, Marcel; Armen, Roger S

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").

Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

PubMed Central

Schumann, Marcel; Armen, Roger S.

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”). PMID:24391782

Improved cardiovascular diagnostic accuracy by pocket size imaging device in non-cardiologic outpatients: the NaUSiCa (Naples Ultrasound Stethoscope in Cardiology) study

PubMed Central

2010-01-01

Miniaturization has evolved in the creation of a pocket-size imaging device which can be utilized as an ultrasound stethoscope. This study assessed the additional diagnostic power of pocket size device by both experts operators and trainees in comparison with physical examination and its appropriateness of use in comparison with standard echo machine in a non-cardiologic population. Three hundred four consecutive non cardiologic outpatients underwent a sequential assessment including physical examination, pocket size imaging device and standard Doppler-echo exam. Pocket size device was used by both expert operators and trainees (who received specific training before the beginning of the study). All the operators were requested to give only visual, qualitative insights on specific issues. All standard Doppler-echo exams were performed by expert operators. One hundred two pocket size device exams were performed by experts and two hundred two by trainees. The time duration of the pocket size device exam was 304 ± 117 sec. Diagnosis of cardiac abnormalities was made in 38.2% of cases by physical examination and in 69.7% of cases by physical examination + pocket size device (additional diagnostic power = 31.5%, p < 0.0001). The overall K between pocket size device and standard Doppler-echo was 0.67 in the pooled population (0.84 by experts and 0.58 by trainees). K was suboptimal for trainees in the eyeball evaluation of ejection fraction, left atrial dilation and right ventricular dilation. Overall sensitivity was 91% and specificity 76%. Sensitivity and specificity were lower in trainees than in experts. In conclusion, pocket size device showed a relevant additional diagnostic value in comparison with physical examination. Sensitivity and specificity were good in experts and suboptimal in trainees. Specificity was particularly influenced by the level of experience. Training programs are needed for pocket size device users. PMID:21110840

Effects of 980 diode laser treatment combined with scaling and root planing on periodontal pockets in chronic periodontitis patients

NASA Astrophysics Data System (ADS)

Fallah, Alireza

2010-02-01

Objective: This study compared the effect of 980 Diode laser + scaling and root planing (SRP) versus SRP alone in the treatment of chronic periodontitis. Method: 21 healthy patients with moderate periodontitis with a probing depth of at least 5mm were included in the study. A total of 42 sites were treated during 6weeks with a combination of 980 Diode laser and SRP (21 sites) or SRP alone (21 sites). The gingival index (GI), probing pocket depth (PPD) and bleeding on probing (BOP) were examined at the baseline and after 6 weeks after the start of treatment. Results: Both groups showed statistically significant improvements in GI, BOP and PPD after treatment. The results also showed significant improvement from laser+ SRP group to SRP alone group. Conclusion: The present data suggest that treatment of chronic periodontitis with either 980 Diode laser + SRP or SRP alone results in statistically significant improvements in the clinical parameters. The combination of 980 Diode laser irradiation in the gingival sulcus and SRP, was significantly better as compared to SRP alone.

DRoP: a water analysis program identifies Ras-GTP-specific pathway of communication between membrane-interacting regions and the active site.

PubMed

Kearney, Bradley M; Johnson, Christian W; Roberts, Daniel M; Swartz, Paul; Mattos, Carla

2014-02-06

Ras GTPase mediates several cellular signal transduction pathways and is found mutated in a large number of cancers. It is active in the GTP-bound state, where it interacts with effector proteins, and at rest in the GDP-bound state. The catalytic domain is tethered to the membrane, with which it interacts in a nucleotide-dependent manner. Here we present the program Detection of Related Solvent Positions (DRoP) for crystallographic water analysis on protein surfaces and use it to study Ras. DRoP reads and superimposes multiple Protein Data Bank coordinates, transfers symmetry-related water molecules to the position closest to the protein surface, and ranks the waters according to how well conserved and tightly clustered they are in the set of structures. Coloring according to this rank allows visualization of the results. The effector-binding region of Ras is hydrated with highly conserved water molecules at the interface between the P-loop, switch I, and switch II, as well as at the Raf-RBD binding pocket. Furthermore, we discovered a new conserved water-mediated H-bonding network present in Ras-GTP, but not in Ras-GDP, that links the nucleotide sensor residues R161 and R164 on helix 5 to the active site. The double mutant RasN85A/N86A, where the final link between helix 5 and the nucleotide is not possible, is a severely impaired enzyme, while the single mutant RasN86A, with partial connection to the active site, has a wild-type hydrolysis rate. DRoP was instrumental in determining the water-mediated connectivity networks that link two lobes of the catalytic domain in Ras. Copyright © 2013 Elsevier Ltd. All rights reserved.