Web Content Accessibility of Consumer Health Information Web Sites for People with Disabilities: A Cross Sectional Evaluation

PubMed Central

Parmanto, Bambang

2004-01-01

Background The World Wide Web (WWW) has become an increasingly essential resource for health information consumers. The ability to obtain accurate medical information online quickly, conveniently and privately provides health consumers with the opportunity to make informed decisions and participate actively in their personal care. Little is known, however, about whether the content of this online health information is equally accessible to people with disabilities who must rely on special devices or technologies to process online information due to their visual, hearing, mobility, or cognitive limitations. Objective To construct a framework for an automated Web accessibility evaluation; to evaluate the state of accessibility of consumer health information Web sites; and to investigate the possible relationships between accessibility and other features of the Web sites, including function, popularity and importance. Methods We carried out a cross-sectional study of the state of accessibility of health information Web sites to people with disabilities. We selected 108 consumer health information Web sites from the directory service of a Web search engine. A measurement framework was constructed to automatically measure the level of Web Accessibility Barriers (WAB) of Web sites following Web accessibility specifications. We investigated whether there was a difference between WAB scores across various functional categories of the Web sites, and also evaluated the correlation between the WAB and Alexa traffic rank and Google Page Rank of the Web sites. Results We found that none of the Web sites we looked at are completely accessible to people with disabilities, i.e., there were no sites that had no violation of Web accessibility rules. However, governmental and educational health information Web sites do exhibit better Web accessibility than the other categories of Web sites (P < 0.001). We also found that the correlation between the WAB score and the popularity of a

The impact of target site accessibility on the design of effective siRNAs.

PubMed

Tafer, Hakim; Ameres, Stefan L; Obernosterer, Gregor; Gebeshuber, Christoph A; Schroeder, Renée; Martinez, Javier; Hofacker, Ivo L

2008-05-01

Small-interfering RNAs (siRNAs) assemble into RISC, the RNA-induced silencing complex, which cleaves complementary mRNAs. Despite their fluctuating efficacy, siRNAs are widely used to assess gene function. Although this limitation could be ascribed, in part, to variations in the assembly and activation of RISC, downstream events in the RNA interference (RNAi) pathway, such as target site accessibility, have so far not been investigated extensively. In this study we present a comprehensive analysis of target RNA structure effects on RNAi by computing the accessibility of the target site for interaction with the siRNA. Based on our observations, we developed a novel siRNA design tool, RNAxs, by combining known siRNA functionality criteria with target site accessibility. We calibrated our method on two data sets comprising 573 siRNAs for 38 genes, and tested it on an independent set of 360 siRNAs targeting four additional genes. Overall, RNAxs proves to be a robust siRNA selection tool that substantially improves the prediction of highly efficient siRNAs.

Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes.

PubMed

Urban, Philippe; Truan, Gilles; Pompon, Denis

2015-04-01

A cytochrome P450 active site is buried within the protein molecule and several channels connect the catalytic cavity to the protein surface. Their role in P450 catalysis is still matter of debate. The aim of this study was to understand the possible relations existing between channels and substrate specificity. Time course studies were carried out with a collection of polycyclic substrates of increasing sizes assayed with a library of wild-type and chimeric CYP1A enzymes. This resulted in a matrix of activities sufficiently large to allow statistical analysis. Multivariate statistical tools were used to decipher the correlation between observed activity shifts and sequence segment swaps. The global kinetic behavior of CYP1A enzymes toward polycyclic substrates is significantly different depending on the size of the substrate. Mutations which are close or lining the P450 channels significantly affect this discrimination, whereas mutations distant from the P450 channels do not. Size discrimination is taking place for polycyclic substrates at the entrance of the different P450 access channels. It is thus hypothesized that channels differentiate small from large substrates in CYP1A enzymes, implying that residues located at the surface of the protein may be implied in this differential recognition. Catalysis thus occurs after a two-step recognition process, one at the surface of the protein and the second within the catalytic cavity in enzymes with a buried active site. Copyright © 2014 Elsevier B.V. All rights reserved.

How Accessible Are Public Libraries' Web Sites? A Study of Georgia Public Libraries

ERIC Educational Resources Information Center

Ingle, Emma; Green, Ravonne A.; Huprich, Julia

2009-01-01

One issue that public librarians must consider when planning Web site design is accessibility for patrons with disabilities. This article reports a study of Web site accessibility of public libraries in Georgia. The focus of the report is whether public libraries use accessible guidelines and standards in making their Web sites accessible. An…

Eisenhower National Historic Site visitor transportation and access study

DOT National Transportation Integrated Search

2017-11-01

This study evaluates the current shuttle system and Eisenhower National Historic Site, which is currently the sole access to the site. Visitation at Eisenhower has been declining since the site opened, and the study looks at the impacts of this trend...

Investigation of prototypal MOFs consisting of polyhedral cages with accessible Lewis-acid sites for quinoline synthesis.

PubMed

Gao, Wen-Yang; Leng, Kunyue; Cash, Lindsay; Chrzanowski, Matthew; Stackhouse, Chavis A; Sun, Yinyong; Ma, Shengqian

2015-03-21

A series of prototypal metal-organic frameworks (MOFs) consisting of polyhedral cages with accessible Lewis-acid sites, have been systematically investigated for Friedländer annulation reaction, a straightforward approach to synthesizing quinoline and its derivatives. Amongst them MMCF-2 demonstrates significantly enhanced catalytic activity compared with the benchmark MOFs, HKUST-1 and MOF-505, as a result of a high-density of accessible Cu(II) Lewis acid sites and large window size in the cuboctahedral cage-based nanoreactor of MMCF-2.

Challenges in Obtaining Property Access: The FUSRAP Maywood Site Experience - 13433

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

Kollar, William

2013-07-01

The Formerly Utilized Sites Remedial Action Program (FUSRAP) is the US government program started in 1974 to identify, investigate and clean up or control sites that became contaminated as a result of the nation's early atomic programs. Many of these sites are not owned by the federal government and therefore require owner permission to enter. The experience in pursuing such access at the FUSRAP Maywood Superfund Site (the Maywood Site or the Site) in Bergen County, New Jersey, is extensive. Since the US Army Corps of Engineers (the Corps) assumed responsibility for the Maywood Site from the US Department ofmore » Energy in 1997, at least 186 separate property access agreements (known in FUSRAP as a Real Estate Right-of- Entry or ROE) have been executed between the Corps and approximately 55 different land owners and tenant occupants at the Maywood Site (agreement renewals with the same owners over time account for the difference). Maywood's experience during the Corps' tenure, reflected here in three case studies of representative property access efforts, offers some lessons and best practices that may apply to other remedial programs. While the Site Community Relations Manager (the author of this paper) managed the property access task, multi-disciplinary support from across the project was also critical to success in this endeavor. (authors)« less

Total Access: Making College Web Sites Accessible to Students with Disabilities

ERIC Educational Resources Information Center

Bruyere, Susanne

2008-01-01

Colleges increasingly rely on the Web to attract, inform, and interact with students. This makes Web site accessibility and usability critical concerns, particularly for public community colleges, which educate sizable numbers of students with disabilities. As committed providers of postsecondary education to students with disabilities and thus a…

Geographic approaches to quantifying the risk environment: a focus on syringe exchange program site access and drug-related law enforcement activities

PubMed Central

Cooper, Hannah LF; Bossak, Brian; Tempalski, Barbara; Des Jarlais, Don C.; Friedman, Samuel R.

2009-01-01

The concept of the “risk environment” – defined as the “space … [where] factors exogenous to the individual interact to increase the chances of HIV transmission” – draws together the disciplines of public health and geography. Researchers have increasingly turned to geographic methods to quantify dimensions of the risk environment that are both structural and spatial (e.g., local poverty rates). The scientific power of the intersection between public health and geography, however, has yet to be fully mined. In particular, research on the risk environment has rarely applied geographic methods to create neighbourhood-based measures of syringe exchange programs (SEPs) or of drug-related law enforcement activities, despite the fact that these interventions are widely conceptualized as structural and spatial in nature and are two of the most well-established dimensions of the risk environment. To strengthen research on the risk environment, this paper presents a way of using geographic methods to create neighbourhood-based measures of (1) access to SEP sites and (2) exposure to drug-related arrests, and then applies these methods to one setting (New York City). NYC-based results identified substantial cross-neighbourhood variation in SEP site access and in exposure to drug-related arrest rates (even within the subset of neighbourhoods nominally experiencing the same drug-related police strategy). These geographic measures – grounded as they are in conceptualizations of SEPs and drug-related law enforcement strategies – can help develop new arenas of inquiry regarding the impact of these two dimensions of the risk environment on injectors’ health, including exploring whether and how neighbourhood-level access to SEP sites and exposure to drug-related arrests shape a range of outcomes among local injectors. PMID:18963907

Quantifying accessible sites and reactivity on titania–silica (photo)catalysts: Refining TOF calculations

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

Eaton, Todd R.; Campos, Michael P.; Gray, Kimberly A.

2014-01-01

It can be difficult to determine the number of active atoms accessible to the fluid phase in mixed oxide catalysts, as required for obtaining true turnover frequencies (TOF). Here, we utilize the selective titration of surface Ti atoms with phenylphosphonic acid (PPA) on TiO 2–SiO 2 materials to estimate the number of reactant-accessible sites. TiO 2–SiO 2 composites were synthesized over a range of Ti loadings from grafting of titanocene dichloride (Cp 2TiCl 2) or tetraethoxy orthotitanate (TEOT) on SiO 2 and sol–gel co-hydrolysis of Si and Ti alkoxides. The materials were characterized by DRUV–vis, XRD, BET, and XANES. Despitemore » the significant morphological and electronic differences, materials prepared by Cp 2TiCl 2 and TEOT yielded a near-constant TOF of 0.14 h -1 (±0.04) across Ti loadings, for benzyl alcohol photooxidation, when normalizing rates by sites titrated by PPA. The fraction of Ti atoms titrated by PPA was strongly dependent on synthesis method and surface density. PPA titration and benzyl alcohol photooxidation may be useful measures of surface accessibility in other supported oxides.« less

Ocean Drilling Program: Web Site Access Statistics

Science.gov Websites

and products Drilling services and tools Online Janus database Search the ODP/TAMU web site ODP's main See statistics for JOIDES members. See statistics for Janus database. 1997 October November December accessible only on www-odp.tamu.edu. ** End of ODP, start of IODP. Privacy Policy ODP | Search | Database

A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

PubMed

Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

2016-10-04

Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

40 CFR 1400.5 - Internet access to certain off-site consequence analysis data elements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 33 2011-07-01 2011-07-01 false Internet access to certain off-site... DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.5 Internet access to certain off... elements in the risk management plan database available on the Internet: (a) The concentration of the...

Evaluating existing access opportunities for disabled persons at remote shoreline recreation sites

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

Bley, M.R.; Kearns, M.T.

1995-12-31

Draft guidelines for providing outdoor recreation access opportunities for disabled persons have been recommended by the Recreation Access Advisory Committee and in the Universal Access to Outdoor Recreation: A Design Guide. The Federal Energy Regulatory Commission requires applicants for new hydropower licenses to consider access opportunities for disabled persons at existing hydropower projects. A process for evaluating existing access opportunities for disabled persons at remote shoreline recreation sites at hydropower projects is described. The process includes five steps: (1) preparing a preliminary map of existing recreation sites; (2) data collection in the field; (3) evaluating compliance of existing facilities; (4)more » feasibility of enhancing existing facilities; and (5) designing enhancements. The process will be refined when final standards and processes are approved by the appropriate agencies and organizations.« less

40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

Code of Federal Regulations, 2012 CFR

2012-07-01

...-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.3 Public access to paper copies of off-site consequence analysis information. (a) General. The Administrator and the...

40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

Code of Federal Regulations, 2013 CFR

2013-07-01

...-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.3 Public access to paper copies of off-site consequence analysis information. (a) General. The Administrator and the...

40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

Code of Federal Regulations, 2011 CFR

2011-07-01

...-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.3 Public access to paper copies of off-site consequence analysis information. (a) General. The Administrator and the...

40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

Code of Federal Regulations, 2014 CFR

2014-07-01

...-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.3 Public access to paper copies of off-site consequence analysis information. (a) General. The Administrator and the...

40 CFR 1400.8 - Access to off-site consequence analysis information by Federal government officials.

Code of Federal Regulations, 2011 CFR

2011-07-01

... analysis information by Federal government officials. 1400.8 Section 1400.8 Protection of Environment... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.8 Access to off-site consequence analysis information by Federal...

40 CFR 1400.8 - Access to off-site consequence analysis information by Federal government officials.

Code of Federal Regulations, 2013 CFR

2013-07-01

... analysis information by Federal government officials. 1400.8 Section 1400.8 Protection of Environment... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.8 Access to off-site consequence analysis information by Federal...

40 CFR 1400.8 - Access to off-site consequence analysis information by Federal government officials.

Code of Federal Regulations, 2012 CFR

2012-07-01

... analysis information by Federal government officials. 1400.8 Section 1400.8 Protection of Environment... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.8 Access to off-site consequence analysis information by Federal...

40 CFR 1400.8 - Access to off-site consequence analysis information by Federal government officials.

Code of Federal Regulations, 2014 CFR

2014-07-01

... analysis information by Federal government officials. 1400.8 Section 1400.8 Protection of Environment... INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.8 Access to off-site consequence analysis information by Federal...

Hand Society and Matching Program Web Sites Provide Poor Access to Information Regarding Hand Surgery Fellowship.

PubMed

Hinds, Richard M; Klifto, Christopher S; Naik, Amish A; Sapienza, Anthony; Capo, John T

2016-08-01

The Internet is a common resource for applicants of hand surgery fellowships, however, the quality and accessibility of fellowship online information is unknown. The objectives of this study were to evaluate the accessibility of hand surgery fellowship Web sites and to assess the quality of information provided via program Web sites. Hand fellowship Web site accessibility was evaluated by reviewing the American Society for Surgery of the Hand (ASSH) on November 16, 2014 and the National Resident Matching Program (NRMP) fellowship directories on February 12, 2015, and performing an independent Google search on November 25, 2014. Accessible Web sites were then assessed for quality of the presented information. A total of 81 programs were identified with the ASSH directory featuring direct links to 32% of program Web sites and the NRMP directory directly linking to 0%. A Google search yielded direct links to 86% of program Web sites. The quality of presented information varied greatly among the 72 accessible Web sites. Program description (100%), fellowship application requirements (97%), program contact email address (85%), and research requirements (75%) were the most commonly presented components of fellowship information. Hand fellowship program Web sites can be accessed from the ASSH directory and, to a lesser extent, the NRMP directory. However, a Google search is the most reliable method to access online fellowship information. Of assessable programs, all featured a program description though the quality of the remaining information was variable. Hand surgery fellowship applicants may face some difficulties when attempting to gather program information online. Future efforts should focus on improving the accessibility and content quality on hand surgery fellowship program Web sites.

Accredited hand surgery fellowship Web sites: analysis of content and accessibility.

PubMed

Trehan, Samir K; Morrell, Nathan T; Akelman, Edward

2015-04-01

To assess the accessibility and content of accredited hand surgery fellowship Web sites. A list of all accredited hand surgery fellowships was obtained from the online database of the American Society for Surgery of the Hand (ASSH). Fellowship program information on the ASSH Web site was recorded. All fellowship program Web sites were located via Google search. Fellowship program Web sites were analyzed for accessibility and content in 3 domains: program overview, application information/recruitment, and education. At the time of this study, there were 81 accredited hand surgery fellowships with 169 available positions. Thirty of 81 programs (37%) had a functional link on the ASSH online hand surgery fellowship directory; however, Google search identified 78 Web sites. Three programs did not have a Web site. Analysis of content revealed that most Web sites contained contact information, whereas information regarding the anticipated clinical, research, and educational experiences during fellowship was less often present. Furthermore, information regarding past and present fellows, salary, application process/requirements, call responsibilities, and case volume was frequently lacking. Overall, 52 of 81 programs (64%) had the minimal online information required for residents to independently complete the fellowship application process. Hand fellowship program Web sites could be accessed either via the ASSH online directory or Google search, except for 3 programs that did not have Web sites. Although most fellowship program Web sites contained contact information, other content such as application information/recruitment and education, was less frequently present. This study provides comparative data regarding the clinical and educational experiences outlined on hand fellowship program Web sites that are of relevance to residents, fellows, and academic hand surgeons. This study also draws attention to various ways in which the hand surgery fellowship application

Novel Use of a Pneumatic Compression Device for Haemostasis of Haemodialysis Fistula Access Catheterisation Sites

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

O’Reilly, Michael K., E-mail: moreilly1@mater.ie; Ryan, David; Sugrue, Gavin

PurposeTransradial pneumatic compression devices can be used to achieve haemostasis following radial artery puncture. This article describes a novel technique for acquiring haemostasis of arterio-venous haemodialysis fistula access sites without the need for suture placement using one such compression device.Materials and MethodsA retrospective review of fistulograms with or without angioplasty/thrombectomy in a single institution was performed. 20 procedures performed on 12 patients who underwent percutaneous intervention of failing or thrombosed arterio-venous fistulas (AVF) had 27 puncture sites. Haemostasis was achieved using a pneumatic compression device at all access sites. Procedure details including size of access sheath, heparin administration and complicationsmore » were recorded.ResultsTwo diagnostic fistulograms, 14 fistulograms and angioplasties and four thrombectomies were performed via access sheaths with an average size (±SD) of 6 Fr (±1.12). IV unfractionated heparin was administered in 11 of 20 procedures. Haemostasis was achieved in 26 of 27 access sites following 15–20 min of compression using the pneumatic compression device. One case experienced limited bleeding from an inflow access site that was successfully treated with reinflation of the device for a further 5 min. No other complication was recorded.ConclusionsHaemostasis of arterio-venous haemodialysis fistula access sites can be safely and effectively achieved using a pneumatic compression device. This is a technically simple, safe and sutureless technique for acquiring haemostasis after AVF intervention.« less

Impact of prolonged storm activity on the Ecological Status of intertidal benthic habitats within oyster (Crassostrea gigas) trestle cultivation sites.

PubMed

O'Carroll, Jack P J; Quinn, Christina; Forde, James; Patterson, Adrian; O'Beirn, Francis X; Kennedy, Robert

2016-09-15

The Ecological Status (ES; sensu the Water Framework Directive) of intertidal benthic communities within six oyster trestle cultivation sites was found to be negatively impacted along the access routes to trestles in a 2013 study. All cultivation sites occur within Natura 2000 sites. The current study revisited four of the 2013 cultivation sites in February 2014 one month after the storm activity of winter 2013/14 to test if the compaction effect along access routes persisted after the storms. Three levels of the fixed factor treatment were sampled; immediately below the trestles, along the access route and 300m away from any anthropogenic activity. The compaction effect at the Access treatment persisted in spite of the major storm activity. The current study showed the IQI to be effective for assessing the impacts of aquaculture and highlights the IQI as a tool for monitoring Conservation Status of intertidal communities under the Habitats Directive. Copyright © 2016 Elsevier Ltd. All rights reserved.

40 CFR 1400.5 - Internet access to certain off-site consequence analysis data elements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.5 Internet access to certain off... consequence analysis data elements. 1400.5 Section 1400.5 Protection of Environment ENVIRONMENTAL PROTECTION...

40 CFR 1400.5 - Internet access to certain off-site consequence analysis data elements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.5 Internet access to certain off... consequence analysis data elements. 1400.5 Section 1400.5 Protection of Environment ENVIRONMENTAL PROTECTION...

40 CFR 1400.5 - Internet access to certain off-site consequence analysis data elements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.5 Internet access to certain off... consequence analysis data elements. 1400.5 Section 1400.5 Protection of Environment ENVIRONMENTAL PROTECTION...

Content and Accessibility of Shoulder and Elbow Fellowship Web Sites in the United States.

PubMed

Young, Bradley L; Oladeji, Lasun O; Cichos, Kyle; Ponce, Brent

2016-01-01

Increasing numbers of training physicians are using the Internet to gather information about graduate medical education programs. The content and accessibility of web sites that provide this information have been demonstrated to influence applicants' decisions. Assessments of orthopedic fellowship web sites including sports medicine, pediatrics, hand and spine have found varying degrees of accessibility and material. The purpose of this study was to evaluate the accessibility and content of the American Shoulder and Elbow Surgeons (ASES) fellowship web sites (SEFWs). A complete list of ASES programs was obtained from a database on the ASES web site. The accessibility of each SEFWs was assessed by the existence of a functioning link found in the database and through Google®. Then, the following content areas of each SEFWs were evaluated: fellow education, faculty/previous fellow information, and recruitment. At the time of the study, 17 of the 28 (60.7%) ASES programs had web sites accessible through Google®, and only five (17.9%) had functioning links in the ASES database. Nine programs lacked a web site. Concerning web site content, the majority of SEFWs contained information regarding research opportunities, research requirements, case descriptions, meetings and conferences, teaching responsibilities, attending faculty, the application process, and a program description. Fewer than half of the SEFWs provided information regarding rotation schedules, current fellows, previous fellows, on-call expectations, journal clubs, medical school of current fellows, residency of current fellows, employment of previous fellows, current research, and previous research. A large portion of ASES fellowship programs lacked functioning web sites, and even fewer provided functioning links through the ASES database. Valuable information for potential applicants was largely inadequate across present SEFWs.

Frequency and prognostic significance of access site and non-access site bleeding and impact of choice of antithrombin therapy in patients undergoing primary percutaneous coronary intervention. The EUROMAX trial.

PubMed

Kilic, Sinem; Van't Hof, Arnoud W J; Ten Berg, Jurrien; Lopez, Ana Ayesta; Zeymer, Uwe; Hamon, Martial; Soulat, Louis; Bernstein, Debra; Deliargyris, Efthymios N; Steg, Phillippe Gabriel

2016-05-15

The overall impact of post percutaneous coronary intervention (PCI) bleeding on long term prognosis after acute coronary syndromes (ACS) has been established, but it may differ between access and non-access related bleeding events. The impact of antithrombin choice on bleeding may also differ according to the origin of the bleed. We sought to determine the origin of bleeding relative to the access site, its prognostic significance and the respective impact of antithrombin therapy in the EUROMAX trial. We performed a blinded review of the case records of all TIMI major or minor bleeds in the EUROMAX trial and assigned them in one of 2 categories: access site bleeds (ASB), or rest of bleeds (ROB). Incidence of bleeding for each category was assessed according to randomization to antithrombotic treatment. A total of 231 out of 2198 patients suffered a TIMI major/minor bleed (10.5%) and ASB accounted for 48.5%, while ROB for 51.5% of the bleeds. Thirty day mortality was 2.5% (50/1967) for patients without a bleed, 2.7% (3/112, p=0.76 vs. no bleed) for patients with ASB, and 10.9% (13/119, p<0.0001 vs. no bleed) for ROB patients. The use of bivalirudin reduced both ASB and ROB with relative risk reductions of 34% and 46% respectively. In contemporary primary PCI, bleeding originates with equal frequency either at or away from the access site. Access site bleeds were not associated with an excess in 30day mortality, but the rest of the bleeds were. Bivalirudin is associated with a lower risk of bleeding irrespective of origin. ClinicalTrials.gov identifier NCT01087723. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Robotic single-access splenectomy using the Da Vinci Single-Site® platform: a case report.

PubMed

Corcione, Francesco; Bracale, Umberto; Pirozzi, Felice; Cuccurullo, Diego; Angelini, Pier Luigi

2014-03-01

Single-access laparoscopic splenectomy can offer patients some advantages. It has many difficulties, such as instrument clashing, lack of triangulation, odd angles and lack of space. The Da Vinci Single-Site® robotic surgery platform could decrease these difficulties. We present a case of single-access robotic splenectomy using this device. A 37 year-old female with idiopathic thrombocytopenic purpura was operated on with a single-site approach, using the Da Vinci Single-Site robotic surgery device. The procedure was successfully completed in 140 min. No intraoperative and postoperative complications occurred. The patient was discharged from hospital on day 3. Single-access robotic splenectomy seems to be feasible and safe using the new robotic single-access platform, which seems to overcome certain limits of previous robotic or conventional single-access laparoscopy. We think that additional studies should also be performed to explore the real cost-effectiveness of the platform. Copyright © 2013 John Wiley & Sons, Ltd.

Ultrafast Light-Driven Substrate Expulsion from the Active Site of a Photoswitchable Catalyst.

PubMed

Pescher, Manuel D; van Wilderen, Luuk J G W; Grützner, Susanne; Slavov, Chavdar; Wachtveitl, Josef; Hecht, Stefan; Bredenbeck, Jens

2017-09-25

The photoswitchable piperidine general base catalyst is a prototype structure for light control of catalysis. Its azobenzene moiety moves sterically shielding groups to either protect or expose the active site, thereby changing the basicity and hydrogen-bonding affinity of the compound. The reversible switching dynamics of the catalyst is probed in the infrared spectral range by monitoring hydrogen bond (HB) formation between its active site and methanol (MeOH) as HB donor. Steady-state infrared (IR) and ultrafast IR and UV/Vis spectroscopies are used to uncover ultrafast expulsion of MeOH from the active site within a few picoseconds. Thus, the force generated by the azobenzene moiety even in the final phase of its isomerization is sufficient to break a strong HB within 3 ps and to shut down access to the active site. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

PubMed

Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

2015-05-01

Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, Inc.

41 CFR 60-1.43 - Access to records and site of employment.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 41 Public Contracts and Property Management 1 2011-07-01 2009-07-01 true Access to records and site of employment. 60-1.43 Section 60-1.43 Public Contracts and Property Management Other Provisions..., DEPARTMENT OF LABOR 1-OBLIGATIONS OF CONTRACTORS AND SUBCONTRACTORS Ancillary Matters § 60-1.43 Access to...

41 CFR 60-1.43 - Access to records and site of employment.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Access to records and site of employment. 60-1.43 Section 60-1.43 Public Contracts and Property Management Other Provisions..., DEPARTMENT OF LABOR 1-OBLIGATIONS OF CONTRACTORS AND SUBCONTRACTORS Ancillary Matters § 60-1.43 Access to...

40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

Code of Federal Regulations, 2014 CFR

2014-07-01

... analysis information by State and local government officials. 1400.9 Section 1400.9 Protection of... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

Code of Federal Regulations, 2011 CFR

2011-07-01

... analysis information by State and local government officials. 1400.9 Section 1400.9 Protection of... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

Code of Federal Regulations, 2010 CFR

2010-07-01

... analysis information by State and local government officials. 1400.9 Section 1400.9 Protection of... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

Code of Federal Regulations, 2013 CFR

2013-07-01

... analysis information by State and local government officials. 1400.9 Section 1400.9 Protection of... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

Code of Federal Regulations, 2012 CFR

2012-07-01

... analysis information by State and local government officials. 1400.9 Section 1400.9 Protection of... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites.

PubMed

Ribeiro, António J M; Holliday, Gemma L; Furnham, Nicholas; Tyzack, Jonathan D; Ferris, Katherine; Thornton, Janet M

2018-01-04

M-CSA (Mechanism and Catalytic Site Atlas) is a database of enzyme active sites and reaction mechanisms that can be accessed at www.ebi.ac.uk/thornton-srv/m-csa. Our objectives with M-CSA are to provide an open data resource for the community to browse known enzyme reaction mechanisms and catalytic sites, and to use the dataset to understand enzyme function and evolution. M-CSA results from the merging of two existing databases, MACiE (Mechanism, Annotation and Classification in Enzymes), a database of enzyme mechanisms, and CSA (Catalytic Site Atlas), a database of catalytic sites of enzymes. We are releasing M-CSA as a new website and underlying database architecture. At the moment, M-CSA contains 961 entries, 423 of these with detailed mechanism information, and 538 with information on the catalytic site residues only. In total, these cover 81% (195/241) of third level EC numbers with a PDB structure, and 30% (840/2793) of fourth level EC numbers with a PDB structure, out of 6028 in total. By searching for close homologues, we are able to extend M-CSA coverage of PDB and UniProtKB to 51 993 structures and to over five million sequences, respectively, of which about 40% and 30% have a conserved active site. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Direct accessibility of mixed-metal (III/II) acid sites through the rational synthesis of porous metal carboxylates.

PubMed

Wongsakulphasatch, S; Nouar, F; Rodriguez, J; Scott, L; Le Guillouzer, C; Devic, T; Horcajada, P; Grenèche, J-M; Llewellyn, P L; Vimont, A; Clet, G; Daturi, M; Serre, C

2015-06-25

The scalable and environmentally-friendly synthesis of mixed Fe(III)/M(II) (M = Ni, Co, Mg) polycarboxylate porous MOFs based on the Secondary Building Unit approach is reported. A combination of in situ infrared spectroscopy, (57)Fe Mössbauer spectrometry and adsorption microcalorimetry confirms the direct accessibility of the iron(III) and metal(II) sites under low temperature activation conditions.

Prediction of active sites of enzymes by maximum relevance minimum redundancy (mRMR) feature selection.

PubMed

Gao, Yu-Fei; Li, Bi-Qing; Cai, Yu-Dong; Feng, Kai-Yan; Li, Zhan-Dong; Jiang, Yang

2013-01-27

Identification of catalytic residues plays a key role in understanding how enzymes work. Although numerous computational methods have been developed to predict catalytic residues and active sites, the prediction accuracy remains relatively low with high false positives. In this work, we developed a novel predictor based on the Random Forest algorithm (RF) aided by the maximum relevance minimum redundancy (mRMR) method and incremental feature selection (IFS). We incorporated features of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure and solvent accessibility to predict active sites of enzymes and achieved an overall accuracy of 0.885687 and MCC of 0.689226 on an independent test dataset. Feature analysis showed that every category of the features except disorder contributed to the identification of active sites. It was also shown via the site-specific feature analysis that the features derived from the active site itself contributed most to the active site determination. Our prediction method may become a useful tool for identifying the active sites and the key features identified by the paper may provide valuable insights into the mechanism of catalysis.

Endovascular Revascularization of Chronically Thrombosed Arteriovenous Fistulas and Grafts for Hemodialysis: A Retrospective Study in 15 Patients With 18 Access Sites

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

Weng Meijui; Chen, Matt Chiung-Yu, E-mail: jjychen@gmail.com; Chi Wenche

2011-04-15

The current study retrospectively evaluated whether endovascular revascularization of chronically thrombosed and long-discarded vascular access sites for hemodialysis was feasible. Technical and clinical success rates, postintervention primary and secondary patency rates, and complications were reported. During a 1-year period, we reviewed a total of 924 interventions performed for dysfunction and/or failed hemodialysis vascular access sites and permanent catheters in 881 patients. In patients whose vascular access-site problems were considered untreatable or were considered treatable with a high risk of failure and access-site abandonment, we attempted to revascularize (resurrect) the chronically occluded and long-discarded (mummy) vascular access sites. We attempted tomore » resurrect a total of 18 mummy access sites (mean age 46.6 {+-} 38.7 months; range 5-144) in 15 patients (8 women and 7 men; mean age 66.2 {+-} 11.5 years; age range 50-85) and had an overall technical success rate of 77.8%. Resurrection failure occurred in 3 fistulas and in 1 straight graft. The clinical success rate was 100% at 2 months after resurrection. In the 14 resurrected vascular access sites, 6 balloon-assisted maturation procedures were required in 5 fistulas; after access-site maturation, a total of 22 interventions were performed to maintain access-site patency. The mean go-through time for successful resurrection procedures was 146.6 {+-} 34.3 min (range 74-193). Postmaturation primary patency rates were 71.4 {+-} 12.1% at 30 days, 57.1 {+-} 13.2% at 60 days, 28.6 {+-} 13.4% at 90 days, and 19 {+-} 11.8% at 180 days. Postmaturation secondary patency rates were 100% at 30, 60, and 90 days and 81.8 {+-} 11.6% at 180 days. There were 2 major complications consisting of massive venous ruptures in 2 mummy access sites during balloon dilation; in both cases, prolonged balloon inflation failed to achieve hemostasis, but percutaneous N-butyl cyanoacrylate glue seal-off was performed

Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites

NASA Astrophysics Data System (ADS)

Lu, Cheng-Tsung; Chen, Shu-An; Bretaña, Neil Arvin; Cheng, Tzu-Hsiu; Lee, Tzong-Yi

2011-10-01

In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator (http://csb.cse.yzu.edu.tw/Carboxylator/), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.

Evaluation of the content and accessibility of web sites for accredited orthopaedic sports medicine fellowships.

PubMed

Mulcahey, Mary K; Gosselin, Michelle M; Fadale, Paul D

2013-06-19

The Internet is a common source of information for orthopaedic residents applying for sports medicine fellowships, with the web sites of the American Orthopaedic Society for Sports Medicine (AOSSM) and the San Francisco Match serving as central databases. We sought to evaluate the web sites for accredited orthopaedic sports medicine fellowships with regard to content and accessibility. We reviewed the existing web sites of the ninety-five accredited orthopaedic sports medicine fellowships included in the AOSSM and San Francisco Match databases from February to March 2012. A Google search was performed to determine the overall accessibility of program web sites and to supplement information obtained from the AOSSM and San Francisco Match web sites. The study sample consisted of the eighty-seven programs whose web sites connected to information about the fellowship. Each web site was evaluated for its informational value. Of the ninety-five programs, fifty-one (54%) had links listed in the AOSSM database. Three (3%) of all accredited programs had web sites that were linked directly to information about the fellowship. Eighty-eight (93%) had links listed in the San Francisco Match database; however, only five (5%) had links that connected directly to information about the fellowship. Of the eighty-seven programs analyzed in our study, all eighty-seven web sites (100%) provided a description of the program and seventy-six web sites (87%) included information about the application process. Twenty-one web sites (24%) included a list of current fellows. Fifty-six web sites (64%) described the didactic instruction, seventy (80%) described team coverage responsibilities, forty-seven (54%) included a description of cases routinely performed by fellows, forty-one (47%) described the role of the fellow in seeing patients in the office, eleven (13%) included call responsibilities, and seventeen (20%) described a rotation schedule. Two Google searches identified direct links for

Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

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

Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.

2015-06-04

Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiO x domains are titrated on TiO x–SiO 2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiO x, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H 2O 2 activation occurs. We use this method to determine the active sitemore » densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h -1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.« less

Web-Based Geographic Information System Tool for Accessing Hanford Site Environmental Data

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

Triplett, Mark B.; Seiple, Timothy E.; Watson, David J.

Data volume, complexity, and access issues pose severe challenges for analysts, regulators and stakeholders attempting to efficiently use legacy data to support decision making at the U.S. Department of Energy’s (DOE) Hanford Site. DOE has partnered with the Pacific Northwest National Laboratory (PNNL) on the PHOENIX (PNNL-Hanford Online Environmental Information System) project, which seeks to address data access, transparency, and integration challenges at Hanford to provide effective decision support. PHOENIX is a family of spatially-enabled web applications providing quick access to decades of valuable scientific data and insight through intuitive query, visualization, and analysis tools. PHOENIX realizes broad, public accessibilitymore » by relying only on ubiquitous web-browsers, eliminating the need for specialized software. It accommodates a wide range of users with intuitive user interfaces that require little or no training to quickly obtain and visualize data. Currently, PHOENIX is actively hosting three applications focused on groundwater monitoring, groundwater clean-up performance reporting, and in-tank monitoring. PHOENIX-based applications are being used to streamline investigative and analytical processes at Hanford, saving time and money. But more importantly, by integrating previously isolated datasets and developing relevant visualization and analysis tools, PHOENIX applications are enabling DOE to discover new correlations hidden in legacy data, allowing them to more effectively address complex issues at Hanford.« less

Binding site in eag voltage sensor accommodates a variety of ions and is accessible in closed channel.

PubMed

Silverman, William R; Bannister, John P A; Papazian, Diane M

2004-11-01

In ether-a-go-go K+ channels, voltage-dependent activation is modulated by ion binding to a site located in an extracellular-facing crevice between transmembrane segments S2 and S3 in the voltage sensor. We find that acidic residues D278 in S2 and D327 in S3 are able to coordinate a variety of divalent cations, including Mg2+, Mn2+, and Ni2+, which have qualitatively similar functional effects, but different half-maximal effective concentrations. Our data indicate that ions binding to individual voltage sensors in the tetrameric channel act without cooperativity to modulate activation gating. We have taken advantage of the unique phenotype of Ni2+ in the D274A channel, which contains a mutation of a nonbinding site residue, to demonstrate that ions can access the binding site from the extracellular solution when the voltage sensor is in the resting conformation. Our results are difficult to reconcile with the x-ray structure of the KvAP K+ channel, in which the binding site residues are widely separated, and with the hydrophobic paddle model for voltage-dependent activation, in which the voltage sensor domain, including the S3-S4 loop, is near the cytoplasmic side of the membrane in the closed channel.

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

PubMed Central

2016-01-01

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1.

PubMed

Blaha-Nelson, David; Krüger, Dennis M; Szeler, Klaudia; Ben-David, Moshe; Kamerlin, Shina Caroline Lynn

2017-01-25

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the k cat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.

Molecular dynamics explorations of active site structure in designed and evolved enzymes.

PubMed

Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

2015-04-21

, a noncatalytic arrangement of the catalytic triad is dominant. Unnatural truncated substrates are inactive because of the lack of protein-protein interactions provided by the ACP. Directed evolution is able to gradually restore the catalytic organization of the active site by motion of the protein backbone that alters the active site geometry. In the third case, we demonstrate the key role of MD in combination with crystallography to identify the origins of substrate-dependent stereoselectivities in a number of Codexis-engineered ketoreductases, one of which is used commercially for the production of the antibiotic sulopenem. Here, mutations alter the shape of the active site as well as the accessibility of water to different regions of it. Each of these examples reveals something different about how mutations can influence enzyme activity and shows that directed evolution, like natural evolution, can increase catalytic activity in a variety of remarkable and often subtle ways.

Access site-related complications after transradial catheterization can be reduced with smaller sheath size and statins.

PubMed

Honda, Tsuyoshi; Fujimoto, Kazuteru; Miyao, Yuji; Koga, Hidenobu; Hirata, Yoshihiro

2012-09-01

The aim of this study was to investigate the risk factors for access site-related complications after transradial coronary angiography (CAG) or percutaneous coronary intervention (PCI). Transradial PCI has been shown to reduce access site-related bleeding complications compared with procedures performed through a femoral approach. Although previous studies focused on risk factors for access site-related complications after a transfemoral approach or transfemoral and transradial approaches, it is uncertain which factors affect vascular complications after transradial catheterization. We enrolled 500 consecutive patients who underwent transradial CAG or PCI. We determined the incidence and risk factors for access site-related complications such as radial artery occlusion and bleeding complications. Age, sheath size, the dose of heparin and the frequency of PCI (vs. CAG) were significantly greater in patients with than without bleeding complications. However, body mass index (BMI) was significantly lower in patients with than without bleeding complications. Sheath size was significantly higher and the frequency of statin use was significantly lower in patients with than without radial artery occlusion. Multiple logistic analysis revealed that sheath size [odds ratio (OR) 5.5; P < 0.05] and BMI (OR 0.86; P < 0.01) were risk factors for bleeding complications; and sheath size (OR 5.2; P < 0.05) and the lack of statin pretreatment (OR 0.50; P < 0.05) were risk factors for occlusive complications. In conclusion, these findings indicate that down-sizing of the devices used in transradial procedures might attenuate access site-related complications after transradial CAG or PCI. Statin pretreatment might also be a strategy that could prevent radial artery occlusion after transradial procedures.

22 CFR 502.6 - Terms of use for accessing program materials available on agency Web sites.

Code of Federal Regulations, 2014 CFR

2014-04-01

... available on agency Web sites. 502.6 Section 502.6 Foreign Relations BROADCASTING BOARD OF GOVERNORS... program materials available on agency Web sites. (a) By accessing Agency Web sites, Requestors agree to all the Terms of Use available on those Web sites. (b) All Requestors are advised that Agency program...

LASIC: Light Activated Site-Specific Conjugation of Native IgGs.

PubMed

Hui, James Z; Tamsen, Shereen; Song, Yang; Tsourkas, Andrew

2015-08-19

Numerous biological applications, from diagnostic assays to immunotherapies, rely on the use of antibody-conjugates. The efficacy of these conjugates can be significantly influenced by the site at which Immunoglobulin G (IgG) is modified. Current methods that provide control over the conjugation site, however, suffer from a number of shortfalls and often require large investments of time and cost. We have developed a novel adapter protein that, when activated by long wavelength UV light, can covalently and site-specifically label the Fc region of nearly any native, full-length IgG, including all human IgG subclasses. Labeling occurs with unprecedented efficiency and speed (>90% after 30 min), with no effect on IgG affinity. The adapter domain can be bacterially expressed and customized to contain a variety of moieties (e.g., biotin, azide, fluorophores), making reliable and efficient conjugation of antibodies widely accessible to researchers at large.

Simple Enough--Even for Web Virgins: Lisa Mitten's Access to Native American Web Sites. Web Site Review Essay.

ERIC Educational Resources Information Center

Belgarde, Mary Jiron

1998-01-01

A mixed-blood Mohawk urban Indian and university librarian, Lisa Mitten provides access to Web sites with solid information about American Indians. Links are provided to 10 categories--Native nations, Native organizations, Indian education, Native media, powwows and festivals, Indian music, Native arts, Native businesses, and Indian-oriented home…

Beyond Section 508: The Spectrum of Legal Requirements for Accessible e-Government Web Sites in the United States

ERIC Educational Resources Information Center

Jaeger, Paul T.

2004-01-01

In the United States, a number of federal laws establish requirements that electronic government (e-government) information and services be accessible to individuals with disabilities. These laws affect e-government Web sites at the federal, state, and local levels. To this point, research about the accessibility of e-government Web sites has…

47 CFR 79.109 - Activating accessibility features.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ACCESSIBILITY OF VIDEO PROGRAMMING Apparatus § 79.109 Activating accessibility features. (a) Requirements... video programming transmitted in digital format simultaneously with sound, including apparatus designed to receive or display video programming transmitted in digital format using Internet protocol, with...

Single or Multiple Access Channels to the CYP450s Active Site? An Answer from Free Energy Simulations of the Human Aromatase Enzyme.

PubMed

Magistrato, Alessandra; Sgrignani, Jacopo; Krause, Rolf; Cavalli, Andrea

2017-05-04

Cytochromes P450 (CYP450s), in particular, CYP19A1 and CYP17A1, are key clinical targets of breast and prostate anticancer therapies, critical players in drug metabolism, and their overexpression in tumors is associated with drug resistance. In these enzymes, ligand (substrates, drugs) metabolism occurs in deeply buried active sites accessible only via several grueling channels, whose exact biological role remains unclear. Gaining direct insights on the mechanism by which ligands travel in and out is becoming increasingly important given that channels are involved in the modulation of binding/dissociation kinetics and the specificity of ligands toward a CYP450. This has profound implications for enzymatic efficiency and drug efficacy/toxicity. Here, by applying free energy methods, for a cumulative simulation time of 20 μs, we provide detailed atomistic characterization and free energy profiles of the entry/exit routes preferentially followed by a substrate (androstenedione) and a last-generation inhibitor (letrozole) to/from the catalytic site of CYP19A1 (the human aromatase (HA) enzyme), a key clinical target against breast cancer, studied here as prototypical CYP450. Despite the remarkably different size/shape/hydrophobicity of the ligands, two channels appear accessible to their entrance, while only one exit route appears to be preferential. Our study shows that the preferential paths may be conserved among different CYP450s. Moreover, our results highlight that, at least in the case of HA, ligand channeling is associated with large enzyme structural rearrangements. A wise choice of the computational method and very long simulations are, thus, required to obtain fully converged quantitative free energy profiles, which might be used to design novel biocatalysts or next-generation cytochrome inhibitors with an in silico tuned K m .

Vascular access site complication in transfemoral coronary angiography between uninterrupted warfarin and heparin bridging.

PubMed

Wongcharoen, Wanwarang; Pinyosamosorn, Kittipong; Gunaparn, Siriluck; Boonnayhun, Suchada; Thonghong, Tasalak; Suwannasom, Pannipa; Phrommintikul, Arintaya

2017-08-01

Warfarin discontinuation with heparin bridging is a common practice in patients receiving warfarin prior to elective coronary angiography (CAG). The uninterrupted warfarin strategy has been suggested to be alternative option for patients with high thromboembolic risk. Therefore, we aimed to assess the safety of elective transfemoral CAG during uninterrupted warfarin therapy compared to heparin bridging. This study was a randomized open-label design with blinded event evaluation. The 110 consecutive patients (age ≥ 18 years) receiving warfarin before the planned transfemoral CAG were randomly assigned to either heparin bridging or uninterrupted warfarin with targeted INR (2.0-3.0). The primary outcome was the incidence of major vascular access site complications. The baseline characteristics were comparable between two groups (mean age was 60.1 ± 7.8 years, 49 males). The mean INR on the day of CAG of heparin bridging and uninterrupted warfarin groups was 1.2 ± 0.3 and 2.2 ± 0.5 (P < 0.001). The major vascular access site complications occurred in 3 of 55 (5.5%) heparin-bridging patients and in none of 55 uninterrupted warfarin patients (P = 0.243). The total vascular access site complications occurred in 6 (10.9%) heparin-bridging and one (1.8%) uninterrupted warfarin patients (P = 0.113). No patient developed either other bleeding or thromboembolic events during 7 days after CAG. We demonstrated that an uninterrupted warfarin strategy did not increase vascular access site complications in patients undergoing transfemoral CAG compared to heparin bridging therapy. Due to the safety and the ease of uninterrupted warfarin strategy, this approach should be encouraged in patients receiving long-term warfarin who undergo elective transfemoral CAG. © 2017, Wiley Periodicals, Inc.

Role of the Conserved Valine 236 in Access of Ligands to the Active Site of Thermus thermophilus ba3 Cytochrome Oxidase.

PubMed

Funatogawa, Chie; Li, Yang; Chen, Ying; McDonald, William; Szundi, Istvan; Fee, James A; Stout, C David; Einarsdóttir, Ólöf

2017-01-10

Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba 3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O 2 , and NO binding were investigated. O 2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a 3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by Cu A . This interpretation is supported by classical molecular dynamics simulations of diffusion of O 2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a 3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba 3 .

Exploring the Active Site of the Tungsten, Iron-Sulfur Enzyme Acetylene Hydratase▿ †

PubMed Central

tenBrink, Felix; Schink, Bernhard; Kroneck, Peter M. H.

2011-01-01

The soluble tungsten, iron-sulfur enzyme acetylene hydratase (AH) from mesophilic Pelobacter acetylenicus is a member of the dimethyl sulfoxide (DMSO) reductase family. It stands out from its class as it catalyzes a nonredox reaction, the addition of H2O to acetylene (H—C☰C—H) to form acetaldehyde (CH3CHO). Caught in its active W(IV) state, the high-resolution three-dimensional structure of AH offers an excellent starting point to tackle its unique chemistry and to identify catalytic amino acid residues within the active site cavity: Asp13 close to W(IV) coordinated to two molybdopterin-guanosine-dinucleotide ligands, Lys48 which couples the [4Fe-4S] cluster to the W site, and Ile142 as part of a hydrophobic ring at the end of the substrate access channel designed to accommodate the substrate acetylene. A protocol was developed to express AH in Escherichia coli and to produce active-site variants which were characterized with regard to activity and occupancy of the tungsten and iron-sulfur centers. By this means, fusion of the N-terminal chaperone binding site of the E. coli nitrate reductase NarG to the AH gene improved the yield and activity of AH and its variants significantly. Results from site-directed mutagenesis of three key residues, Asp13, Lys48, and Ile142, document their important role in catalysis of this unusual tungsten enzyme. PMID:21193613

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.

Accessibility Trends among Academic Library and Library School Web Sites in the USA and Canada

ERIC Educational Resources Information Center

Schmetzke, Axel; Comeaux, David

2009-01-01

This paper focuses on the accessibility of North American library and library school Web sites for all users, including those with disabilities. Web accessibility data collected in 2006 are compared to those of 2000 and 2002. The findings of this follow-up study continue to give cause for concern: Despite improvements since 2002, library and…

A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

NASA Astrophysics Data System (ADS)

Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

2015-11-01

A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

An Ecological Approach to Exploring Rural Food Access and Active Living for Families With Preschoolers.

PubMed

Buro, Brandy; Gold, Abby; Contreras, Dawn; Keim, Ann L; Mobley, Amy R; Oscarson, Renee; Peters, Paula; Procter, Sandy; Smathers, Carol

2015-01-01

To identify factors using the Ecological Model of Childhood Overweight related to accessing nutritious foods and physical activity opportunities from the perspectives of rural parents of preschoolers. A mixed-methods study using a quantitative survey (Active Where?) and qualitative interviews. Analyzed interview themes provided context to the survey results. The setting was Head Start centers, county human service offices, and Women, Infants, and Children Program sites in rural counties in the Midwest. Rural parents (n = 377) of preschoolers took part in the survey in 7 Midwestern states; 15 similar participants were interviewed from 1 of the states. Transcribed interviews were coded. Frequencies and chi-square tests were computed; significance was set at P < .05. The Active Where? survey and interviews revealed that close proximity to recreation spaces and traffic safety issues influenced physical activity. For food access, close proximity to full service grocery stores did not influence access to healthy foods because respondents traveled to urban communities to purchase healthy foods. Public transportation solutions and enhanced neighborhood safety are potential community-wide obesity prevention strategies in rural communities. However, interventions should be tailored to the community's stage of readiness. Strong social networks should be considered an asset for community change in these regions. Copyright © 2015 Society for Nutrition Education and Behavior. Published by Elsevier Inc. All rights reserved.

Individual Public Transportation Accessibility is Positively Associated with Self-Reported Active Commuting.

PubMed

Djurhuus, Sune; Hansen, Henning Sten; Aadahl, Mette; Glümer, Charlotte

2014-01-01

Active commuters have lower risk of chronic disease. Understanding which of the, to some extent, modifiable characteristics of public transportation that facilitate its use is thus important in a public health perspective. The aim of the study was to examine the association between individual public transportation accessibility and self-reported active commuting, and whether the associations varied with commute distance, age, and gender. Twenty-eight thousand nine hundred twenty-eight commuters in The Capital Region of Denmark reported self-reported time spent either walking or cycling to work or study each day and the distance to work or study. Data were obtained from the Danish National Health Survey collected in February to April 2010. Individual accessibility by public transportation was calculated using a multi-modal network in a GIS. Multilevel logistic regression was used to analyze the association between accessibility, expressed as access area, and being an active commuter. Public transport accessibility area based on all stops within walking and cycling distance was positively associated with being an active commuter. Distance to work, age, and gender modified the associations. Residing within 10 km commute distance and in areas of high accessibility was associated with being an active commuter and meeting the recommendations of physical activity. For the respondents above 29 years, individual public transportation accessibility was positively associated with being an active commuter. Women having high accessibility had significantly higher odds of being an active commuter compared to having a low accessibility. For men, the associations were insignificant. This study extends the knowledge about the driving forces of using public transportation for commuting by examining the individual public transportation accessibility. Findings suggest that transportation accessibility supports active commuting and planning of improved public transit accessibility

Individual Public Transportation Accessibility is Positively Associated with Self-Reported Active Commuting

PubMed Central

Djurhuus, Sune; Hansen, Henning Sten; Aadahl, Mette; Glümer, Charlotte

2014-01-01

Background: Active commuters have lower risk of chronic disease. Understanding which of the, to some extent, modifiable characteristics of public transportation that facilitate its use is thus important in a public health perspective. The aim of the study was to examine the association between individual public transportation accessibility and self-reported active commuting, and whether the associations varied with commute distance, age, and gender. Methods: Twenty-eight thousand nine hundred twenty-eight commuters in The Capital Region of Denmark reported self-reported time spent either walking or cycling to work or study each day and the distance to work or study. Data were obtained from the Danish National Health Survey collected in February to April 2010. Individual accessibility by public transportation was calculated using a multi-modal network in a GIS. Multilevel logistic regression was used to analyze the association between accessibility, expressed as access area, and being an active commuter. Results: Public transport accessibility area based on all stops within walking and cycling distance was positively associated with being an active commuter. Distance to work, age, and gender modified the associations. Residing within 10 km commute distance and in areas of high accessibility was associated with being an active commuter and meeting the recommendations of physical activity. For the respondents above 29 years, individual public transportation accessibility was positively associated with being an active commuter. Women having high accessibility had significantly higher odds of being an active commuter compared to having a low accessibility. For men, the associations were insignificant. Conclusion: This study extends the knowledge about the driving forces of using public transportation for commuting by examining the individual public transportation accessibility. Findings suggest that transportation accessibility supports active commuting and planning

Partisan Activism and Access to Welfare in Lebanon

PubMed Central

2014-01-01

How do welfare regimes function when state institutions are weak and ethnic or sectarian groups control access to basic services? This paper explores how people gain access to basic services in Lebanon, where sectarian political parties from all major religious communities are key providers of social assistance and services. Based on analyses of an original national survey (n= 1,911) as well as in-depth interviews with providers and other elites (n= 175) and beneficiaries of social programs (n= 135), I make two main empirical claims in the paper. First, political activism and a demonstrated commitment to a party are associated with access to social assistance; and second, higher levels of political activism may facilitate access to higher levels or quantities of aid, including food baskets and financial assistance for medical and educational costs. These arguments highlight how politics can mediate access to social assistance in direct ways and add new dimensions to scholarly debates about clientelism by focusing on contexts with politicized religious identities and by problematizing the actual goods and services exchanged. PMID:24904187

Enabling Remote Access to Fieldwork: Gaining Insight into the Pedagogic Effectiveness of "Direct" and "Remote" Field Activities

ERIC Educational Resources Information Center

Stokes, Alison; Collins, Trevor; Maskall, John; Lea, John; Lunt, Paul; Davies, Sarah

2012-01-01

This study considers the pedagogical effectiveness of remote access to fieldwork locations. Forty-one students from across the GEES disciplines (geography, earth and environmental sciences) undertook a fieldwork exercise, supported by two lecturers. Twenty students accessed the field site directly and the remainder accessed the site remotely using…

UTSA-74: A MOF-74 Isomer with Two Accessible Binding Sites per Metal Center for Highly Selective Gas Separation.

PubMed

Luo, Feng; Yan, Changsheng; Dang, Lilong; Krishna, Rajamani; Zhou, Wei; Wu, Hui; Dong, Xinglong; Han, Yu; Hu, Tong-Liang; O'Keeffe, Michael; Wang, Lingling; Luo, Mingbiao; Lin, Rui-Biao; Chen, Banglin

2016-05-04

A new metal-organic framework Zn2(H2O)(dobdc)·0.5(H2O) (UTSA-74, H4dobdc = 2,5-dioxido-1,4-benzenedicarboxylic acid), Zn-MOF-74/CPO-27-Zn isomer, has been synthesized and structurally characterized. It has a novel four coordinated fgl topology with one-dimensional channels of about 8.0 Å. Unlike metal sites in the well-established MOF-74 with a rod-packing structure in which each of them is in a five coordinate square pyramidal coordination geometry, there are two different Zn(2+) sites within the binuclear secondary building units in UTSA-74 in which one of them (Zn1) is in a tetrahedral while another (Zn2) in an octahedral coordination geometry. After activation, the two axial water molecules on Zn2 sites can be removed, generating UTSA-74a with two accessible gas binding sites per Zn2 ion. Accordingly, UTSA-74a takes up a moderately high and comparable amount of acetylene (145 cm(3)/cm(3)) to Zn-MOF-74. Interestingly, the accessible Zn(2+) sites in UTSA-74a are bridged by carbon dioxide molecules instead of being terminally bound in Zn-MOF-74, so UTSA-74a adsorbs a much smaller amount of carbon dioxide (90 cm(3)/cm(3)) than Zn-MOF-74 (146 cm(3)/cm(3)) at room temperature and 1 bar, leading to a superior MOF material for highly selective C2H2/CO2 separation. X-ray crystal structures, gas sorption isotherms, molecular modeling, and simulated and experimental breakthroughs comprehensively support this result.

Patients undergoing PCI from the femoral route by default radial operators are at high risk of vascular access-site complications.

PubMed

Rafie, Ihsan M; Uddin, Muez M; Ossei-Gerning, Nicholas; Anderson, Richard A; Kinnaird, Timothy D

2014-02-01

Radial artery (RA) access for PCI has a lower incidence of vascular access-site (VAS) complications than the femoral artery (FA) approach. However, even for default radial operators certain patients are intervened upon from the FA. We examined the demographics and incidence of VAS complications when default radial operators resort to the FA for PCI. The demographics and VAS complications were compared by access site retrospectively for all PCI cases performed by default radial operators (n=1,392). A modified ACUITY trial definition of major VAS complication was used. FA puncture occurred in 25.2% (351/1,392) of cases. Patients were more likely to be female, older and weigh less than patients undergoing PCI from the RA. The FA procedure was likely to be more complex with larger sheaths, more left main stem, graft and multivessel intervention, and there was a greater proportion of emergency cases. Despite increased case complexity, glycoprotein inhibitors were used less frequently in femoral cases (26.5% vs. 36.8%, p<0.001). A VAS complication occurred in 12.5% (44/351) of cases. The risk factors for access-site bleeding are disproportionately high in the population requiring FA puncture by default radial operators, and as a result such patients have a high rate of vascular access-site complications.

Preliminary access routes and cost study analyses for seven potentially acceptable salt sites: Final report, October 1984

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

Not Available

1987-02-01

This report analyzes highway and railroad access to seven potentially acceptable salt repository sites: Richton Dome and Cypress Creek Dome in Mississippi, Vacherie Dome in Louisiana, Swisher County and Deaf Smith County in Texas, and Davis Canyon and Lavender Canyon in utah. The objectives of the study were to investigate the routing of reasonable access corridors to the sites, describe major characteristics of each route, and estimate the costs for constructing or upgrading highways and railroads. The routes used in the analysis are not necessarily recommended or preferred over other routes, nor do they represent an implied final selection. Detailedmore » engineering studies must be performed for the Davis Canyon and Lavender Canyon highway access before the analyzed routes can be considered to be viable. 20 refs., 7 figs., 3 tabs.« less

Digital Accessible Knowledge and well-inventoried sites for birds in Mexico: baseline sites for measuring faunistic change

PubMed Central

Navarro-Sigüenza, Adolfo G.; Martínez-Meyer, Enrique

2016-01-01

Background Faunal change is a basic and fundamental element in ecology, biogeography, and conservation biology, yet vanishingly few detailed studies have documented such changes rigorously over decadal time scales. This study responds to that gap in knowledge, providing a detailed analysis of Digital Accessible Knowledge of the birds of Mexico, designed to marshal DAK to identify sites that were sampled and inventoried rigorously prior to the beginning of major global climate change (1980). Methods We accumulated DAK records for Mexican birds from all relevant online biodiversity data portals. After extensive cleaning steps, we calculated completeness indices for each 0.05° pixel across the country; we also detected ‘hotspots’ of sampling, and calculated completeness indices for these broader areas as well. Sites were designated as well-sampled if they had completeness indices above 80% and >200 associated DAK records. Results We identified 100 individual pixels and 20 broader ‘hotspots’ of sampling that were demonstrably well-inventoried prior to 1980. These sites are catalogued and documented to promote and enable resurvey efforts that can document events of avifaunal change (and non-change) across the country on decadal time scales. Conclusions Development of repeated surveys for many sites across Mexico, and particularly for sites for which historical surveys document their avifaunas prior to major climate change processes, would pay rich rewards in information about distributional dynamics of Mexican birds. PMID:27651986

Digital Accessible Knowledge and well-inventoried sites for birds in Mexico: baseline sites for measuring faunistic change.

PubMed

Peterson, A Townsend; Navarro-Sigüenza, Adolfo G; Martínez-Meyer, Enrique

2016-01-01

Faunal change is a basic and fundamental element in ecology, biogeography, and conservation biology, yet vanishingly few detailed studies have documented such changes rigorously over decadal time scales. This study responds to that gap in knowledge, providing a detailed analysis of Digital Accessible Knowledge of the birds of Mexico, designed to marshal DAK to identify sites that were sampled and inventoried rigorously prior to the beginning of major global climate change (1980). We accumulated DAK records for Mexican birds from all relevant online biodiversity data portals. After extensive cleaning steps, we calculated completeness indices for each 0.05° pixel across the country; we also detected 'hotspots' of sampling, and calculated completeness indices for these broader areas as well. Sites were designated as well-sampled if they had completeness indices above 80% and >200 associated DAK records. We identified 100 individual pixels and 20 broader 'hotspots' of sampling that were demonstrably well-inventoried prior to 1980. These sites are catalogued and documented to promote and enable resurvey efforts that can document events of avifaunal change (and non-change) across the country on decadal time scales. Development of repeated surveys for many sites across Mexico, and particularly for sites for which historical surveys document their avifaunas prior to major climate change processes, would pay rich rewards in information about distributional dynamics of Mexican birds.

Local health department activities to ensure access to care.

PubMed

Luo, Huabin; Sotnikov, Sergey; Shah, Gulzar

2013-12-01

Local health departments (LHDs) can play an important role in linking people to personal health services and ensuring the provision of health care when it is otherwise unavailable. However, the extent to which LHDs are involved in ensuring access to health care in its jurisdictions is not well known. To provide nationally representative estimates of LHD involvement in specific activities to ensure access to healthcare services and to assess their association with macro-environment/community and LHD capacity and process characteristics. Data used were from the 2010 National Profile of Local Health Departments Study, Area Resource Files, and the Association of State and Territorial Health Officials' 2010 Profile of State Public Health Agencies Survey. Data were analyzed in 2012. Approximately 66.0% of LHDs conducted activities to ensure access to medical care, 45.9% to dental care, and 32.0% to behavioral health care. About 28% of LHDs had not conducted activities to ensure access to health care in their jurisdictions in 2010. LHDs with higher per capita expenditures and larger jurisdiction population sizes were more likely to provide access to care services (p <0.05). There is substantial variation in LHD engagement in activities to ensure access to care. Differences in LHD capacity and the needs of the communities in which they are located may account for this variation. Further research is needed to determine whether this variation is associated with adverse population health outcomes. © 2013 American Journal of Preventive Medicine Published by American Journal of Preventive Medicine All rights reserved.

Web Accessibility and Accessibility Instruction

ERIC Educational Resources Information Center

Green, Ravonne A.; Huprich, Julia

2009-01-01

Section 508 of the Americans with Disabilities Act (ADA) mandates that programs and services be accessible to people with disabilities. While schools of library and information science (SLIS*) and university libraries should model accessible Web sites, this may not be the case. This article examines previous studies about the Web accessibility of…

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?

Ty1-copia elements reveal diverse insertion sites linked to polymorphisms among flax (Linum usitatissimum L.) accessions.

PubMed

Galindo-González, Leonardo; Mhiri, Corinne; Grandbastien, Marie-Angèle; Deyholos, Michael K

2016-12-07

Initial characterization of the flax genome showed that Ty1-copia retrotransposons are abundant, with several members being recently inserted, and in close association with genes. Recent insertions indicate a potential for ongoing transpositional activity that can create genomic diversity among accessions, cultivars or varieties. The polymorphisms generated constitute a good source of molecular markers that may be associated with phenotype if the insertions alter gene activity. Flax, where accessions are bred mainly for seed nutritional properties or for fibers, constitutes a good model for studying the relationship of transpositional activity with diversification and breeding. In this study, we estimated copy number and used a type of transposon display known as Sequence-Specific Amplification Polymorphisms (SSAPs), to characterize six families of Ty1-copia elements across 14 flax accessions. Polymorphic insertion sites were sequenced to find insertions that could potentially alter gene expression, and a preliminary test was performed with selected genes bearing transposable element (TE) insertions. Quantification of six families of Ty1-copia elements indicated different abundances among TE families and between flax accessions, which suggested diverse transpositional histories. SSAPs showed a high level of polymorphism in most of the evaluated retrotransposon families, with a trend towards higher levels of polymorphism in low-copy number families. Ty1-copia insertion polymorphisms among cultivars allowed a general distinction between oil and fiber types, and between spring and winter types, demonstrating their utility in diversity studies. Characterization of polymorphic insertions revealed an overwhelming association with genes, with insertions disrupting exons, introns or within 1 kb of coding regions. A preliminary test on the potential transcriptional disruption by TEs of four selected genes evaluated in three different tissues, showed one case of significant

41 CFR 60-300.81 - Access to records.

Code of Federal Regulations, 2014 CFR

2014-07-01

... REGARDING DISABLED VETERANS, RECENTLY SEPARATED VETERANS, ACTIVE DUTY WARTIME OR CAMPAIGN BADGE VETERANS... conducting on-site compliance evaluations and complaint investigations and inspecting and copying such books... provide OFCCP access to these materials, including electronic records, off-site for purposes of conducting...

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

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

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

Impact of arterial access site on outcomes after primary percutaneous coronary intervention: prespecified subgroup analysis from the EUROMAX trial.

PubMed

Hamon, Martial; Coste, Pierre; Van't Hof, Arnoud; Ten Berg, Jurrien; Clemmensen, Peter; Tabone, Xavier; Benamer, Hakim; Kristensen, Steen D; Cavallini, Claudio; Marzocchi, Antonio; Hamm, Christian; Kanic, Vojko; Bernstein, Debra; Anthopoulos, Prodromos; Deliargyris, Efthymios N; Steg, Philippe Gabriel

2015-06-01

In European Ambulance Acute Coronary Syndrome Angiography (EUROMAX), bivalirudin improved 30-day clinical outcomes with reduced major bleeding compared with heparins plus optional glycoprotein IIb/IIIa inhibitors. We assessed whether choice of access site (radial or femoral) had an impact on 30-day outcomes and whether it interacted with the benefit of bivalirudin. In EUROMAX, choice of arterial access was left to operator discretion. Overall, 47% of patients underwent radial and 53% femoral access. Baseline risk was higher in the femoral access group. Unadjusted proportions for the primary outcome (death or noncoronary artery bypass graft protocol major bleeding at 30 days) were lower with radial access, however, without differences in major or major plus minor bleeding proportions. After multivariable adjustment, ischemic outcomes were no longer different between access site groups, except for a lower risk of stroke in radial patients. Bivalirudin was associated with lower proportions of the primary outcome in both the radial (odds ratio, 0.58; 95% CI, 0.33-1.03; P=0.058) and the femoral groups (odds ratio, 0.59; 95% CI, 0.37-0.93; P=0.022; interaction P=0.97). Bleeding was significantly lower in the bivalirudin group both in the radial- and femoral-treated patients but no significant difference was observed in ischemic outcomes. In multivariable analysis, bivalirudin emerged as the only independent predictor of reduced major bleeding (odds ratio, 0.45; 95% CI, 0.27-0.74; P=0.002). In this prespecified analysis from EUROMAX, radial access was preferred in lower risk patients and did not improve clinical outcomes. Bivalirudin was associated with less bleeding irrespective of access site. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01087723. © 2015 American Heart Association, Inc.

Site-selective and stereoselective functionalization of non-activated tertiary C-H bonds

NASA Astrophysics Data System (ADS)

Liao, Kuangbiao; Pickel, Thomas C.; Boyarskikh, Vyacheslav; Bacsa, John; Musaev, Djamaladdin G.; Davies, Huw M. L.

2017-11-01

The synthesis of complex organic compounds usually relies on controlling the reactions of the functional groups. In recent years, it has become possible to carry out reactions directly on the C-H bonds, previously considered to be unreactive. One of the major challenges is to control the site-selectivity because most organic compounds have many similar C-H bonds. The most well developed procedures so far rely on the use of substrate control, in which the substrate has one inherently more reactive C-H bond or contains a directing group or the reaction is conducted intramolecularly so that a specific C-H bond is favoured. A more versatile but more challenging approach is to use catalysts to control which site in the substrate is functionalized. p450 enzymes exhibit C-H oxidation site-selectivity, in which the enzyme scaffold causes a specific C-H bond to be functionalized by placing it close to the iron-oxo haem complex. Several studies have aimed to emulate this enzymatic site-selectivity with designed transition-metal catalysts but it is difficult to achieve exceptionally high levels of site-selectivity. Recently, we reported a dirhodium catalyst for the site-selective functionalization of the most accessible non-activated (that is, not next to a functional group) secondary C-H bonds by means of rhodium-carbene-induced C-H insertion. Here we describe another dirhodium catalyst that has a very different reactivity profile. Instead of the secondary C-H bond, the new catalyst is capable of precise site-selectivity at the most accessible tertiary C-H bonds. Using this catalyst, we modify several natural products, including steroids and a vitamin E derivative, indicating the applicability of this method of synthesis to the late-stage functionalization of complex molecules. These studies show it is possible to achieve site-selectivity at different positions within a substrate simply by selecting the appropriate catalyst. We hope that this work will inspire the design of

Access to parks and physical activity: an eight country comparison.

PubMed

Schipperijn, Jasper; Cerin, Ester; Adams, Marc A; Reis, Rodrigo; Smith, Graham; Cain, Kelli; Christiansen, Lars B; van Dyck, Delfien; Gidlow, Christopher; Frank, Lawrence D; Mitáš, Josef; Pratt, Michael; Salvo, Deborah; Schofield, Grant; Sallis, James F

2017-10-01

Several systematic reviews have reported mixed associations between access to parks and physical activity, and suggest that this is due to inconsistencies in the study methods or differences across countries. An international study using consistent methods is needed to investigate the association between access to parks and physical activity. The International Physical Activity and Environment Network (IPEN) Adult Study is a multi-country cross-sectional study using a common design and consistent methods. Accelerometer, survey and Geographic Information Systems (GIS) data for 6,181 participants from 12 cities in 8 countries (Belgium, Brazil, Czech Republic, Denmark, Mexico, New Zealand, UK, USA) were used to estimate the strength and shape of associations of 11 measures of park access (1 perceived and 10 GIS-based measures) with accelerometer-based moderate-to-vigorous physical activity (MVPA) and four types of self-reported leisure-time physical activity. Associations were estimated using generalized additive mixed models. More parks within 1 km from participants' homes were associated with greater leisure-time physical activity and accelerometer-measured MVPA. Respondents who lived in the neighborhoods with the most parks did on average 24 minutes more MVPA per week than those living in the neighborhoods with the lowest number of parks. Perceived proximity to a park was positively associated with multiple leisure-time physical activity outcomes. Associations were homogeneous across all cities studied. Living in neighborhoods with many parks could contribute with up to 1/6 of the recommended weekly Having multiple parks nearby was the strongest positive correlate of PA. To increase comparability and validity of park access measures, we recommend that researchers, planners and policy makers use the number of parks within 1 km travel distance of homes as an objective indicator for park access in relation to physical activity.

78 FR 67881 - Nondiscrimination on the Basis of Disability in Air Travel: Accessibility of Web Sites and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-11-12

... corresponding accessible pages on a mobile Web site by one year after the final rule's effective date; and (3... Mobile Web site conformant with any of the following standards: WCAG 1.0, WCAG 2.0 at Level A, existing Section 508 standards, or Mobile Web Best Practices (MWBP) 1.0 (if applicable). Two of the options they...

Former UST Site Reused as Greenspace and Access to Ancient Ceremonial Nikwasi Mound in North Carolina

EPA Pesticide Factsheets

Read about a former underground storage tank site fronting on the Little Tennessee River in Franklin, NC that is now reused as an attractive greenspace with parking and Main Street access to the Nikwasi Mound.

40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Public access to paper copies of off-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY AND DEPARTMENT OF JUSTICE ACCIDENTAL RELEASE PREVENTION REQUIREMENTS; RISK MANAGEMENT...

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules

PubMed Central

Davidge, Kelly S; Singh, Sandip; Bowman, Lesley AH; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

2015-01-01

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and •NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels interrupted by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify •NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, •NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations affect both the tunnels accessibility as well as the affinity of distal site water molecules, thus modifying the ligand access to the iron. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site. PMID:26478812

Ligand uptake in Mycobacterium tuberculosis truncated hemoglobins is controlled by both internal tunnels and active site water molecules.

PubMed

Boron, Ignacio; Bustamante, Juan Pablo; Davidge, Kelly S; Singh, Sandip; Bowman, Lesley Ah; Tinajero-Trejo, Mariana; Carballal, Sebastián; Radi, Rafael; Poole, Robert K; Dikshit, Kanak; Estrin, Dario A; Marti, Marcelo A; Boechi, Leonardo

2015-01-01

Mycobacterium tuberculosis, the causative agent of human tuberculosis, has two proteins belonging to the truncated hemoglobin (trHb) family. Mt-trHbN presents well-defined internal hydrophobic tunnels that allow O 2 and • NO to migrate easily from the solvent to the active site, whereas Mt-trHbO possesses tunnels interrupted by a few bulky residues, particularly a tryptophan at position G8. Differential ligand migration rates allow Mt-trHbN to detoxify • NO, a crucial step for pathogen survival once under attack by the immune system, much more efficiently than Mt-trHbO. In order to investigate the differences between these proteins, we performed experimental kinetic measurements, • NO decomposition, as well as molecular dynamics simulations of the wild type Mt-trHbN and two mutants, VG8F and VG8W. These mutations affect both the tunnels accessibility as well as the affinity of distal site water molecules, thus modifying the ligand access to the iron. We found that a single mutation allows Mt-trHbN to acquire ligand migration rates comparable to those observed for Mt-trHbO, confirming that ligand migration is regulated by the internal tunnel architecture as well as by water molecules stabilized in the active site.

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.

Molecular investigation of active binding site of isoniazid (INH) and insight into resistance mechanism of S315T-MtKatG in Mycobacterium tuberculosis.

PubMed

Srivastava, Gaurava; Tripathi, Shubhandra; Kumar, Akhil; Sharma, Ashok

2017-07-01

Multi drug resistant tuberculosis is a major threat for mankind. Resistance against Isoniazid (INH), targeting MtKatG protein, is one of the most commonly occurring resistances in MDR TB strains. S315T-MtKatG mutation is widely reported for INH resistance. Despite having knowledge about the mechanism of INH, exact binding site of INH to MtKatG is still uncertain and proposed to have three presumable binding sites (site-1, site-2, and site-3). In the current study docking, molecular dynamics simulation, binding free energy estimation, principal component analysis and free energy landscape analysis were performed to get molecular level details of INH binding site on MtKatG, and to probe the effect of S315T mutation on INH binding. Molecular docking and MD analysis suggested site-1 as active binding site of INH, where the effects of S315T mutation were observed on both access tunnel as well as molecular interaction between INH and its neighboring residues. MMPBSA also supported site-1 as potential binding site with lowest binding energy of -44.201 kJ/mol. Moreover, PCA and FEL revealed that S315T mutation not only reduces the dimension of heme access tunnel but also showed that extra methyl group at 315 position altered heme cavity, enforcing heme group distantly from INH, and thus preventing INH activation. The present study not only investigated the active binding site of INH but also provides a new insight about the conformational changes in the binding site of S315T-MtKatG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Spatial accessibility to vaccination sites in a campaign against rabies in São Paulo city, Brazil.

PubMed

Polo, Gina; Acosta, Carlos Mera; Dias, Ricardo Augusto

2013-08-01

It is estimated that the city of São Paulo has over 2.5 million dogs and 560 thousand cats. These populations are irregularly distributed throughout the territory, making it difficult to appropriately allocate health services focused on these species. To reasonably allocate vaccination sites, it is necessary to identify social groups and their access to the referred service. Rabies in dogs and cats has been an important zoonotic health issue in São Paulo and the key component of rabies control is vaccination. The present study aims to introduce an approach to quantify the potential spatial accessibility to the vaccination sites of the 2009 campaign against rabies in the city of São Paulo and solve the overestimation associated with the classic methodology that applies buffer zones around vaccination sites based on Euclidean (straight-line) distance. To achieve this, a Gaussian-based two-step floating catchment area method with a travel-friction coefficient was adapted in a geographic information system environment, using distances along a street network based on Dijkstra's algorithm (short path method). The choice of the distance calculation method affected the results in terms of the population covered. In general, areas with low accessibility for both dogs and cats were observed, especially in densely populated areas. The eastern zone of the city had higher accessibility values compared with peripheral and central zones. The Gaussian-based two-step floating catchment method with a travel-friction coefficient was used to assess the overestimation of the straight-line distance method, which is the most widely used method for coverage analysis. We conclude that this approach has the potential to improve the efficiency of resource use when planning rabies control programs in large urban environments such as São Paulo. The findings emphasize the need for surveillance and intervention in isolated areas. Copyright © 2013 Elsevier B.V. All rights reserved.

Neighbourhood access to open spaces and the physical activity of residents: a national study.

PubMed

Witten, Karen; Hiscock, Rosemary; Pearce, Jamie; Blakely, Tony

2008-09-01

Increasing population levels of physical activity is high on the health agenda in many countries. There is some evidence that neighbourhood access to public open space can increase physical activity by providing easier and more direct access to opportunities for exercise. This national study examines the relationship between travel time access to parks and beaches, BMI and physical activity in New Zealand neighbourhoods. Access to parks and beaches, measured in minutes taken by a car, was calculated for 38,350 neighbourhoods nationally using Geographic Information Systems. Multilevel regression analyses were used to establish the significance of access to these recreational amenities as a predictor of BMI, and levels of physical activity and sedentary behaviour in the 12,529 participants, living in 1178 neighbourhoods, of the New Zealand Health Survey 2002/3. Neighbourhood access to parks was not associated with BMI, sedentary behaviour or physical activity, after controlling for individual-level socio-economic variables, and neighbourhood-level deprivation and urban/rural status. There was some evidence of a relationship between beach access and BMI and physical activity in the expected direction. This study found little evidence of an association between locational access to open spaces and physical activity.

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.

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

Atomistic characterization of the active-site solvation dynamics of a model photocatalyst

DOE PAGES

van Driel, Tim B.; Kjær, Kasper S.; Hartsock, Robert W.; ...

2016-11-28

The interactions between the reactive excited state of molecular photocatalysts and surrounding solvent dictate reaction mechanisms and pathways, but are not readily accessible to conventional optical spectroscopic techniques. Here we report an investigation of the structural and solvation dynamics following excitation of a model photocatalytic molecular system [Ir 2(dimen) 4] 2+, where dimen is para-diisocyanomenthane. The time-dependent structural changes in this model photocatalyst, as well as the changes in the solvation shell structure, have been measured with ultrafast diffuse X-ray scattering and simulated with Born-Oppenheimer Molecular Dynamics. Both methods provide direct access to the solute–solvent pair distribution function, enabling themore » solvation dynamics around the catalytically active iridium sites to be robustly characterized. Our results provide evidence for the coordination of the iridium atoms by the acetonitrile solvent and demonstrate the viability of using diffuse X-ray scattering at free-electron laser sources for studying the dynamics of photocatalysis.« less

Experience with ActiveX control for simple channel access

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

Timossi, C.; Nishimura, H.; McDonald, J.

2003-05-15

Accelerator control system applications at Berkeley Lab's Advanced Light Source (ALS) are typically deployed on operator consoles running Microsoft Windows 2000 and utilize EPICS[2]channel access for data access. In an effort to accommodate the wide variety of Windows based development tools and developers with little experience in network programming, ActiveX controls have been deployed on the operator stations. Use of ActiveX controls for use in the accelerator control environment has been presented previously[1]. Here we report on some of our experiences with the use and development of these controls.

Accessibility for persons with mobility impairments within an informal trading site: A case study on the markets of Warwick, South Africa

PubMed Central

Koch, Helga Elke; Anderson, Jassmine; Ghela, Prashika; Govender, Perusha; Hoosen, Nausheena; Khan, Halima

2014-01-01

Background There are a number of informal trading sites across cities in sub-Saharan Africa, of which the markets of Warwick is one example. Since the informal economy is an important contributor to a city’s economy as well as a source of employment, it is important for these sites to be accessible for all persons. Whilst the South African government has put structures in place to identify and remove environmental barriers in order to meet the individual needs of persons with mobility impairments and improve their quality of life, persons with mobility impairments still face barriers and restricting environments that prevent them from participating in society and its social and economic activities. Objectives This case study aimed at exploring accessibility within the markets of Warwick for persons with mobility impairments by an ergonomic assessment, augmented by voices of participants within the market. Method A qualitative, instrumental, single case study design was utilised with purposive sampling of the markets of Warwick as the study setting. Multiple sources of data were gathered, such as semi-structured interviews, direct observations of an environmental survey supported by photographs, and the authors’ review of relevant documents. Transcriptions were analysed using NVivo 10 software programme with inductive coding. Results Whilst policies have been in place since 1996 to adjust infrastructure, the markets of Warwick still remain inaccessible to persons with mobility impairments and do not meet the standardised infrastructural design. Conclusion The findings of this study may offer a significant understanding of the complexity of accessibility within an informal trading site and create an awareness of the limitations this has for persons with mobility impairments. Additionally, these findings may assist in effecting a positive change in terms of the infrastructure of the Markets and in continuous advocating for the rights of persons with all

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

77 FR 69454 - Emergency Access Advisory Committee; Announcement of Date of Next Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-19

... subcommittees and other activities needed to ensure access to 911 by individuals with disabilities. DATES: The... Generation 911 (NG 9-1-1) emergency services by individuals with disabilities, and to make recommendations to... and other activities needed to ensure access to 911 by individuals with disabilities. The meeting site...

78 FR 23564 - Emergency Access Advisory Committee; Announcement of Date of Next Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-04-19

... activities needed to ensure access to 911 by individuals with disabilities. DATES: The Committee's next... Generation 911 (NG 9-1-1) emergency services by individuals with disabilities, and to make recommendations to... activities needed to ensure access to 911 by individuals with disabilities. The meeting site is fully...

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.

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes.

PubMed

Endrizzi, James A; Beernink, Peter T

2017-11-01

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non-polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild-type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate-analog bound conformation and the third hinge angle is intermediate to the other two. The active-site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active-site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active-site conformations. © 2017 The Protein Society.

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.

Characteristics, Access, Utilization, Satisfaction, and Outcomes of Healthy Start Participants in Eight Sites

PubMed Central

O’Neil, So; Cook, Benjamin; Trebino, Lisa; Walker, Deborah Klein

2009-01-01

To describe the characteristics, access, utilization, satisfaction, and outcomes of Healthy Start participants in eight selected sites, a survey of Healthy Start participants with infants ages 6–12-months-old at time of interview was conducted between October 2006 and January 2007. The response rate was 66% (n = 646), ranging from 37% in one site to >70% in seven sites. Healthy Start participants’ outcomes were compared to two national benchmarks. Healthy Start participants reported that they were satisfied with the program (>90% on five measures). Level of unmet need was 6% or less for most services, except for dental appointments (11%), housing (13%), and child care (11%). Infants had significantly better access to medical care than did their mothers, with higher rates of insurance coverage, medical homes, and checkups, and fewer unmet needs for health care. Healthy Start participants’ rates of ever breastfeeding (72%) and putting infants to sleep on their backs (70%) were at or near the Healthy People 2010 objectives, and considerably higher than rates among low-income mothers in the ECLS. The high rate of health education (>90%) may have contributed to these outcomes. Elimination of smoking among Healthy Start participants (46%) fell short of the Healthy People 2010 objective (99%). The low-birth weight (LBW) rate among Black Healthy Start participants (14%) was three times higher than the rate for Whites and Hispanics (5% each). Overall, the LBW rate in the eight sites (7.5%) was similar to the rate for low-income mothers in the ECLS, but both rates were above the Healthy People 2010 objective (5%). Challenges remain in reducing disparities in maternal and child health outcomes. Further attention to risk factors associated with LBW (especially smoking) may help close the gaps. The life course theory suggests that improved outcomes may require longer-term investments. Healthy Start’s emerging focus on interconception care has the potential to address

Evaluation of the Content and Accessibility of Web Sites for Accredited Orthopaedic Trauma Surgery Fellowships.

PubMed

Shaath, M Kareem; Yeranosian, Michael G; Ippolito, Joseph A; Adams, Mark R; Sirkin, Michael S; Reilly, Mark C

2018-05-02

Orthopaedic trauma fellowship applicants use online-based resources when researching information on potential U.S. fellowship programs. The 2 primary sources for identifying programs are the Orthopaedic Trauma Association (OTA) database and the San Francisco Match (SF Match) database. Previous studies in other orthopaedic subspecialty areas have demonstrated considerable discrepancies among fellowship programs. The purpose of this study was to analyze content and availability of information on orthopaedic trauma surgery fellowship web sites. The online databases of the OTA and SF Match were reviewed to determine the availability of embedded program links or external links for the included programs. Thereafter, a Google search was performed for each program individually by typing the program's name, followed by the term "orthopaedic trauma fellowship." All identified fellowship web sites were analyzed for accessibility and content. Web sites were evaluated for comprehensiveness in mentioning key components of the orthopaedic trauma surgery curriculum. By consensus, we refined the final list of variables utilizing the methodology of previous studies on the topic. We identified 54 OTA-accredited fellowship programs, offering 87 positions. The majority (94%) of programs had web sites accessible through a Google search. Of the 51 web sites found, all (100%) described their program. Most commonly, hospital affiliation (88%), operative experiences (76%), and rotation overview (65%) were listed, and, least commonly, interview dates (6%), selection criteria (16%), on-call requirements (20%), and fellow evaluation criteria (20%) were listed. Programs with ≥2 fellows provided more information with regard to education content (p = 0.0001) and recruitment content (p = 0.013). Programs with Accreditation Council for Graduate Medical Education (ACGME) accreditation status also provided greater information with regard to education content (odds ratio, 4.0; p = 0.0001). Otherwise

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

STS-61B Astronaut Ross During ACCESS Extravehicular Activity

NASA Technical Reports Server (NTRS)

1985-01-01

The crew assigned to the STS-61B mission included Bryan D. O'Conner, pilot; Brewster H. Shaw, commander; Charles D. Walker, payload specialist; mission specialists Jerry L. Ross, Mary L. Cleave, and Sherwood C. Spring; and Rodolpho Neri Vela, payload specialist. Launched aboard the Space Shuttle Atlantis November 28, 1985 at 7:29:00 pm (EST), the STS-61B mission's primary payload included three communications satellites: MORELOS-B (Mexico); AUSSAT-2 (Australia); and SATCOM KU-2 (RCA Americom). Two experiments were conducted to test assembling erectable structures in space: EASE (Experimental Assembly of Structures in Extravehicular Activity), and ACCESS (Assembly Concept for Construction of Erectable Space Structure). In a joint venture between NASA/Langley Research Center in Hampton, VA and the Marshall Space Flight Center (MSFC), ACCESS and EASE were developed and demonstrated at MSFC's Neutral Buoyancy Simulator (NBS). In this STS-61B onboard photo, astronaut Ross was working on the ACCESS experiment during an Extravehicular Activity (EVA). The primary objective of this experiment was to test the ACCESS structural assembly concept for suitability as the framework for larger space structures and to identify ways to improve the productivity of space construction.

Polycomb Group Repression Reduces DNA Accessibility

PubMed Central

Fitzgerald, Daniel P.; Bender, Welcome

2001-01-01

The Polycomb group proteins are responsible for long-term repression of a number of genes in Drosophila melanogaster, including the homeotic genes of the bithorax complex. The Polycomb protein is thought to alter the chromatin structure of its target genes, but there has been little direct evidence for this model. In this study, the chromatin structure of the bithorax complex was probed with three separate assays for DNA accessibility: (i) activation of polymerase II (Pol II) transcription by Gal4, (ii) transcription by the bacteriophage T7 RNA polymerase (T7RNAP), and (iii) FLP-mediated site-specific recombination. All three processes are restricted or blocked in Polycomb-repressed segments. In contrast, control test sites outside of the bithorax complex permitted Gal4, T7RNAP, and FLP activities throughout the embryo. Several P insertions in the bithorax complex were tested, providing evidence that the Polycomb-induced effect is widespread over target genes. This accessibility effect is similar to that seen for SIR silencing in Saccharomyces cerevisiae. In contrast to SIR silencing, however, episomes excised from Polycomb-repressed chromosomal sites do not show an altered superhelix density. PMID:11533246

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

The Decision to Access Patient Information from a Social Media Site: What Would You Do?

PubMed Central

Jent, Jason F.; Eaton, Cyd K.; Merrick, Melissa T.; Englebert, Nicole E.; Dandes, Susan K.; Chapman, Ana V.; Hershorin, Eugene R.

2011-01-01

Purpose The current study examined the prevalence with which healthcare providers use a social media site account (e.g., Facebook), the extent to which they utilize social media sites in clinical practice, and their decision-making process after accessing patient information from a social media site. Methods Pediatric faculty and trainees from a medical school campus were provided a social media site history form and seven fictional social media site adolescent profile vignettes that depicted concerning information. Participants were instructed to rate their personal use and beliefs about social media sites and to report how they would respond if they obtained concerning information about an adolescent patient from their public social media site profile. Results Healthcare providers generally believed it not to be an invasion of privacy to conduct an Internet/social media site search of someone they know. A small percentage of trainees reported a personal history of conducting an Internet search (18%) or a social media site search (14%) for a patient. However, no faculty endorsed a history of conducting searches for patients. Faculty and trainees also differed in how they would respond to concerning social media site adolescent profile information. Conclusions The findings that trainees are conducting Internet/social media site searches of patients and that faculty and trainees differ in how they would respond to concerning profile information suggest the need for specific guidelines regarding the role of social media sites in clinical practice. Practice, policy, and training implications are discussed. PMID:21939873

48 CFR 311.7001 - Section 508 accessibility standards for HHS Web site content and communications materials.

Code of Federal Regulations, 2011 CFR

2011-10-01

... standards, and resolve any related issues. (c) Based on those discussions, the Project Officer shall provide... communication must meet the accessibility standards in 36 CFR 1194.22, “Web-based intranet and Internet... standards for HHS Web site content and communications materials. 311.7001 Section 311.7001 Federal...

48 CFR 311.7001 - Section 508 accessibility standards for HHS Web site content and communications materials.

Code of Federal Regulations, 2013 CFR

2013-10-01

... standards, and resolve any related issues. (c) Based on those discussions, the Project Officer shall provide... communication must meet the accessibility standards in 36 CFR 1194.22, “Web-based intranet and Internet... standards for HHS Web site content and communications materials. 311.7001 Section 311.7001 Federal...

48 CFR 311.7001 - Section 508 accessibility standards for HHS Web site content and communications materials.

Code of Federal Regulations, 2012 CFR

2012-10-01

... standards, and resolve any related issues. (c) Based on those discussions, the Project Officer shall provide... communication must meet the accessibility standards in 36 CFR 1194.22, “Web-based intranet and Internet... standards for HHS Web site content and communications materials. 311.7001 Section 311.7001 Federal...

48 CFR 311.7001 - Section 508 accessibility standards for HHS Web site content and communications materials.

Code of Federal Regulations, 2014 CFR

2014-10-01

... standards, and resolve any related issues. (c) Based on those discussions, the Project Officer shall provide... communication must meet the accessibility standards in 36 CFR 1194.22, “Web-based intranet and Internet... standards for HHS Web site content and communications materials. 311.7001 Section 311.7001 Federal...

Predictors of patient non-attendance at Improving Access to Psychological Therapy services demonstration sites

PubMed Central

Di Bona, Laura; Saxon, David; Barkham, Michael; Dent-Brown, Kim; Parry, Glenys

2014-01-01

Background Improving Access to Psychological Therapy (IAPT) services have increased the number of people with common mental health disorders receiving psychological therapy in England, but concerns remain about how equitably these services are accessed. Method Using cohort patient data (N=363) collected as part of the independent evaluation of the two demonstration sites, logistic regression was utilised to identify socio-demographic, clinical and service factors predictive of IAPT non-attendance. Results Significant predictors of IAPT first session non-attendance by patients were: lower non-risk score on the Clinical Outcomes in Routine Evaluation-Outcome Measure (CORE-OM); more frequent thoughts of “being better off dead” (derived from the CORE-OM); either a very recent onset of common mental health disorder (1 month or less) or a long term condition (more than 2 years); and site. Limitations The small sample and low response rate are limitations, as the sample may not be representative of all those referred to IAPT services. The predictive power of the logistic regression model is limited and suggests other variables not available in the dataset may also be important predictors. Conclusions The clinical characteristics of risk to self, severity of emotional distress, and illness duration, along with site, were more predictive of IAPT non-attendance than socio-demographic characteristics. Further testing of the relationship between these variables and IAPT non-attendance is recommended. Clinicians should monitor IAPT uptake in those they refer and implement strategies to increase their engagement with services, particularly when referring people presenting with suicidal ideation or more chronic illness. PMID:25194784

Patient Factors But Not the Use of Novel Anticoagulants or Warfarin Are Associated With Internal Jugular Vein Access-Site Hematoma After Right Heart Catheterization.

PubMed

Dasa, Osama; Shafiq, Qaiser; Ruzieh, Mohammed; Alhazmi, Luai; Al-Dabbas, Maen; Ammari, Zaid; Khouri, Samer; Moukarbel, George

2017-12-01

Right heart catheterization (RHC) is routinely performed to assess hemodynamics. Generally, anticoagulants are held prior to the procedure. At our center, anticoagulants are continued and ultrasound guidance is always used for internal jugular vein access. A micropuncture access kit is used to place a 5 or 6 Fr sheath using the modified Seldinger technique. Manual compression is applied for 10-15 min and the patient is observed for at least 2 hours after the procedure. In a retrospective analysis, we investigated the risk of bleeding complications associated with RHC via the internal jugular vein in patients with and without full anticoagulation. Our catheterization laboratory database was searched for adult patients who underwent RHC by a single operator between January 2012 and December 2015. A total of 571 patients were included in the analysis. Baseline characteristics, labs, relevant invasive hemodynamics, co-morbid conditions, and incidence of access-site hematoma are presented. Multivariable binary logistic regression was performed using IBM SPSS v. 23.0 software. Statistically significant associations with access-site hematoma were observed with body mass index (P=.02; 95% confidence interval [CI], 1.0-1.1), right atrial pressure (P=.03; 95% CI, 0.7-0.9), and dialysis dependence (P<.01; 95% CI, 0.1-0.6). There was no association of access-site hematoma with the use of anticoagulants (P>.99). The incidence of internal jugular vein access-site hematoma is small when using careful access techniques for RHC even with the continued use of novel oral anticoagulants and warfarin. Patient characteristics and co-morbid conditions are related to bleeding complications.

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.

High Plains Regional Ground-water Study web site

USGS Publications Warehouse

Qi, Sharon L.

2000-01-01

Now available on the Internet is a web site for the U.S. Geological Survey's (USGS) National Water-Quality Assessment (NAWQA) Program-High Plains Regional Ground-Water Study. The purpose of the web site is to provide public access to a wide variety of information on the USGS investigation of the ground-water resources within the High Plains aquifer system. Typical pages on the web site include the following: descriptions of the High Plains NAWQA, the National NAWQA Program, the study-area setting, current and past activities, significant findings, chemical and ancillary data (which can be downloaded), listing and access to publications, links to other sites about the High Plains area, and links to other web sites studying High Plains ground-water resources. The High Plains aquifer is a regional aquifer system that underlies 174,000 square miles in parts of eight States (Colorado, Kansas, Nebraska, New Mexico, Oklahoma, South Dakota, Texas, and Wyoming). Because the study area is so large, the Internet is an ideal way to provide project data and information on a near real-time basis. The web site will be a collection of living documents where project data and information are updated as it becomes available throughout the life of the project. If you have an interest in the High Plains area, you can check this site periodically to learn how the High Plains NAWQA activities are progressing over time and access new data and publications as they become available.

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.

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

PubMed Central

Tien, Jason; Peters, Christian J; Wong, Xiu Ming; Cheng, Tong; Jan, Yuh Nung; Jan, Lily Yeh; Yang, Huanghe

2014-01-01

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001 PMID:24980701

The Association between Access to Public Transportation and Self-Reported Active Commuting

PubMed Central

Djurhuus, Sune; Hansen, Henning S.; Aadahl, Mette; Glümer, Charlotte

2014-01-01

Active commuting provides routine-based regular physical activity which can reduce the risk of chronic diseases. Using public transportation involves some walking or cycling to a transit stop, transfers and a walk to the end location and users of public transportation have been found to accumulate more moderate physical activity than non-users. Understanding how public transportation characteristics are associated with active transportation is thus important from a public health perspective. This study examines the associations between objective measures of access to public transportation and self-reported active commuting. Self-reported time spent either walking or cycling commuting each day and the distance to workplace were obtained for adults aged 16 to 65 in the Danish National Health Survey 2010 (n = 28,928). Access to public transportation measures were computed by combining GIS-based road network distances from home address to public transit stops an integrating their service level. Multilevel logistic regression was used to examine the association between access to public transportation measures and active commuting. Distance to bus stop, density of bus stops, and number of transport modes were all positively associated with being an active commuter and with meeting recommendations of physical activity. No significant association was found between bus services at the nearest stop and active commuting. The results highlight the importance of including detailed measurements of access to public transit in order to identify the characteristics that facilitate the use of public transportation and active commuting. PMID:25489998

The association between access to public transportation and self-reported active commuting.

PubMed

Djurhuus, Sune; Hansen, Henning S; Aadahl, Mette; Glümer, Charlotte

2014-12-05

Active commuting provides routine-based regular physical activity which can reduce the risk of chronic diseases. Using public transportation involves some walking or cycling to a transit stop, transfers and a walk to the end location and users of public transportation have been found to accumulate more moderate physical activity than non-users. Understanding how public transportation characteristics are associated with active transportation is thus important from a public health perspective. This study examines the associations between objective measures of access to public transportation and self-reported active commuting. Self-reported time spent either walking or cycling commuting each day and the distance to workplace were obtained for adults aged 16 to 65 in the Danish National Health Survey 2010 (n = 28,928). Access to public transportation measures were computed by combining GIS-based road network distances from home address to public transit stops an integrating their service level. Multilevel logistic regression was used to examine the association between access to public transportation measures and active commuting. Distance to bus stop, density of bus stops, and number of transport modes were all positively associated with being an active commuter and with meeting recommendations of physical activity. No significant association was found between bus services at the nearest stop and active commuting. The results highlight the importance of including detailed measurements of access to public transit in order to identify the characteristics that facilitate the use of public transportation and active commuting.

Downgrade of the Savannah River Sites FB-Line

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

SADOWSKI, ED; YOURCHAK, RANDY; PRETZELLO MARJI

2005-07-05

This paper will discuss the Safeguards & Security (S&S) activities that resulted in the downgrade of the Savannah River Site's FB-Line (FBL) from a Category I Material Balance Area (MBA) in a Material Access Area (MAA) to a Category IV MBA in a Property Protection Area (PPA). The Safeguards activities included measurement of final product items, transferal of nuclear material to other Savannah River Site (SRS) facilities, discard of excess nuclear material items, and final measurements of holdup material. The Security activities included relocation and destruction of classified documents and repositories, decertification of a classified computer, access control changes, updatesmore » to planning documents, deactivation and removal of security systems, Human Reliability Program (HRP) removals, and information security training for personnel that will remain in the FBL PPA.« less

IAU astroEDU: an open-access platform for peer-reviewed astronomy education activities

NASA Astrophysics Data System (ADS)

Heenatigala, Thilina; Russo, Pedro; Strubbe, Linda; Gomez, Edward

2015-08-01

astroEDU is an open access platform for peer-reviewed astronomy education activities. It addresses key problems in educational repositories such as variability in quality, not maintained or updated regularly, limited content review, and more. This is achieved through a peer-review process similar to what scholarly articles are based on. Activities submitted are peer-reviewed by an educator and a professional astronomer which gives the credibility to the activities. astroEDU activities are open-access in order to make the activities accessible to educators around the world while letting them discover, review, distribute and remix the activities. The activity submission process allows authors to learn how to apply enquiry-based learning into the activity, identify the process skills required, how to develop core goals and objectives, and how to evaluate the activity to determine the outcome. astroEDU is endorsed by the International Astronomical Union meaning each activity is given an official stamp by the international organisation for professional astronomers.

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

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

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.

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.

76 FR 71914 - Nondiscrimination on the Basis of Disability in Air Travel: Accessibility of Web Sites and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-21

... Disability in Air Travel: Accessibility of Web Sites and Automated Kiosks at U.S. Airports AGENCY: Office of... January 9, 2012. This extension is a result of requests from a number of parties for additional time to... constructive comments for the Department's consideration. The Interactive Travel Services Association requested...

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

Accessing Electronic Journals.

ERIC Educational Resources Information Center

McKay, Sharon Cline

1999-01-01

Discusses issues librarians need to consider when providing access to electronic journals. Topics include gateways; index and abstract services; validation and pay-per-view; title selection; integration with OPACs (online public access catalogs)or Web sites; paper availability; ownership versus access; usage restrictions; and services offered…

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.

Chapter 6: Children's Environmental Access in Relation to Motor Competence, Physical Activity, and Fitness

ERIC Educational Resources Information Center

Erwin, Heather E.; Woods, Amelia Mays; Woods, Martha K.; Castelli, Darla M.

2007-01-01

The purpose of this study was to examine levels of physical activity engagement, motor competence, and physical fitness as related to child access to physical activity facilities in the home and school environments. The present investigation attempts to further efforts to examine the relationship between physical activity levels and access.…

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.

STS-61B Astronaut Ross During ACCESS Extravehicular Activity

NASA Technical Reports Server (NTRS)

1985-01-01

The crew assigned to the STS-61B mission included Bryan D. O'Conner, pilot; Brewster H. Shaw, commander; Charles D. Walker, payload specialist; mission specialists Jerry L. Ross, Mary L. Cleave, and Sherwood C. Spring; and Rodolpho Neri Vela, payload specialist. Launched aboard the Space Shuttle Atlantis November 28, 1985 at 7:29:00 pm (EST), the STS-61B mission's primary payload included three communications satellites: MORELOS-B (Mexico); AUSSAT-2 (Australia); and SATCOM KU-2 (RCA Americom). Two experiments were conducted to test assembling erectable structures in space: EASE (Experimental Assembly of Structures in Extravehicular Activity), and ACCESS (Assembly Concept for Construction of Erectable Space Structure). In a joint venture between NASA/Langley Research Center in Hampton, Virginia and the Marshall Space Flight Center (MSFC), EASE and ACCESS were developed and demonstrated at MSFC's Neutral Buoyancy Simulator (NBS). The primary objective of this experiment was to test the structural assembly concepts for suitability as the framework for larger space structures and to identify ways to improve the productivity of space construction. In this STS-61B onboard photo, astronaut Ross was working on the ACCESS experiment during an Extravehicular Activity (EVA).

Policy for Establishing and Maintaining Publicly Accessible Department of Defense Web Information Service

DTIC Science & Technology

1997-07-18

the Marine Corps Exchange. If these sites contain commercial advertisements or sponsorships, the appropriate disclaimer below shall be given...or the information, products or services contained therein. For other than authorized activities such as military exchanges and Morale...posted to the commercial site. 4.9. Design Standards and Non-standard Features ( ActiveX and Java) 4.9.1. Design of publicly accessible web

Nevada National Security Site Environmental Report 2011 Attachment A: Site Description

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

Cathy Wills, ed.

2012-09-12

This attachment expands on the general description of the Nevada National Security Site (NNSS) presented in the Introduction to the Nevada National Security Site Environmental Report 2011. Included are subsections that summarize the site's geological, hydrological, climatological, and ecological setting and the cultural resources of the NNSS. The subsections are meant to aid the reader in understanding the complex physical and biological environment of the NNSS. An adequate knowledge of the site's environment is necessary to assess the environmental impacts of new projects, design and implement environmental monitoring activities for current site operations, and assess the impacts of site operationsmore » on the public residing in the vicinity of the NNSS. The NNSS environment contributes to several key features of the site that afford protection to the inhabitants of adjacent areas from potential exposure to radioactivity or other contaminants resulting from NNSS operations. These key features include the general remote location of the NNSS, restricted access, extended wind transport times, the great depths to slow-moving groundwater, little or no surface water, and low population density. This attachment complements the annual summary of monitoring program activities and dose assessments presented in the main body of this report.« less

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...

Synthetic Active Site Model of the [NiFeSe] Hydrogenase.

PubMed

Wombwell, Claire; Reisner, Erwin

2015-05-26

A dinuclear synthetic model of the [NiFeSe] hydrogenase active site and a structural, spectroscopic and electrochemical analysis of this complex is reported. [NiFe('S2Se2')(CO)3] (H2'S2Se2' = 1,2-bis(2-thiabutyl-3,3-dimethyl-4-selenol)benzene) has been synthesized by reacting the nickel selenolate complex [Ni('S2Se2')] with [Fe(CO)3bda] (bda = benzylideneacetone). X-ray crystal structure analysis confirms that [NiFe('S2Se2')(CO)3] mimics the key structural features of the enzyme active site, including a doubly bridged heterobimetallic nickel and iron center with a selenolate terminally coordinated to the nickel center. Comparison of [NiFe('S2Se2')(CO)3] with the previously reported thiolate analogue [NiFe('S4')(CO)3] (H2'S4' = H2xbsms = 1,2-bis(4-mercapto-3,3-dimethyl-2-thiabutyl)benzene) showed that the selenolate groups in [NiFe('S2Se2')(CO)3] give lower carbonyl stretching frequencies in the IR spectrum. Electrochemical studies of [NiFe('S2Se2')(CO)3] and [NiFe('S4')(CO)3] demonstrated that both complexes do not operate as homogenous H2 evolution catalysts, but are precursors to a solid deposit on an electrode surface for H2 evolution catalysis in organic and aqueous solution. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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...

Affective regulation of stereotype activation: It’s the (accessible) thought that counts

PubMed Central

Huntsinger, Jeffrey R.; Sinclair, Stacey; Dunn, Elizabeth; Clore, Gerald L.

2010-01-01

Extant research demonstrates that positive affect, compared to negative affect, increases stereotyping. In four experiments we explore whether the link between affect and stereotyping depends, critically, on the relative accessibility of stereotype-relevant thoughts and response tendencies. As well as manipulating mood, we measured or manipulated the accessibility of egalitarian response tendencies (Experiments 1-2) and counter-stereotypic thoughts (Experiments 3-4). In the absence of such response tendencies and thoughts, people in positive moods displayed greater stereotype activation —consistent with past research. By contrast, in the presence of accessible egalitarian response tendencies or counter-stereotypic thoughts, people in positive moods exhibited less stereotype activation than those in negative moods. PMID:20363909

Two signs indicative of successful access in nuclear medicine cerebrospinal fluid diversionary shunt studies.

PubMed

Bermo, Mohammed S; Khalatbari, Hedieh; Parisi, Marguerite T

2018-05-08

Successful shunt access is the first step in a properly performed nuclear medicine cerebrospinal fluid (CSF) shunt study. To determine the significance of the radiotracer configuration at the injection site during initial nuclear medicine CSF shunt imaging and the lack of early systemic radiotracer activity as predictors of successful shunt access. With Institutional Review Board approval, three nuclear medicine physicians performed a retrospective review of all consecutive CSF shunt studies performed in children at our institution in 2015. Antecedent nuclear medicine CSF shunt studies in these patients were also assessed and included in the review. The appearance of the reservoir site immediately after radiotracer injection was classified as either figure-of-eight or round/ovoid configuration. The presence or absence of early systemic distribution of the tracer on the 5-min static images was noted and separately evaluated. A total of 98 nuclear medicine ventriculoperitoneal CSF shunt studies were evaluated. Figure-of-eight configuration was identified in 87% of studies and, when present, had 93% sensitivity, 78% specificity, 92% accuracy, 98% positive predictive value (PPV) and 54% negative predictive value (NPV) as a predictor of successful shunt access. Early systemic activity was absent in 89 of 98 studies. Lack of early systemic distribution of the radiotracer had 98% sensitivity, 78% specificity, 96% accuracy, 98% PPV and 78% NPV as a predictor of successful shunt access. Figure-of-eight configuration in conjunction with the absence of early systemic tracer activity had 99% PPV for successful shunt access. Figure-of-eight configuration at the injection site or lack of early systemic radiotracer activity had moderate specificity for successful shunt access. Specificity and PPV significantly improved when both signs were combined in assessment.

Computer simulation of the active site of human serum cholinesterase

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

Kefang Jiao; Song Li; Zhengzheng Lu

1996-12-31

The first 3D-structure of acetylchelinesterase from Torpedo California electric organ (T.AChE) was published by JL. Sussman in 1991. We have simulated 3D-structure of human serum cholinesterase (H.BuChE) and the active site of H.BuChE. It is discovered by experiment that the residue of H.BuChE is still active site after a part of H.BuChE is cut. For example, the part of 21KD + 20KD is active site of H.BuChE. The 20KD as it is. Studies on these peptides by Hemelogy indicate that two active peptides have same negative electrostatic potential maps diagram. These negative electrostatic areas attached by acetyl choline with positivemore » electrostatic potency. We predict that 147...236 peptide of AChE could be active site because it was as 20KD as with negative electrostatic potential maps. We look forward to proving from other ones.« less

Transcription factor target site search and gene regulation in a background of unspecific binding sites.

PubMed

Hettich, J; Gebhardt, J C M

2018-06-02

Response time and transcription level are vital parameters of gene regulation. They depend on how fast transcription factors (TFs) find and how efficient they occupy their specific target sites. It is well known that target site search is accelerated by TF binding to and sliding along unspecific DNA and that unspecific associations alter the occupation frequency of a gene. However, whether target site search time and occupation frequency can be optimized simultaneously is mostly unclear. We developed a transparent and intuitively accessible state-based formalism to calculate search times to target sites on and occupation frequencies of promoters of arbitrary state structure. Our formalism is based on dissociation rate constants experimentally accessible in live cell experiments. To demonstrate our approach, we consider promoters activated by a single TF, by two coactivators or in the presence of a competitive inhibitor. We find that target site search time and promoter occupancy differentially vary with the unspecific dissociation rate constant. Both parameters can be harmonized by adjusting the specific dissociation rate constant of the TF. However, while measured DNA residence times of various eukaryotic TFs correspond to a fast search time, the occupation frequencies of target sites are generally low. Cells might tolerate low target site occupancies as they enable timely gene regulation in response to a changing environment. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

De novo active sites for resurrected Precambrian enzymes

NASA Astrophysics Data System (ADS)

Risso, Valeria A.; Martinez-Rodriguez, Sergio; Candel, Adela M.; Krüger, Dennis M.; Pantoja-Uceda, David; Ortega-Muñoz, Mariano; Santoyo-Gonzalez, Francisco; Gaucher, Eric A.; Kamerlin, Shina C. L.; Bruix, Marta; Gavira, Jose A.; Sanchez-Ruiz, Jose M.

2017-07-01

Protein engineering studies often suggest the emergence of completely new enzyme functionalities to be highly improbable. However, enzymes likely catalysed many different reactions already in the last universal common ancestor. Mechanisms for the emergence of completely new active sites must therefore either plausibly exist or at least have existed at the primordial protein stage. Here, we use resurrected Precambrian proteins as scaffolds for protein engineering and demonstrate that a new active site can be generated through a single hydrophobic-to-ionizable amino acid replacement that generates a partially buried group with perturbed physico-chemical properties. We provide experimental and computational evidence that conformational flexibility can assist the emergence and subsequent evolution of new active sites by improving substrate and transition-state binding, through the sampling of many potentially productive conformations. Our results suggest a mechanism for the emergence of primordial enzymes and highlight the potential of ancestral reconstruction as a tool for protein engineering.

Access to pedestrian roads, daily activities, and physical performance of adolescents.

PubMed

Sjolie, A N

2000-08-01

A cross-sectional study using a questionnaire and physical tests was performed. To study how access to pedestrian roads and daily activities are related to low back strength, low back mobility, and hip mobility in adolescents. Although many authorities express concern about the passive lifestyle of adolescents, little is known about associations between daily activities and physical performance. This study compared 38 youths in a community lacking access to pedestrian roads with 50 youths in nearby area providing excellent access to pedestrian roads. A standardized questionnaire was used to obtain data about pedestrian roads, school journeys, and activities from the local authorities and the pupils. Low back strength was tested as static endurance strength, low back mobility by modified Schober techniques, and hip mobility by goniometer. For statistical analyses, a P value of 0.05 or less determined significance. In the area using school buses, the pupils had less low back extension, less hamstring flexibility, and less hip abduction, flexion, and extension than pupils in the area with pedestrian roads. Multivariate analyses showed no associations between walking or bicycling to school and anatomic function, but regular walking or bicycling to leisure-time activities associated positively with low back strength, low back extension, hip flexion, and extension. Distance by school bus associated negatively with hip abduction, hip flexion, hip extension, and hamstring flexibility (P<0.001). Time spent on television or computer associated negatively but insignificantly with low back strength, hamstring flexibility, hip abduction, and flexion (P<0.1). The results indicate that access to pedestrian roads and other lifestyle factors are associated with physical performance.

A unique H2A histone variant occupies the transcriptional start site of active genes.

PubMed

Soboleva, Tatiana A; Nekrasov, Maxim; Pahwa, Anuj; Williams, Rohan; Huttley, Gavin A; Tremethick, David J

2011-12-04

Transcriptional activation is controlled by chromatin, which needs to be unfolded and remodeled to ensure access to the transcription start site (TSS). However, the mechanisms that yield such an 'open' chromatin structure, and how these processes are coordinately regulated during differentiation, are poorly understood. We identify the mouse (Mus musculus) H2A histone variant H2A.Lap1 as a previously undescribed component of the TSS of active genes expressed during specific stages of spermatogenesis. This unique chromatin landscape also includes a second histone variant, H2A.Z. In the later stages of round spermatid development, H2A.Lap1 dynamically loads onto the inactive X chromosome, enabling the transcriptional activation of previously repressed genes. Mechanistically, we show that H2A.Lap1 imparts unique unfolding properties to chromatin. We therefore propose that H2A.Lap1 coordinately regulates gene expression by directly opening the chromatin structure of the TSS at genes regulated during spermatogenesis.

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.

Nevada National Security Site Environmental Report 2012 Attachment A: Site Description

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

Wills, Cathy A

This attachment expands on the general description of the Nevada National Security Site (NNSS) presented in the Introduction to the Nevada National Security Site Environmental Report 2012 (National Security Technologies, LLC [NSTec], 2013). Included are subsections that summarize the site’s geological, hydrological, climatological, and ecological setting and the cultural resources of the NNSS. The subsections are meant to aid the reader in understanding the complex physical and biological environment of the NNSS. An adequate knowledge of the site’s environment is necessary to assess the environmental impacts of new projects, design and implement environmental monitoring activities for current site operations, andmore » assess the impacts of site operations on the public residing in the vicinity of the NNSS. The NNSS environment contributes to several key features of the site that afford protection to the inhabitants of adjacent areas from potential exposure to radioactivity or other contaminants resulting from NNSS operations. These key features include the general remote location of the NNSS, restricted access, extended wind transport times, the great depths to slow-moving groundwater, little or no surface water, and low population density. This attachment complements the annual summary of monitoring program activities and dose assessments presented in the main body of this report.« less

Nevada National Security Site Environmental Report 2013 Attachment A: Site Description

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

Wills, C.

This attachment expands on the general description of the Nevada National Security Site (NNSS) presented in the Introduction to the Nevada National Security Site Environmental Report 2012 (National Security Technologies, LLC [NSTec], 2013). Included are subsections that summarize the site’s geological, hydrological, climatological, and ecological setting and the cultural resources of the NNSS. The subsections are meant to aid the reader in understanding the complex physical and biological environment of the NNSS. An adequate knowledge of the site’s environment is necessary to assess the environmental impacts of new projects, design and implement environmental monitoring activities for current site operations, andmore » assess the impacts of site operations on the public residing in the vicinity of the NNSS. The NNSS environment contributes to several key features of the site that afford protection to the inhabitants of adjacent areas from potential exposure to radioactivity or other contaminants resulting from NNSS operations. These key features include the general remote location of the NNSS, restricted access, extended wind transport times, the great depths to slow-moving groundwater, little or no surface water, and low population density. This attachment complements the annual summary of monitoring program activities and dose assessments presented in the main body of this report.« less

10 CFR 960.5-2-2 - Site ownership and control.

Code of Federal Regulations, 2010 CFR

2010-01-01

... NUCLEAR WASTE REPOSITORY Preclosure Guidelines Preclosure Radiological Safety § 960.5-2-2 Site ownership... control of access that are required in order that surface and subsurface activities during repository...

Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

PubMed

Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

2016-08-01

Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

Active Sites Environmental Monitoring Program: Mid-FY 1991 report

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

Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

1991-10-01

This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading ofmore » vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.« less

Polyphenolic Composition and Antioxidant Activities of 6 New Turmeric (Curcuma Longa L.) Accessions.

PubMed

Chinedum, Eleazu; Kate, Eleazu; Sonia, Chukwuma; Ironkwe, Adanma; Andrew, Igwe

2015-01-01

The phytochemical composition and antioxidant capacities of 6 new NRCRI turmeric (Curcuma longa L.) accessions (39, 35, 60, 30, 50 and 41) were determined using standard techniques. The moisture contents of the tumeric samples ranged from 15.75 to 47.80% and the curcumin contents of the turmeric samples fell within the range of curcumin obtained from turmeric in other countries of the world. Furthermore, the turmeric accessions contained considerable amounts of antioxidants (measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and reducing power assays), alkaloids, flavonoids, anthocyanins, and phenolics. There was significant correlation between the anthocyanin contents of the tumeric accessions versus their alkaloid (0.744) and flavonoid contents (0.986) suggesting an additive effect between the anthocyanins and alkaloids in turmeric; significant correlation between the inhibition of the turmeric accessions on DPPH radical versus their flavonoid (0.892) and anthocyanin (0.949) contents and significant correlation between the reducing power of the turmeric accessions versus their flavonoid (0.973) and anthocyanin (0.974) contents suggesting that anthocyanins as flavonoids largely contribute to the antioxidant activities of turmeric. The positive regression recorded between inhibition of DPPH radical by the turmeric accessions and quercetin versus reducing power (R2 = 0.852) suggest that any of these methods could be used to assess the antioxidant activities of tumeric. Finally, the study indicated the potentials of the turmeric accessions especially accessions 30 and 50 as promising sources of antioxidants.

AMSARA: Accession Medical Standards Analysis and Research Activity. Report of 2006 Attrition and Morbidity Data for 2005 Accessions

DTIC Science & Technology

2007-12-17

and Research Activity (AMSARA) NUMBER Department of Epidemiology Division of Preventive Medicine Walter Reed Army Institute of Research 503 Robert...Hospitalizations, Recruits, Epidemiology, Attrition, Disability, Statistics, Preventive Medicine , Physical Fitness, Motivation, Accession, Waiver, Existing Prior to...an active duty Army enlistee. These findings are presented in abstract form in this report and as a full manuscript submitted to Military Medicine in

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants.

PubMed

Meyer, Thomas; Melin, Frédéric; Richter, Oliver-M H; Ludwig, Bernd; Kannt, Aimo; Müller, Hanne; Michel, Hartmut; Hellwig, Petra

2015-02-27

Two different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn(2+) ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Effect of Vascular Access Site Choice on Radiation Exposure During Coronary Angiography: The REVERE Trial (Randomized Evaluation of Vascular Entry Site and Radiation Exposure).

PubMed

Pancholy, Samir B; Joshi, Pankaj; Shah, Sanjay; Rao, Sunil V; Bertrand, Olivier F; Patel, Tejas M

2015-08-17

This study sought to perform a randomized noninferiority trial of radiation exposure during cardiac catheterization comparing femoral access (FA) with left radial access (LRA) and right radial access (RRA). Increased radiation exposure with radial approach compared with femoral approach remains a controversial issue. This study randomized 1,493 patients undergoing cardiac catheterization at a tertiary care center to FA, LRA, and RRA in a 1:1:1 fashion. The primary endpoint was air kerma. The secondary endpoints included dose-area product, fluoroscopy time and operator dose per procedure, number of cineangiograms, and number of catheters. Baseline and procedural characteristics were similar among groups. No significant differences were observed in air kerma (medians: FA: 421 mGy [interquartile range (IQR): 337 to 574 mGy], LRA: 454 mGy [IQR: 331 to 643 mGy], and RRA: 483 mGy [IQR: 382 to 592 mGy], p = 0.146), dose-area product (medians: FA: 25.5 Gy cm(2) [IQR: 19.6 to 34.5 Gy cm(2)], LRA: 26.6 Gy cm(2) [IQR: 19.5 to 37.5 Gy cm(2)], and RRA: 27.7 Gy cm(2) [IQR: 21.9 to 34.4 Gy cm(2)], p = 0.40), or fluoroscopy time (medians: FA: 1.3 min [IQR: 1.0 to 1.7 min], LRA: 1.3 min [IQR: 1.0 to 1.7 min], and RRA: 1.32 min [IQR: 1.0 to 1.7 min], p = 0.19) among the 3 access sites. Median operator exposure was higher in the LRA group (3 mrem [IQR: 2 to 5 mrem], p = 0.001 vs. FA, and p = 0.0001 vs. RRA) compared with the FA (2 mrem [IQR: 2 to 4 mrem] and RRA groups (3 mrem [IQR: 2 to 5 mrem]). Radiation exposure to patients was similar during diagnostic coronary angiography with FA, RRA, and LRA. However, LRA was associated with significantly higher operator radiation exposure than were FA and RRA procedures. (Randomized Evaluation of Vascular Entry Site and Radiation Exposure [REVERE]; NCT01677481). Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

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.

Site Planning and Layout.

ERIC Educational Resources Information Center

Moore, Gary T.

1998-01-01

Examines five issues related to child care facility design: (1) siting the building, outdoor play, and service areas; (2) creating favorable microclimates; (3) developmentally appropriate play yards; (4) pedestrian access and site circulation; and (5) vehicular access and parking away from pedestrians and play. (KB)

Accessibility of U.S. Federal Government Home Pages: Section 508 Compliance and Site Accessibility Statements

ERIC Educational Resources Information Center

Olalere, Abiodun; Lazar, Jonathan

2011-01-01

U.S. federal websites are required to be accessible for people with impairments. However, despite the existing regulations and guidelines, many federal websites continue to be inaccessible, and accessibility policy statements available on federal websites often do not provide any useful information. This paper provides three contributions to the…

Exploring the Structure of the Voltage-gated Na+ Channel by an Engineered Drug Access Pathway to the Receptor Site for Local Anesthetics*

PubMed Central

Lukacs, Peter; Gawali, Vaibhavkumar S.; Cervenka, Rene; Ke, Song; Koenig, Xaver; Rubi, Lena; Zarrabi, Touran; Hilber, Karlheinz; Stary-Weinzinger, Anna; Todt, Hannes

2014-01-01

Despite the availability of several crystal structures of bacterial voltage-gated Na+ channels, the structure of eukaryotic Na+ channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na+ channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na+ channels. PMID:24947510

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism.

PubMed

Singh, Appu Kumar; Ekka, Mary Krishna; Kaushik, Abhishek; Pandya, Vaibhav; Singh, Ravi P; Banerjee, Shrijita; Mittal, Monica; Singh, Vijay; Kumaran, S

2017-09-19

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10 4 -10 6 ) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

All the catalytic active sites of MoS 2 for hydrogen evolution

DOE PAGES

Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

2016-11-29

MoS 2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS 2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated tomore » be 7.5 s –1 (65–75 mV/dec), 3.2 s –1 (65–85 mV/dec), and 0.1 s –1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS 2 is higher than that in low crystalline quality MoS 2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

DOE PAGES

Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping; ...

2017-07-27

Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

Machine-Learning Methods Enable Exhaustive Searches for Active Bimetallic Facets and Reveal Active Site Motifs for CO 2 Reduction

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

Ulissi, Zachary W.; Tang, Michael T.; Xiao, Jianping

Bimetallic catalysts are promising for the most difficult thermal and electrochemical reactions, but modeling the many diverse active sites on polycrystalline samples is an open challenge. Here, we present a general framework for addressing this complexity in a systematic and predictive fashion. Active sites for every stable low-index facet of a bimetallic crystal are enumerated and cataloged, yielding hundreds of possible active sites. The activity of these sites is explored in parallel using a neural-network-based surrogate model to share information between the many density functional theory (DFT) relaxations, resulting in activity estimates with an order of magnitude fewer explicit DFTmore » calculations. Sites with interesting activity were found and provide targets for follow-up calculations. This process was applied to the electrochemical reduction of CO 2 on nickel gallium bimetallics and indicated that most facets had similar activity to Ni surfaces, but a few exposed Ni sites with a very favorable on-top CO configuration. This motif emerged naturally from the predictive modeling and represents a class of intermetallic CO 2 reduction catalysts. These sites rationalize recent experimental reports of nickel gallium activity and why previous materials screens missed this exciting material. Most importantly these methods suggest that bimetallic catalysts will be discovered by studying facet reactivity and diversity of active sites more systematically.« less

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.

COMPARISON OF LAPAROSCOPIC SKILLS PERFORMANCE USING SINGLE-SITE ACCESS (SSA) DEVICES VS. AN INDEPENDENT-PORT SSA APPROACH

PubMed Central

Schill, Matthew R.; Varela, J. Esteban; Frisella, Margaret M.; Brunt, L. Michael

2015-01-01

Background We compared performance of validated laparoscopic tasks on four commercially available single site access (SSA) access devices (AD) versus an independent port (IP) SSA set-up. Methods A prospective, randomized comparison of laparoscopic skills performance on four AD (GelPOINT™, SILS™ Port, SSL Access System™, TriPort™) and one IP SSA set-up was conducted. Eighteen medical students (2nd–4th year), four surgical residents, and five attending surgeons were trained to proficiency in multi-port laparoscopy using four laparoscopic drills (peg transfer, bean drop, pattern cutting, extracorporeal suturing) in a laparoscopic trainer box. Drills were then performed in random order on each IP-SSA and AD-SSA set-up using straight laparoscopic instruments. Repetitions were timed and errors recorded. Data are mean ± SD, and statistical analysis was by two-way ANOVA with Tukey HSD post-hoc tests. Results Attending surgeons had significantly faster total task times than residents or students (p< 0.001), but the difference between residents and students was NS. Pair-wise comparisons revealed significantly faster total task times for the IP-SSA set-up compared to all four AD-SSA’s within the student group only (p<0.05). Total task times for residents and attending surgeons showed a similar profile, but the differences were NS. When data for the three groups was combined, the total task time was less for the IP-SSA set-up than for each of the four AD-SSA set-ups (p < 0.001). Similarly,, the IP-SSA set-up was significantly faster than 3 of 4 AD-SSA set-ups for peg transfer, 3 of 4 for pattern cutting, and 2 of 4 for suturing. No significant differences in error rates between IP-SSA and AD-SSA set-ups were detected. Conclusions When compared to an IP-SSA laparoscopic set-up, single site access devices are associated with longer task performance times in a trainer box model, independent of level of training. Task performance was similar across different SSA

Accessibility of School Districts' Web Sites: A Descriptive Study

ERIC Educational Resources Information Center

Bray, Marty; Flowers, Claudia; Gibson, Patricia

2003-01-01

Many school districts (SDs) use the World Wide Web (WWW or Web) to disseminate a wide variety of information about things such as district events, policies, and a wide variety of student information. On-line barriers limit the accessibility of the WWW for persons and students with disabilities and thus can limit their access to vital information.…

Carnegie Science Academy Web Site

NASA Astrophysics Data System (ADS)

Kotwicki, John; Atzinger, Joe; Turso, Denise

1997-11-01

The Carnegie Science Academy is a professional society "For Teens...By Teens" at the Carnegie Science Center in Pittsburgh. The CSA Web Site [ http://csa.clpgh.org ] is designed for teens who have an interest in science and technology. This online or virtual science academy provides resources for teens in high school science classes. The Web site also allows students around the world to participate and communicate with other students, discuss current events in science, share opinions, find answers to questions, or make online friends. Visitors can enjoy the main components of the site or sign up for a free membership which allows access to our chat room for monthly meeting, online newsletter, members forum, and much more. Main components to the site include a spot for cool links and downloads, available for any visitor to download or view. Online exhibits are created by students to examine and publish an area of study and also allow teachers to easily post classroom activities as exhibits by submitting pictures and text. Random Access, the interactive part of the academy, allows users to share ideas and opinions. Planet CSA focuses on current events in science and the academy. In the future the CSA Web site will become a major resource for teens and science teachers providing materials that will allow students to further enhance their interest and experiences in science.

Functional Evolution of PLP-dependent Enzymes based on Active-Site Structural Similarities

PubMed Central

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-01-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5’-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the Comparison of Protein Active Site Structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. PMID:24920327

Chapter 12: Daily Patterns of Marbled Murrelet Activity at Inland Sites

Treesearch

Nancy L. Naslund; Brian P. O’Donnell

1995-01-01

Patterns in the daily activity of Marbled Murrelets (Brachyramphus marmoratus) at inland sites has been studied throughout their range from California to Alaska. Murrelets are most active at inland sites around dawn, and to a lesser degree, at dusk. Throughout their range, peak levels of activity (detections) occur in the hour around dawn, but...

An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

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

Eren, Elif; Murphy, Megan; Goguen, Jon

2010-08-13

The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changesmore » of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.« less

The New USGS Volcano Hazards Program Web Site

NASA Astrophysics Data System (ADS)

Venezky, D. Y.; Graham, S. E.; Parker, T. J.; Snedigar, S. F.

2008-12-01

The U.S. Geological Survey's (USGS) Volcano Hazard Program (VHP) has launched a revised web site that uses a map-based interface to display hazards information for U.S. volcanoes. The web site is focused on better communication of hazards and background volcano information to our varied user groups by reorganizing content based on user needs and improving data display. The Home Page provides a synoptic view of the activity level of all volcanoes for which updates are written using a custom Google® Map. Updates are accessible by clicking on one of the map icons or clicking on the volcano of interest in the adjacent color-coded list of updates. The new navigation provides rapid access to volcanic activity information, background volcano information, images and publications, volcanic hazards, information about VHP, and the USGS volcano observatories. The Volcanic Activity section was tailored for emergency managers but provides information for all our user groups. It includes a Google® Map of the volcanoes we monitor, an Elevated Activity Page, a general status page, information about our Volcano Alert Levels and Aviation Color Codes, monitoring information, and links to monitoring data from VHP's volcano observatories: Alaska Volcano Observatory (AVO), Cascades Volcano Observatory (CVO), Long Valley Observatory (LVO), Hawaiian Volcano Observatory (HVO), and Yellowstone Volcano Observatory (YVO). The YVO web site was the first to move to the new navigation system and we are working on integrating the Long Valley Observatory web site next. We are excited to continue to implement new geospatial technologies to better display our hazards and supporting volcano information.

The Influence of High Computer Access on Schoolwork and Student Empowerment: An Exploratory Study of the Nashville ACOT Site.

ERIC Educational Resources Information Center

Fisher, Charles W.

This study examines the relationship between high computer access and "student empowerment" at the Nashville, Tennessee, site of the Apple Classroom of Tomorrow (ACOT) project. The study rests on the premise that school learning is a function of the work carried out by students in school, and that schoolwork is experienced by students as…

State and Local Health Department Activities Related to Abortion: A Web Site Content Analysis.

PubMed

Berglas, Nancy F; Johns, Nicole E; Rosenzweig, Caroline; Hunter, Lauren A; Roberts, Sarah C M

Recent legislation in states across the United States has required governmental health agencies to take on new and different roles in relation to abortion. While there has been media attention to health department roles in regulating abortion providers, there has been no systematic investigation of the range of activities in which state and local health departments are engaged. To systematically investigate health department activities related to abortion. We searched state health department Web sites of the 50 states and District of Columbia using key words such as "abortion" and "pregnancy termination". Two trained coders categorized 6093 documents using the 10 Essential Public Health Services (EPHS) framework. We then applied these methods to 671 local health department documents. State and local health department Web sites. N/A. On average, states engaged in 5.1 of 10 Essential Services related to abortion. Most (76%-98%) state health departments engaged in activities to Monitor Health Status (EPHS1), Enforce Laws (EPHS6), and Evaluate Effectiveness, Accessibility, and Quality (EPHS9). Many (47%-69%) engaged in activities to Inform and Educate (EPHS3), Develop Policies (EPHS5), and Link to Services (EPHS7). A minority (4%-29%) engaged in activities to Diagnose and Investigate Health Problems (EPHS2), Mobilize Community Partnerships (EPHS4), and Assure Competent Workforce (EPHS8). No state engaged in Innovative Research (EPHS10). Few local health departments engaged in abortion-related activities. While most state health departments engage in abortion-related activities, they appear to reflect what the law requires rather than the range of core public health activities. Additional research is needed to assess whether these services meet quality standards for public health services and determine how best to support governmental health agencies in their growing tasks. These findings raise important questions about the role of public health agencies and

Nevada National Security Site Environmental Report 2016, Attachment A: Site Description

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

Wills, Cathy

This attachment expands on the general description of the Nevada National Security Site (NNSS) presented in the Introduction to the Nevada National Security Site Environmental Report 2016 (prepared by National Security Technologies, LLC [NSTec], 2017). Included are subsections that summarize the site’s geological, hydrological, climatological, and ecological settings and the cultural resources of the NNSS. The subsections are meant to aid the reader in understanding the complex physical and biological environment of the NNSS. An adequate knowledge of the site’s environment is necessary to assess the environmental impacts of new projects, design and implement environmental monitoring activities for current sitemore » operations, and assess the impacts of site operations on the public residing in the vicinity of the NNSS. The NNSS environment contributes to several key features of the site that afford protection to the inhabitants of adjacent areas from potential exposure to radioactivity or other contaminants resulting from NNSS operations. These key features include the general remote location of the NNSS, restricted access, extended wind transport times, the great depths to slow-moving groundwater, little or no surface water, and low population density. This attachment complements the annual summary of monitoring program activities and dose assessments presented in the main body of this report.« less

Anisotropic Covalency Contributions to Superexchange Pathways in Type One Copper Active Sites

PubMed Central

2015-01-01

Type one (T1) Cu sites deliver electrons to catalytic Cu active sites: the mononuclear type two (T2) Cu site in nitrite reductases (NiRs) and the trinuclear Cu cluster in the multicopper oxidases (MCOs). The T1 Cu and the remote catalytic sites are connected via a Cys-His intramolecular electron-transfer (ET) bridge, which contains two potential ET pathways: P1 through the protein backbone and P2 through the H-bond between the Cys and the His. The high covalency of the T1 Cu–S(Cys) bond is shown here to activate the T1 Cu site for hole superexchange via occupied valence orbitals of the bridge. This covalency-activated electronic coupling (HDA) facilitates long-range ET through both pathways. These pathways can be selectively activated depending on the geometric and electronic structure of the T1 Cu site and thus the anisotropic covalency of the T1 Cu–S(Cys) bond. In NiRs, blue (π-type) T1 sites utilize P1 and green (σ-type) T1 sites utilize P2, with P2 being more efficient. Comparing the MCOs to NiRs, the second-sphere environment changes the conformation of the Cys-His pathway, which selectively activates HDA for superexchange by blue π sites for efficient turnover in catalysis. These studies show that a given protein bridge, here Cys-His, provides different superexchange pathways and electronic couplings depending on the anisotropic covalencies of the donor and acceptor metal sites. PMID:25310460

Active site specificity profiling datasets of matrix metalloproteinases (MMPs) 1, 2, 3, 7, 8, 9, 12, 13 and 14.

PubMed

Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Auf dem Keller, Ulrich; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

2016-06-01

The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites) to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015) [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number PXD002265.

Functional evolution of PLP-dependent enzymes based on active-site structural similarities.

PubMed

Catazaro, Jonathan; Caprez, Adam; Guru, Ashu; Swanson, David; Powers, Robert

2014-10-01

Families of distantly related proteins typically have very low sequence identity, which hinders evolutionary analysis and functional annotation. Slowly evolving features of proteins, such as an active site, are therefore valuable for annotating putative and distantly related proteins. To date, a complete evolutionary analysis of the functional relationship of an entire enzyme family based on active-site structural similarities has not yet been undertaken. Pyridoxal-5'-phosphate (PLP) dependent enzymes are primordial enzymes that diversified in the last universal ancestor. Using the comparison of protein active site structures (CPASS) software and database, we show that the active site structures of PLP-dependent enzymes can be used to infer evolutionary relationships based on functional similarity. The enzymes successfully clustered together based on substrate specificity, function, and three-dimensional-fold. This study demonstrates the value of using active site structures for functional evolutionary analysis and the effectiveness of CPASS. © 2014 Wiley Periodicals, Inc.

Determination of protein secondary structure and solvent accessibility using site-directed fluorescence labeling. Studies of T4 lysozyme using the fluorescent probe monobromobimane.

PubMed

Mansoor, S E; McHaourab, H S; Farrens, D L

1999-12-07

We report an investigation of how much protein structural information could be obtained using a site-directed fluorescence labeling (SDFL) strategy. In our experiments, we used 21 consecutive single-cysteine substitution mutants in T4 lysozyme (residues T115-K135), located in a helix-turn-helix motif. The mutants were labeled with the fluorescent probe monobromobimane and subjected to an array of fluorescence measurements. Thermal stability measurements show that introduction of the label is substantially perturbing only when it is located at buried residue sites. At buried sites (solvent surface accessibility of <40 A(2)), the destabilizations are between 3 and 5.5 kcal/mol, whereas at more exposed sites, DeltaDeltaG values of < or = 1.5 kcal/mol are obtained. Of all the fluorescence parameters that were explored (excitation lambda(max), emission lambda(max), fluorescence lifetime, quantum yield, and steady-state anisotropy), the emission lambda(max) and the steady-state anisotropy values most accurately reflect the solvent surface accessibility at each site as calculated from the crystal structure of cysteine-less T4 lysozyme. The parameters we identify allow the classification of each site as buried, partially buried, or exposed. We find that the variations in these parameters as a function of residue number reflect the sequence-specific secondary structure, the determination of which is a key step for modeling a protein of unknown structure.

Active RNAP pre-initiation sites are highly mutated by cytidine deaminases in yeast, with AID targeting small RNA genes

PubMed Central

Taylor, Benjamin JM; Wu, Yee Ling; Rada, Cristina

2014-01-01

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors. DOI: http://dx.doi.org/10.7554/eLife.03553.001 PMID:25237741

NLM Emergency Access Initiative: FAQs

Science.gov Websites

provide temporary free access to full-text articles from over 650 biomedical serial titles and over 4,000 specified on the EAI welcome page can access the free full text resources during the period indicated to will I have access to this site? The dates of the free access period are listed on the home page along

Active-site solvent replenishment observed during human carbonic anhydrase II catalysis.

PubMed

Kim, Jin Kyun; Lomelino, Carrie L; Avvaru, Balendu Sankara; Mahon, Brian P; McKenna, Robert; Park, SangYoun; Kim, Chae Un

2018-01-01

Human carbonic anhydrase II (hCA II) is a zinc metalloenzyme that catalyzes the reversible hydration/dehydration of CO 2 /HCO 3 - . Although hCA II has been extensively studied to investigate the proton-transfer process that occurs in the active site, its underlying mechanism is still not fully understood. Here, ultrahigh-resolution crystallographic structures of hCA II cryocooled under CO 2 pressures of 7.0 and 2.5 atm are presented. The structures reveal new intermediate solvent states of hCA II that provide crystallographic snapshots during the restoration of the proton-transfer water network in the active site. Specifically, a new intermediate water (W I ') is observed next to the previously observed intermediate water W I , and they are both stabilized by the five water molecules at the entrance to the active site (the entrance conduit). Based on these structures, a water network-restructuring mechanism is proposed, which takes place at the active site after the nucleophilic attack of OH - on CO 2 . This mechanism explains how the zinc-bound water (W Zn ) and W1 are replenished, which are directly responsible for the reconnection of the His64-mediated proton-transfer water network. This study provides the first 'physical' glimpse of how a water reservoir flows into the hCA II active site during its catalytic activity.

76 FR 64379 - Agency Information Collection Activities: Proposed Collection; Comments Requested; eForm 6 Access...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-10-18

...] Agency Information Collection Activities: Proposed Collection; Comments Requested; eForm 6 Access Request... Form/Collection: eForm 6 Access Request. (3) Agency form number, if any, and the applicable component...: Respondents must complete the eForm 6 Access Request form in order to receive a user ID and password to obtain...

Not Just a Walk in the Park: Methodological Improvements for Determining Environmental Justice Implications of Park Access in New York City for the Promotion of Physical Activity

PubMed Central

Miyake, Keith K.; Maroko, Andrew R.; Grady, Kristen L.; Maantay, Juliana A.; Arno, Peter S.

2011-01-01

The purpose of this study is to test the hypothesis that access to parks in New York City is not equitable across racial and ethnic categories. It builds on previous research that has linked access to parks and open space with increased physical activity, which in turn may reduce the risk for adverse health outcomes related to obesity. Systematic patterns of uneven access to parks might help to explain disparities in these health outcomes across sociodemographic populations that are not fully explained by individual-level risk factors and health behaviors, and therefore access to parks becomes an environmental justice issue. This study is designed to shed light on the “unpatterned inequities” of park distributions identified in previous studies of New York City park access. It uses a combination of network analysis and a cadastral-based expert dasymetric system (CEDS) to estimate the racial/ethnic composition of populations within a reasonable walking distance of 400m from parks. The distance to the closest park, number of parks within walking distance, amount of accessible park space, and number of physical activity sites are then evaluated across racial/ethnic categories, and are compared to the citywide populations using odds ratios. The odds ratios revealed patterns that at first glance appear to contradict the notion of distributional inequities. However, discussion of the results points to the need for reassessing what is meant by “access” to more thoroughly consider the aspects of parks that are most likely to contribute to physical activity and positive health outcomes. PMID:21874148

Real-time Data Access From Remote Observatories

NASA Astrophysics Data System (ADS)

Detrick, D. L.; Lutz, L. F.; Etter, J. E.; Rosenberg, T. J.; Weatherwax, A. T.

2006-12-01

Real-time access to solar-terrestrial data is becoming increasingly important, not only because it is now possible to acquire and access data rapidly via the internet, but also because of the need for timely publication of real-time data for analysis and modeling efforts. Currently, engineering-scaled summary data are available routinely on a daily basis from many observatories, but only when the observatories have continuous, or at least daily network access. Increasingly, the upgrading of remote data acquisition hardware makes it possible to provide data in real-time, and it is becoming normal to expect timely access to data products. The NSF- supported PENGUIn/AGO constellation of autonomous Antarctic research observatories has provided real-time data since December, 2002, when Iridium satellite modems were installed at three sites. The Iridium telecommunications links are maintained continuously, transferring data between the remote observatories and a U.S.-based data acquisition site. The time-limiting factor with this scenario is now the delay in completing a data record before transmission, which can be as short as minutes depending on the sampling rate. The single-channel data throughput of the current systems is 20-MB/day (megabytes per day), but planned installations will be capable of operating with multiple modem channels. The data records are currently posted immediately to a web site accessible by anonymous FTP client software, for use by the instruments' principal investigators, and survey plots of selected signals are published daily. The web publication facilities are being upgraded, in order to allow other interested researchers rapid access to engineering-scaled data products, in several common formats, as well as providing interactive plotting capabilities. The web site will provide access to data from other collaborating observatories (including South Pole and McMurdo Stations), as well as ancillary data accessible from public sites (e.g., Kp

40 CFR 1400.6 - Enhanced local access.

Code of Federal Regulations, 2011 CFR

2011-07-01

...); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.6 Enhanced local access. (a) OCA data elements. Consistent with 42 U.S.C...) OCA information. (1) LEPCs and related local government agencies are authorized and encouraged to...

40 CFR 1400.6 - Enhanced local access.

Code of Federal Regulations, 2012 CFR

2012-07-01

...); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.6 Enhanced local access. (a) OCA data elements. Consistent with 42 U.S.C...) OCA information. (1) LEPCs and related local government agencies are authorized and encouraged to...

40 CFR 1400.6 - Enhanced local access.

Code of Federal Regulations, 2013 CFR

2013-07-01

...); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.6 Enhanced local access. (a) OCA data elements. Consistent with 42 U.S.C...) OCA information. (1) LEPCs and related local government agencies are authorized and encouraged to...

40 CFR 1400.6 - Enhanced local access.

Code of Federal Regulations, 2014 CFR

2014-07-01

...); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.6 Enhanced local access. (a) OCA data elements. Consistent with 42 U.S.C...) OCA information. (1) LEPCs and related local government agencies are authorized and encouraged to...

The relationship of physical activity and overweight to objectively measured green space accessibility and use

PubMed Central

2013-01-01

This study examines the association between objectively measured access to green space, frequency of green space use, physical activity, and the probability of being overweight or obese in the city of Bristol, England. Data from the Bristol Quality of Life in your Neighbourhood survey for 6,821 adults were combined with a comprehensive GIS database of neighbourhood and green space characteristics.. A range of green space accessibility measures were computed. Associations between accessibility and the odds of respondents achieving a recommended 30 minutes or more of moderate activity five times a week, or being overweight or obese, were examined using logistic regression. Results showed that the reported frequency of green space use declined with increasing distance. The study also found that respondents living closest to the type of green space classified as a Formal park were more likely to achieve the physical activity recommendation and less likely to be overweight or obese. The association with physical activity, but not with overweight or obesity, remained after adjustment for respondent characteristics, area deprivation, and a range of characteristics of the neighbourhood environment. The findings suggest that the provision of good access to green spaces in urban areas may help promote population physical activity. PMID:20060635

Active Site Gate Dynamics Modulate the Catalytic Activity of the Ubiquitination Enzyme E2-25K.

PubMed

Rout, Manoj K; Lee, Brian L; Lin, Aiyang; Xiao, Wei; Spyracopoulos, Leo

2018-05-03

The ubiquitin proteasome system (UPS) signals for degradation of proteins through attachment of K48-linked polyubiquitin chains, or alterations in protein-protein recognition through attachment of K63-linked chains. Target proteins are ubiquitinated in three sequential chemical steps by a three-component enzyme system. Ubiquitination, or E2 enzymes, catalyze the central step by facilitating reaction of a target protein lysine with the C-terminus of Ub that is attached to the active site cysteine of the E2 through a thioester bond. E2 reactivity is modulated by dynamics of an active site gate, whose central residue packs against the active site cysteine in a closed conformation. Interestingly, for the E2 Ubc13, which specifically catalyzes K63-linked ubiquitination, the central gate residue adopts an open conformation. We set out to determine if active site gate dynamics play a role in catalysis for E2-25K, which adopts the canonical, closed gate conformation, and which selectively synthesizes K48-linked ubiquitin chains. Gate dynamics were characterized using mutagenesis of key residues, combined with enzyme kinetics measurements, and main chain NMR relaxation. The experimental data were interpreted with all atom MD simulations. The data indicate that active site gate opening and closing rates for E2-25K are precisely balanced.

An operational data access infrastructure for accessing integrated environmental and socio-economic data from the Dutch Wadden Sea

NASA Astrophysics Data System (ADS)

De Bruin, T.

2012-12-01

The Wadden Sea, an UNESCO World Heritage Site along the Northern coasts of The Netherlands, Germany and Denmark, is a very valuable, yet also highly vulnerable tidal flats area. Knowledge is key to the sustainable management of the Wadden Sea. This knowledge should be reliable, founded on promptly accessible information and sufficiently broad to integrate both ecological and economic analyses. The knowledge is gained from extensive monotoring of both ecological and socio-economic parameters. Even though many organisations, research institutes, government agencies and NGOs carry out monitoring, there is no central overview of monitoring activities, nor easy access to the resulting data. The 'Wadden Sea Long-Term Ecosystem Research' (WaLTER) project (2011-2015) aims to set-up an integrated monitoring plan for the main environmental and management issues relevant to the Wadden Sea, such as sea-level rise, fisheries management, recreation and industry activities. The WaLTER data access infrastructure will be a distributed system of data providers, with a centralized data access portal. It is based on and makes use of the existing data access infrastructure of the Netherlands National Oceanographic Data Committee (NL-NODC), which has been operational since early 2009. The NL-NODC system is identical to and in fact developed by the European SeaDataNet project, furthering standardisation on a pan-European scale. The presentation will focus on the use of a distributed data access infrastructure to address the needs of different user groups such as policy makers, scientists and the general public.

SABER: A computational method for identifying active sites for new reactions

PubMed Central

Nosrati, Geoffrey R; Houk, K N

2012-01-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644–1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were l-Ala d/l-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. PMID:22492397

SABER: a computational method for identifying active sites for new reactions.

PubMed

Nosrati, Geoffrey R; Houk, K N

2012-05-01

A software suite, SABER (Selection of Active/Binding sites for Enzyme Redesign), has been developed for the analysis of atomic geometries in protein structures, using a geometric hashing algorithm (Barker and Thornton, Bioinformatics 2003;19:1644-1649). SABER is used to explore the Protein Data Bank (PDB) to locate proteins with a specific 3D arrangement of catalytic groups to identify active sites that might be redesigned to catalyze new reactions. As a proof-of-principle test, SABER was used to identify enzymes that have the same catalytic group arrangement present in o-succinyl benzoate synthase (OSBS). Among the highest-scoring scaffolds identified by the SABER search for enzymes with the same catalytic group arrangement as OSBS were L-Ala D/L-Glu epimerase (AEE) and muconate lactonizing enzyme II (MLE), both of which have been redesigned to become effective OSBS catalysts, demonstrated by experiments. Next, we used SABER to search for naturally existing active sites in the PDB with catalytic groups similar to those present in the designed Kemp elimination enzyme KE07. From over 2000 geometric matches to the KE07 active site, SABER identified 23 matches that corresponded to residues from known active sites. The best of these matches, with a 0.28 Å catalytic atom RMSD to KE07, was then redesigned to be compatible with the Kemp elimination using RosettaDesign. We also used SABER to search for potential Kemp eliminases using a theozyme predicted to provide a greater rate acceleration than the active site of KE07, and used Rosetta to create a design based on the proteins identified. Copyright © 2012 The Protein Society.

10 CFR 2.1007 - Access.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Licenses for the Receipt of High-Level Radioactive Waste at a Geologic Repository § 2.1007 Access. (a)(1) A system to provide electronic access to the Licensing Support Network shall be provided at the... provide electronic access to the Licensing Support Network shall be provided at the NRC Web site, http...

10 CFR 2.1007 - Access.

Code of Federal Regulations, 2013 CFR

2013-01-01

... Licenses for the Receipt of High-Level Radioactive Waste at a Geologic Repository § 2.1007 Access. (a)(1) A system to provide electronic access to the Licensing Support Network shall be provided at the... provide electronic access to the Licensing Support Network shall be provided at the NRC Web site, http...

The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative "back door".

PubMed

Simon, S; Le Goff, A; Frobert, Y; Grassi, J; Massoulié, J

1999-09-24

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

'Unconventional' coordination chemistry by metal chelating fragments in a metalloprotein active site.

PubMed

Martin, David P; Blachly, Patrick G; Marts, Amy R; Woodruff, Tessa M; de Oliveira, César A F; McCammon, J Andrew; Tierney, David L; Cohen, Seth M

2014-04-09

The binding of three closely related chelators: 5-hydroxy-2-methyl-4H-pyran-4-thione (allothiomaltol, ATM), 3-hydroxy-2-methyl-4H-pyran-4-thione (thiomaltol, TM), and 3-hydroxy-4H-pyran-4-thione (thiopyromeconic acid, TPMA) to the active site of human carbonic anhydrase II (hCAII) has been investigated. Two of these ligands display a monodentate mode of coordination to the active site Zn(2+) ion in hCAII that is not recapitulated in model complexes of the enzyme active site. This unprecedented binding mode in the hCAII-thiomaltol complex has been characterized by both X-ray crystallography and X-ray spectroscopy. In addition, the steric restrictions of the active site force the ligands into a 'flattened' mode of coordination compared with inorganic model complexes. This change in geometry has been shown by density functional computations to significantly decrease the strength of the metal-ligand binding. Collectively, these data demonstrate that the mode of binding by small metal-binding groups can be significantly influenced by the protein active site. Diminishing the strength of the metal-ligand bond results in unconventional modes of metal coordination not found in typical coordination compounds or even carefully engineered active site models, and understanding these effects is critical to the rational design of inhibitors that target clinically relevant metalloproteins.

Enhanced enzyme kinetic stability by increasing rigidity within the active site.

PubMed

Xie, Yuan; An, Jiao; Yang, Guangyu; Wu, Geng; Zhang, Yong; Cui, Li; Feng, Yan

2014-03-14

Enzyme stability is an important issue for protein engineers. Understanding how rigidity in the active site affects protein kinetic stability will provide new insight into enzyme stabilization. In this study, we demonstrated enhanced kinetic stability of Candida antarctica lipase B (CalB) by mutating the structurally flexible residues within the active site. Six residues within 10 Å of the catalytic Ser(105) residue with a high B factor were selected for iterative saturation mutagenesis. After screening 2200 colonies, we obtained the D223G/L278M mutant, which exhibited a 13-fold increase in half-life at 48 °C and a 12 °C higher T50(15), the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment. Further characterization showed that global unfolding resistance against both thermal and chemical denaturation also improved. Analysis of the crystal structures of wild-type CalB and the D223G/L278M mutant revealed that the latter formed an extra main chain hydrogen bond network with seven structurally coupled residues within the flexible α10 helix that are primarily involved in forming the active site. Further investigation of the relative B factor profile and molecular dynamics simulation confirmed that the enhanced rigidity decreased fluctuation of the active site residues at high temperature. These results indicate that enhancing the rigidity of the flexible segment within the active site may provide an efficient method for improving enzyme kinetic stability.

GASS-WEB: a web server for identifying enzyme active sites based on genetic algorithms.

PubMed

Moraes, João P A; Pappa, Gisele L; Pires, Douglas E V; Izidoro, Sandro C

2017-07-03

Enzyme active sites are important and conserved functional regions of proteins whose identification can be an invaluable step toward protein function prediction. Most of the existing methods for this task are based on active site similarity and present limitations including performing only exact matches on template residues, template size restraints, despite not being capable of finding inter-domain active sites. To fill this gap, we proposed GASS-WEB, a user-friendly web server that uses GASS (Genetic Active Site Search), a method based on an evolutionary algorithm to search for similar active sites in proteins. GASS-WEB can be used under two different scenarios: (i) given a protein of interest, to match a set of specific active site templates; or (ii) given an active site template, looking for it in a database of protein structures. The method has shown to be very effective on a range of experiments and was able to correctly identify >90% of the catalogued active sites from the Catalytic Site Atlas. It also managed to achieve a Matthew correlation coefficient of 0.63 using the Critical Assessment of protein Structure Prediction (CASP 10) dataset. In our analysis, GASS was ranking fourth among 18 methods. GASS-WEB is freely available at http://gass.unifei.edu.br/. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

A study in usability: redesigning a health sciences library's mobile site.

PubMed

Rosario, Jovy-Anne; Ascher, Marie T; Cunningham, Diana J

2012-01-01

A mobile site redesign was conducted at a medium-sized academic health sciences library with the goal of creating a site that meets the mobile information needs of its users. The redesign phases included (1) needs assessment, (2) usability testing, and (3) site design. The survey results showed that Apple devices were the most prevalent; the most desirable activities performed on a mobile site were searching for articles, accessing full-text articles and e-books, searching databases, and searching the catalog. These activities guided the development of the usability testing tasks and the redesign. All phases were completed within six months, and the total project cost was $50 for incentive purchases. Copyright © Taylor & Francis Group, LLC

Strategies of arteriovenous dialysis access.

PubMed

Weiswasser, Jonathan M; Kellicut, Dwight; Arora, Subodh; Sidawy, Anton N

2004-03-01

Surgical management of the patient who requires hemodialysis access, while continuing to demand more attention from the vascular surgeon, suffers from discrepancies of approach and strategy. With the increase in incidence of dialysis dependent renal failure among our population, many have attempted to present a uniform, logical strategy with which the vascular surgeon can most effectively treat the hemodialysis patient in the long term. Most notably, the multidisciplinary Dialysis Outcomes Quality Initiative (DOQI) guidelines present the surgeon with a rough outline of hemodialysis access insertion strategy, and it has become nationally recognized as an acceptable summary of treatment strategy and goals. The decision as to the most appropriate surgical access to offer a patient depends on immediate need for hemodialysis, history and physical examination findings, and suitability of available veins in the extremity. While percutaneous, catheter based access affords the luxury of immediate access, these devices suffer from several complicating factors, such as infection, and damage to large, proximal veins. For long-term access, the autogenous access, while perhaps less successful in the immediate short term, is always the preferred access type given its favorable longevity. The surgeons should focus on sites distally on the extremity, reserving proximal sites for potential future access insertions should the primary access fail. In the absence of suitable vein, prosthetic access may be considered. When both the upper and lower aspects of both upper extremities have been exhausted, the surgeon should consider access insertion elsewhere, such as the lower extremity.

Facebook, Twitter Activities Sites, Location and Students' Interest in Learning

ERIC Educational Resources Information Center

Igbo, J. N.; Ezenwaji, Ifeyinwa; Ajuziogu, Christiana U.

2018-01-01

This study was carried out to ascertain the influence of social networking sites activities (twitter and Facebook) on secondary school students' interest in learning It also considered the impact of these social networking sites activities on location of the students. Two research questions and two null hypotheses guided the study. Mean and…

Solvent Flux Method (SFM): A Case Study of Water Access to Candida antarctica Lipase B.

PubMed

Benson, Sven P; Pleiss, Jürgen

2014-11-11

The solvent flux method (SFM) was developed to comprehensively characterize the influx of solvent molecules from the solvent environment into the active site of a protein in the framework of molecular dynamics simulations. This was achieved by introducing a solvent concentration gradient as well as partially reorienting and rescaling the velocity vector of all solvent molecules contained within a spherical volume enclosing the protein, thus inducing an accelerated solvent influx toward the active site. In addition to the detection of solvent access pathway within the protein structure, it is hereby possible to identify potential amino acid positions relevant to solvent-related enzyme engineering with high statistical significance. The method is particularly aimed at improving the reverse hydrolysis reaction rates in nonaqueous media. Candida antarctica lipase B (CALB) binds to a triglyceride-water interface with its substrate entrance channel oriented toward the hydrophobic substrate interface. The lipase-triglyceride-water system served as a model system for SFM to evaluate the influx of water molecules to the active site. As a proof of principle for SFM, a previously known water access pathway in CALB was identified as the primary water channel. In addition, a secondary water channel and two pathways for water access which contribute to water leakage between the protein and the triglyceride-water interface were identified.

Tool independence for the Web Accessibility Quantitative Metric.

PubMed

Vigo, Markel; Brajnik, Giorgio; Arrue, Myriam; Abascal, Julio

2009-07-01

The Web Accessibility Quantitative Metric (WAQM) aims at accurately measuring the accessibility of web pages. One of the main features of WAQM among others is that it is evaluation tool independent for ranking and accessibility monitoring scenarios. This article proposes a method to attain evaluation tool independence for all foreseeable scenarios. After demonstrating that homepages have a more similar error profile than any other web page in a given web site, 15 homepages were measured with 10,000 different values of WAQM parameters using EvalAccess and LIFT, two automatic evaluation tools for accessibility. A similar procedure was followed with random pages and with several test files obtaining several tuples that minimise the difference between both tools. One thousand four hundred forty-nine web pages from 15 web sites were measured with these tuples and those values that minimised the difference between the tools were selected. Once the WAQM was tuned, the accessibility of 15 web sites was measured with two metrics for web sites, concluding that even if similar values can be produced, obtaining the same scores is undesirable since evaluation tools behave in a different way.

Highlights and Opportunities from Continuous Access to Gas Hydrates Sites at Ocean Networks Canada's NEPTUNE Observatory

NASA Astrophysics Data System (ADS)

Scherwath, M.; Heesemann, M.; Riedel, M.; Thomsen, L.; Roemer, M.; Chatzievangelou, D.; Purser, A.

2017-12-01

Since 2009 Ocean Networks Canada provides permanent access and continuous data in near real-time from two prominent gas hydrates research sites at the Northern Cascadia Margin, Barkley Canyon and Clayoquot Slope off Vancouver Island, through power and communication cables directly from shore. We show data highlights from the seafloor crawler Wally, the world's first internet operated vehicle, in a field of hydrate mounds and outcropping gas hydrates, and its co-located sonars and state-of-the-ocean sensors and Barkley Canyon. For example, spectacular views from the benthic communities and their changes over time are captured by video. At Clayoquot Slope highly active gas seep fields are monitored with a rotating multibeam sonar and various other environmental sensors. In addition, newly installed geodetic sensors as well as an instrumented borehole in that area are now online and provide additional data on subduction-related deformation and potential links to gas discharge. These show-case examples highlight the benefits of co-located experiments that enable interdisciplinary research and also the ability for high-power and -bandwidth long-term monitoring at remote seafloor locations, that over time will provide baselines for environmental monitoring together with natural variability and potential long-term trends.

Characterization of the active site properties of CYP4F12.

PubMed

Eksterowicz, John; Rock, Dan A; Rock, Brooke M; Wienkers, Larry C; Foti, Robert S

2014-10-01

Cytochrome P450 4F12 is a drug-metabolizing enzyme that is primarily expressed in the liver, kidney, colon, small intestine, and heart. The properties of CYP4F12 that may impart an increased catalytic selectivity (decreased promiscuity) were explored through in vitro metabolite elucidation, kinetic isotope effect experiments, and computational modeling of the CYP4F12 active site. By using astemizole as a probe substrate for CYP4F12 and CYP3A4, it was observed that although CYP4F12 favored astemizole O-demethylation as the primary route of metabolism, CYP3A4 was capable of metabolizing astemizole at multiple sites on the molecule. Deuteration of astemizole at the site of O-demethylation resulted in an isotope effect of 7.1 as well as an 8.3-fold decrease in the rate of clearance for astemizole by CYP4F12. Conversely, although an isotope effect of 3.8 was observed for the formation of the O-desmethyl metabolite when deuterated astemizole was metabolized by CYP3A4, there was no decrease in the clearance of astemizole. Development of a homology model of CYP4F12 based on the crystal structure of cytochrome P450 BM3 predicted an active site volume for CYP4F12 that was approximately 76% of the active site volume of CYP3A4. As predicted, multiple favorable binding orientations were available for astemizole docked into the active site of CYP3A4, but only a single binding orientation with the site of O-demethylation oriented toward the heme was identified for CYP4F12. Overall, it appears that although CYP4F12 may be capable of binding similar ligands to other cytochrome P450 enzymes such as CYP3A4, the ability to achieve catalytically favorable orientations may be inherently more difficult because of the increased steric constraints of the CYP4F12 active site. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

Influence of limited proteolysis, detergent treatment and lyophilization on the phenoloxidase activity of Rapana thomasiana hemocyanin.

PubMed

Idakieva, Krassimira; Siddiqui, Nurul Islam; Meersman, Filip; De Maeyer, Marc; Chakarska, Irena; Gielens, Constant

2009-08-01

The intrinsic and inducible phenoloxidase (PO) activity of Rapana thomasiana hemocyanin (RtH) and its substructures were studied. With catechol as substrate, a weak o-diPO activity was measured for the didecameric RtH and its subunits. Some activation of the o-diPO activity of RtH was achieved by limited treatment with subtilisin and by incubation of RtH with 2.9 mM sodium dodecyl sulphate (SDS), suggesting an enhanced substrate access to the active sites. The highest artificial induction of o-diPO activity in RtH, however, was obtained by lyophilization of the protein. This is ascribed to conformational changes during the lyophilization process of the didecameric RtH molecules, affecting the accessibility of the active sites. These conformational changes must be very small, since Fourier-transform infrared and circular dichroism spectroscopies did not reveal any changes in secondary structure of lyophilized RtH. The difference in accessibility of the copper containing active site for substrates between catechol oxidase and functional unit RtH2-e was demonstrated by molecular modeling and surface area accessibility calculations. The low level of intrinsic PO activity in the investigated hemocyanin is related to the inaccessibility of the binuclear copper active sites to the substrates.

Improving School Access Control

ERIC Educational Resources Information Center

National Clearinghouse for Educational Facilities, 2008

2008-01-01

Few things are more important for school safety and security than controlling access to buildings and grounds. It is relatively easy to incorporate effective access control measures in new school designs but more difficult in existing schools, where most building and site features cannot be readily altered or reconfigured. The National…

Deletion of loop fragment adjacent to active site diminishes the stability and activity of exo-inulinase.

PubMed

Arjomand, Maryam Rezaei; Habibi-Rezaei, Mehran; Ahmadian, Gholamreza; Hassanzadeh, Malihe; Karkhane, Ali Asghar; Asadifar, Mandana; Amanlou, Massoud

2016-11-01

Inulinases are classified as hydrolases and widely used in the food and medical industries. Here, we report the deletion of a six-membered adjacent active site loop fragment ( 74 YGSDVT 79 sequence) from third Ω-loop of the exo-inulinase containing aspartate residue from Aspergillus niger to study its structural and functional importance. Site-directed mutagenesis was used to create the mutant of the exo-inulinase (Δ6SL). To investigate the stability of the region spanning this loop, MD simulations were performed 80ns for 20-85 residues. Molecular docking was performed to compare the interactions in the active sites of enzymes with fructose as a ligand. Accordingly, the functional thermostability of the exo-inulinase was significantly decreased upon loop fragment deletion. Evaluation of the kinetics parameters (V max , K m , k cat and, k cat /K m ) and activation energy (E a ) of the catalysis of enzymes indicated the importance of the deleted sequence on the catalytic performance of the enzyme. In conclusion, six-membered adjacent active site loop fragment not only plays a crucial role in the stability of the enzyme, but also it involves in the enzyme catalysis through lowering the activation energy of the catalysis and effective improving the catalytic performance. Copyright © 2016. Published by Elsevier B.V.

A multi-level intervention in subsidized housing sites to increase fruit and vegetable access and intake: Rationale, design and methods of the 'Live Well, Viva Bien' cluster randomized trial.

PubMed

Gans, Kim M; Gorham, Gemma; Risica, Patricia M; Dulin-Keita, Akilah; Dionne, Laura; Gao, Tina; Peters, Sarah; Principato, Ludovica

2016-06-28

Adequate fruit and vegetable (F&V) intake is important for disease prevention. Yet, most Americans, especially low-income and racial/ethnic minorities, do not eat adequate amounts. These disparities are partly attributable to food environments in low-income neighborhoods where residents often have limited access to affordable, healthful food and easy access to inexpensive, unhealthful foods. Increasing access to affordable healthful food in underserved neighborhoods through mobile markets is a promising, year-round strategy for improving dietary behaviors and reducing F&V intake disparities. However, to date, there have been no randomized controlled trials studying their effectiveness. The objective of the 'Live Well, Viva Bien' (LWVB) cluster randomized controlled trial is to evaluate the efficacy of a multicomponent mobile market intervention at increasing F&V intake among residents of subsidized housing complexes. One housing complex served as a pilot site for the intervention group and the remaining 14 demographically-matched sites were randomized into either the intervention or control group. The intervention group received bimonthly, discount, mobile, fresh F&V markets in conjunction with a nutrition education intervention (two F&V campaigns, newsletters, DVDs and cooking demonstrations) for 12 months. The control group received physical activity and stress reduction interventions. Outcome measures include F&V intake (measured by two validated F&V screeners at baseline, six-month and twelve-months) along with potential psychosocial mediating variables. Extensive quantitative and qualitative process evaluation was also conducted throughout the study. Modifying neighborhood food environments in ways that increase access to affordable, healthful food is a promising strategy for improving dietary behaviors among low-income, racial and ethnic minority groups at increased risk for obesity and other food-related chronic diseases. Discount, mobile F&V markets

The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

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

Gopal, B.; Madan, Lalima L.; Betz, Stephen F.

2010-11-10

Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s),more » whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.« less

5 CFR 2606.201 - Requests for access.

Code of Federal Regulations, 2011 CFR

2011-01-01

... on OGE's Web site at http://www.usoge.gov, or upon request from OGE's Office of General Counsel and... Office of Federal Register at the GPO Access Web site (http://www.access.gpo.gov/su_docs/aces/PrivacyAct... individual's full name (including her maiden name, if pertinent), dates of employment, social security number...

18 CFR 16.5 - Site access for a competing applicant.

Code of Federal Regulations, 2014 CFR

2014-04-01

... in providing access, including energy generation lost as a result of modification of project... the Office of Energy Projects for resolution in the manner specified in § 16.8(b)(5) prior to the... access may be referred to the Director of the Office of Energy Projects for resolution in the manner...

18 CFR 16.5 - Site access for a competing applicant.

Code of Federal Regulations, 2012 CFR

2012-04-01

... in providing access, including energy generation lost as a result of modification of project... the Office of Energy Projects for resolution in the manner specified in § 16.8(b)(5) prior to the... access may be referred to the Director of the Office of Energy Projects for resolution in the manner...

18 CFR 16.5 - Site access for a competing applicant.

Code of Federal Regulations, 2013 CFR

2013-04-01

... in providing access, including energy generation lost as a result of modification of project... the Office of Energy Projects for resolution in the manner specified in § 16.8(b)(5) prior to the... access may be referred to the Director of the Office of Energy Projects for resolution in the manner...

18 CFR 16.5 - Site access for a competing applicant.

Code of Federal Regulations, 2010 CFR

2010-04-01

... in providing access, including energy generation lost as a result of modification of project... the Office of Energy Projects for resolution in the manner specified in § 16.8(b)(5) prior to the... access may be referred to the Director of the Office of Energy Projects for resolution in the manner...

18 CFR 16.5 - Site access for a competing applicant.

Code of Federal Regulations, 2011 CFR

2011-04-01

... in providing access, including energy generation lost as a result of modification of project... the Office of Energy Projects for resolution in the manner specified in § 16.8(b)(5) prior to the... access may be referred to the Director of the Office of Energy Projects for resolution in the manner...

A registry-based randomized trial comparing radial and femoral approaches in women undergoing percutaneous coronary intervention: the SAFE-PCI for Women (Study of Access Site for Enhancement of PCI for Women) trial.

PubMed

Rao, Sunil V; Hess, Connie N; Barham, Britt; Aberle, Laura H; Anstrom, Kevin J; Patel, Tejan B; Jorgensen, Jesse P; Mazzaferri, Ernest L; Jolly, Sanjit S; Jacobs, Alice; Newby, L Kristin; Gibson, C Michael; Kong, David F; Mehran, Roxana; Waksman, Ron; Gilchrist, Ian C; McCourt, Brian J; Messenger, John C; Peterson, Eric D; Harrington, Robert A; Krucoff, Mitchell W

2014-08-01

This study sought to determine the effect of radial access on outcomes in women undergoing percutaneous coronary intervention (PCI) using a registry-based randomized trial. Women are at increased risk of bleeding and vascular complications after PCI. The role of radial access in women is unclear. Women undergoing cardiac catheterization or PCI were randomized to radial or femoral arterial access. Data from the CathPCI Registry and trial-specific data were merged into a final study database. The primary efficacy endpoint was Bleeding Academic Research Consortium type 2, 3, or 5 bleeding or vascular complications requiring intervention. The primary feasibility endpoint was access site crossover. The primary analysis cohort was the subgroup undergoing PCI; sensitivity analyses were conducted in the total randomized population. The trial was stopped early for a lower than expected event rate. A total of 1,787 women (691 undergoing PCI) were randomized at 60 sites. There was no significant difference in the primary efficacy endpoint between radial or femoral access among women undergoing PCI (radial 1.2% vs. 2.9% femoral, odds ratio [OR]: 0.39; 95% confidence interval [CI]: 0.12 to 1.27); among women undergoing cardiac catheterization or PCI, radial access significantly reduced bleeding and vascular complications (0.6% vs. 1.7%; OR: 0.32; 95% CI: 0.12 to 0.90). Access site crossover was significantly higher among women assigned to radial access (PCI cohort: 6.1% vs. 1.7%; OR: 3.65; 95% CI: 1.45 to 9.17); total randomized cohort: (6.7% vs. 1.9%; OR: 3.70; 95% CI: 2.14 to 6.40). More women preferred radial access. In this pragmatic trial, which was terminated early, the radial approach did not significantly reduce bleeding or vascular complications in women undergoing PCI. Access site crossover occurred more often in women assigned to radial access. (SAFE-PCI for Women; NCT01406236). Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc

Ligand accessibility to the HIV-1 Env co-receptor binding site can occur prior to CD4 engagement and is independent of viral tier category.

PubMed

Boliar, Saikat; Patil, Shilpa; Shukla, Brihaspati N; Ghobbeh, Ali; Deshpande, Suprit; Chen, Weizao; Guenaga, Javier; Dimitrov, Dimiter S; Wyatt, Richard T; Chakrabarti, Bimal K

2018-06-01

HIV-1 virus entry into target cells requires the envelope glycoprotein (Env) to first bind the primary receptor, CD4 and subsequently the co-receptor. Antibody access to the co-receptor binding site (CoRbs) in the pre-receptor-engaged state, prior to cell attachment, remains poorly understood. Here, we have demonstrated that for tier-1 Envs, the CoRbs is directly accessible to full-length CD4-induced (CD4i) antibodies even before primary receptor engagement, indicating that on these Envs the CoRbs site is either preformed or can conformationally sample post-CD4-bound state. Tier-2 and tier-3 Envs, which are resistant to full-length CD4i antibody, are neutralized by m36.4, a lower molecular mass of CD4i-directed domain antibody. In some tier-2 and tier-3 Envs, CoRbs is accessible to m36.4 even prior to cellular attachment in an Env-specific manner independent of their tier category. These data suggest differential structural arrangements of CoRbs and varied masking of ligand access to the CoRbs in different Env isolates. Copyright © 2018 Elsevier Inc. All rights reserved.

ANALYSIS OF STANDARD MULTI-PORT VS. SINGLE SITE ACCESS FOR LAPAROSCOPIC SKILLS TRAINING

PubMed Central

Cox, Daniel R; Zeng, Wenjing; Frisella, Margaret M; Brunt, L. Michael

2015-01-01

Introduction Single site access (SSA) laparoscopy is more challenging to perform than multi-port(MP) laparoscopy. We examined MP versus SSA skills training on laparoscopic performance in surgically naive individuals. Methods Forty end-of-1st year medical students were randomized into two groups. Both were trained on 4 basic laparoscopic drills (peg, rope, bean drop, pattern cutting) using a standard MP setup (Group 1) or SSA approach (Group 2). Time to proficiency and number of repetitions (reps) were recorded. Each group then crossed over to the alternate approach where the sequence was repeated. Data are mean ± SD and statistical analysis was with two-tailed, unpaired t-test. Results Total times to proficiency for the SSA and MP approaches was not significantly different between groups (Group 1 M-P 234.0 ± 114.9 min vs Group 2 SSA 216.4 ± 106.5 min, p=0.67). The MP-trained group took less time to reach proficiency on the standard MP setup than the SSA group on the SSA approach (119.1 ± 69.7 min vs 178.0 ± 93.4 min, p=0.058) with significantly fewer repetitions (77.6 ± 42.6 vs. 118.8 ± 54.3, p=0.027). The SSA-trained group took significantly less time to reach proficiency on the MP setup than the standard MP-trained group (38.4 ± 29.4 min vs. 119.1 ± 69.7 min; p=0.0013) requiring only a mean of 26.9 total repetitions. When the standard MP group crossed over to the SSA setup, they took significantly less time to reach proficiency with the SSA approach than the SSA-trained group (114.8 ± 50.5 min vs. 178.0 ± 93.4 min, p=0.026) but with more total repetitions than with the M-P approach (86.2 ± 35.2 vs 77.6 ± 42.6, p= NS). Conclusions Laparoscopic single site access skills training results in longer times and more repetitions to achieve proficiency than multi-port training, but the skills acquired transfer well to the multi-port approach. PMID:20872019

A hybrid QM/MM simulation study of intramolecular proton transfer in the pyridoxal 5'-phosphate in the active site of transaminase: influence of active site interaction on proton transfer.

PubMed

Dutta Banik, Sindrila; Chandra, Amalendu

2014-09-25

Pyridoxal 5'-phosphate (PLP) Schiff base, a versatile cofactor, exhibits a tautomeric equilibrium that involves an intramolecular proton transfer between the N-protonated zwitterionic ketoenamine tautomer and the O-protonated covalent enolimine tautomer. It has been postulated that for the catalytic activity, the PLP has to be in the zwitterionic ketoenamine tautomeric form. However, the exact position of the tautomeric equilibrium of Schiff base in the active site of PLP-dependent enzyme is not known yet. In the present work, we investigated the tautomeric equilibrium for the external aldimine state of PLP aspartate (PLP-Asp) Schiff base in the active site of aspartate aminotransferase (AspAT) using combined quantum mechanical and molecular mechanical simulations. The main focus of the present study is to analyze the factors that control the tautomeric equilibrium in the active sites of various PLP-dependent enzymes. The results show that the ketoenamine tautomer is more preferred than the enolimine tautomer both in the gas and aqueous phases as well as in the active site of AspAT. Current simulations show that the active site of AspAT is more suitable for the ketoenamine tautomer compared to the enolimine tautomer. Interestingly, the Tyr225 acts as a proton donor to the phenolic oxygen in the ketoenamine tautomer, while in the covalent enolimine tautomer, it acts as a proton acceptor to the phenolic oxygen. Finally, the metadynamics study confirms this result. The calculated free energy barrier is about 7.5 kcal/mol. A comparative analysis of the microenvironment created by the active site residues of three different PLP-dependent enzymes (aspartate aminotransferase, Dopa decarboxylase, and Ala-racemase) has been carried out to understand the controlling factor(s) of the tautomeric equilibrium. The analysis shows that the intermolecular hydrogen bonding between active site residues and the phenolic oxygen of PLP shifts the tautomeric equilibrium toward the N

The AAS Working Group on Accessibility and Disability (WGAD): Year 1 Highlights

NASA Astrophysics Data System (ADS)

Aarnio, Alicia; Monkiewicz, Jacqueline; Murphy, Nicholas Arnold; Nordhaus, Jason; Tuttle, Sarah E.

2017-01-01

The AAS Working Group on Accessibility and Disability (WGAD) was formed in January of 2016 with the express purpose of seeking equity of opportunity and building inclusive practices for disabled astronomers at all career stages. In our first year, the WGAD has been actively developing resources and the online infrastructure for the dissemination of information and engagement with the astronomy community. Our official WGAD website has gone live, and we have used both the access: astronomy google group and blog to discuss specific issues of disability justice and to raise awareness for less-discussed barriers to access. The WGAD has developed relationships and collaboration with AAS inclusion committees (SGMA, CSMA, CSWA) so our work can recognize and address the intersections of identity astronomers occupy. In this presentation, we summarize our year one activities, focusing on our recently developed set of recommendations for journal accessibility to ensure everyone can engage with journal content and navigate the submission process. We will also discuss ongoing and future endeavors: a best practices guide for accessibility to be available via our website, and a site visit program.

Non-visual Web Browsing: Beyond Web Accessibility

PubMed Central

Ramakrishnan, I.V.; Ashok, Vikas

2017-01-01

People with vision impairments typically use screen readers to browse the Web. To facilitate non-visual browsing, web sites must be made accessible to screen readers, i.e., all the visible elements in the web site must be readable by the screen reader. But even if web sites are accessible, screen-reader users may not find them easy to use and/or easy to navigate. For example, they may not be able to locate the desired information without having to listen to a lot of irrelevant contents. These issues go beyond web accessibility and directly impact web usability. Several techniques have been reported in the accessibility literature for making the Web usable for screen reading. This paper is a review of these techniques. Interestingly, the review reveals that understanding the semantics of the web content is the overarching theme that drives these techniques for improving web usability. PMID:29202137

Non-visual Web Browsing: Beyond Web Accessibility.

PubMed

Ramakrishnan, I V; Ashok, Vikas; Billah, Syed Masum

2017-07-01

People with vision impairments typically use screen readers to browse the Web. To facilitate non-visual browsing, web sites must be made accessible to screen readers, i.e., all the visible elements in the web site must be readable by the screen reader. But even if web sites are accessible, screen-reader users may not find them easy to use and/or easy to navigate. For example, they may not be able to locate the desired information without having to listen to a lot of irrelevant contents. These issues go beyond web accessibility and directly impact web usability. Several techniques have been reported in the accessibility literature for making the Web usable for screen reading. This paper is a review of these techniques. Interestingly, the review reveals that understanding the semantics of the web content is the overarching theme that drives these techniques for improving web usability.

Community Update on Site Activities, July 19, 2013

EPA Pesticide Factsheets

In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites*

PubMed Central

Honaker, Matthew T.; Acchione, Mauro; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

2013-01-01

The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. PMID:23649628

Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

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

Crichlow, G.; Lubetsky, J; Leng, L

Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic datamore » indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.« less

Rac1 GTPase activates the WAVE regulatory complex through two distinct binding sites.

PubMed

Chen, Baoyu; Chou, Hui-Ting; Brautigam, Chad A; Xing, Wenmin; Yang, Sheng; Henry, Lisa; Doolittle, Lynda K; Walz, Thomas; Rosen, Michael K

2017-09-26

The Rho GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization, which underpins diverse cellular processes. Here we report the structure of a WRC-Rac1 complex determined by cryo-electron microscopy. Surprisingly, Rac1 is not located at the binding site on the Sra1 subunit of the WRC previously identified by mutagenesis and biochemical data. Rather, it binds to a distinct, conserved site on the opposite end of Sra1. Biophysical and biochemical data on WRC mutants confirm that Rac1 binds to both sites, with the newly identified site having higher affinity and both sites required for WRC activation. Our data reveal that the WRC is activated by simultaneous engagement of two Rac1 molecules, suggesting a mechanism by which cells may sense the density of active Rac1 at membranes to precisely control actin assembly.

Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

DOE PAGES

Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

2016-08-17

Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [Cu IIOH] + species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [Cu IIOH] + active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu] 2+ and Cu 3O 3 motifs.« less

Helicobacter pylori purine nucleoside phosphorylase shows new distribution patterns of open and closed active site conformations and unusual biochemical features.

PubMed

Narczyk, Marta; Bertoša, Branimir; Papa, Lucija; Vuković, Vedran; Leščić Ašler, Ivana; Wielgus-Kutrowska, Beata; Bzowska, Agnieszka; Luić, Marija; Štefanić, Zoran

2018-04-01

Even with decades of research, purine nucleoside phosphorylases (PNPs) are enzymes whose mechanism is yet to be fully understood. This is especially true in the case of hexameric PNPs, and is probably, in part, due to their complex oligomeric nature and a whole spectrum of active site conformations related to interactions with different ligands. Here we report an extensive structural characterization of the apo forms of hexameric PNP from Helicobacter pylori (HpPNP), as well as its complexes with phosphate (P i ) and an inhibitor, formycin A (FA), together with kinetic, binding, docking and molecular dynamics studies. X-ray structures show previously unseen distributions of open and closed active sites. Microscale thermophoresis results indicate that a two-site model describes P i binding, while a three-site model is needed to characterize FA binding, irrespective of P i presence. The latter may be related to the newly found nonstandard mode of FA binding. The ternary complex of the enzyme with P i and FA shows, however, that P i binding stabilizes the standard mode of FA binding. Surprisingly, HpPNP has low affinity towards the natural substrate adenosine. Molecular dynamics simulations show that P i moves out of most active sites, in accordance with its weak binding. Conformational changes between nonstandard and standard binding modes of nucleoside are observed during the simulations. Altogether, these findings show some unique features of HpPNP and provide new insights into the functioning of the active sites, with implications for understanding the complex mechanism of catalysis of this enzyme. The atomic coordinates and structure factors have been deposited in the Protein Data Bank: with accession codes 6F52 (HpPNPapo_1), 6F5A (HpPNPapo_2), 6F5I (HpPNPapo_3), 5LU0 (HpPNP_PO4), 6F4W (HpPNP_FA) and 6F4X (HpPNP_PO4_FA). Purine nucleoside orthophosphate ribosyl transferase, EC2.4.2.1, UniProtID: P56463. © 2018 Federation of European Biochemical Societies.

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.

Structure of the S. aureus PI-specific phospholipase C reveals modulation of active site access by a titratable π-cation latched loop†

PubMed Central

Goldstein, Rebecca; Cheng, Jiongjia; Stec, Boguslaw; Roberts, Mary F.

2012-01-01

Staphylococcus aureus secretes a phosphatidylinositol-specific phospholipase C (PIPLC) as a virulence factor that is unusual in exhibiting higher activity at acidic pH values than other enzymes in this class. We have determined the crystal structure of this enzyme at pH 4.6 and pH 7.5. Under slightly basic conditions, the S. aureus PI-PLC structure closely follows the conformation of other bacterial PI-PLCs. However, when crystallized under acidic conditions, a large section of mobile loop at the αβ-barrel rim in the vicinity of the active site shows ~10 Å shift. This loop displacement at acidic pH is the result of a titratable intramolecular π-cation interaction between His258 and Phe249. This was verified by a structure of the mutant protein H258Y crystallized at pH 4.6, which does not exhibit the large loop shift. The intramolecular π-cation interaction for S. aureus PI-PLC provides an explanation for the activity of the enzyme at acid pH and also suggests how phosphatidylcholine, as a competitor for Phe249, may kinetically activate this enzyme. PMID:22390775

What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

PubMed

Zhang, Ni; Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

2017-06-22

Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. ©Ni Zhang, Shelly Campo, Jingzhen Yang, Petya Eckler, Linda Snetselaar, Kathleen Janz, Emily Leary. Originally published in the Journal of Medical

What Motivates Young Adults to Talk About Physical Activity on Social Network Sites?

PubMed Central

Campo, Shelly; Yang, Jingzhen; Eckler, Petya; Snetselaar, Linda; Janz, Kathleen; Leary, Emily

2017-01-01

Background Electronic word-of-mouth on social network sites has been used successfully in marketing. In social marketing, electronic word-of-mouth about products as health behaviors has the potential to be more effective and reach more young adults than health education through traditional mass media. However, little is known about what motivates people to actively initiate electronic word-of-mouth about health behaviors on their personal pages or profiles on social network sites, thus potentially reaching all their contacts on those sites. Objective This study filled the gap by applying a marketing theoretical model to explore the factors associated with electronic word-of-mouth on social network sites about leisure-time physical activity. Methods A Web survey link was sent to undergraduate students at one of the Midwestern universities and 439 of them completed the survey. Results The average age of the 439 participants was 19 years (SD=1 year, range: 18-24). Results suggested that emotional engagement with leisure-time physical activity (ie, affective involvement in leisure-time physical activity) predicted providing relevant opinions or information on social network sites. Social network site users who perceived stronger ties with all their contacts were more likely to provide and seek leisure-time physical activity opinions and information. People who provided leisure-time physical activity opinions and information were more likely to seek opinions and information, and people who forwarded information about leisure-time physical activity were more likely to chat about it. Conclusions This study shed light on the application of the electronic word-of-mouth theoretical framework in promoting health behaviors. The findings can also guide the development of future social marketing interventions using social network sites to promote leisure-time physical activity. PMID:28642215

Corrosion Research And Web Site Activities

NASA Technical Reports Server (NTRS)

Heidersbach, Robert H.

2001-01-01

This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

Corrosion Research and Web Site Activities

NASA Technical Reports Server (NTRS)

Heidersbach, Robert H.

2002-01-01

This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

76 FR 40454 - Proposed Information Collection (VSO Access to VHA Electronic Health Records) Activity; Comment...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-08

... Access to VHA Electronic Health Records) Activity; Comment Request AGENCY: Veterans Health Administration... Access to VHA Electronic Health Records, VA Form 10- 0400. OMB Control Number: 2900-0710. Type of Review... were granted power of attorney by veterans who have medical information recorded in VHA electronic...

Channeling by Proximity: The Catalytic Advantages of Active Site Colocalization Using Brownian Dynamics.

PubMed

Bauler, Patricia; Huber, Gary; Leyh, Thomas; McCammon, J Andrew

2010-05-06

Nature often colocalizes successive steps in a metabolic pathway. Such organization is predicted to increase the effective concentration of pathway intermediates near their recipient active sites and to enhance catalytic efficiency. Here, the pathway of a two-step reaction is modeled using a simple spherical approximation for the enzymes and substrate particles. Brownian dynamics are used to simulate the trajectory of a substrate particle as it diffuses between the active site zones of two different enzyme spheres. The results approximate distances for the most effective reaction pathways, indicating that the most effective reaction pathway is one in which the active sites are closely aligned. However, when the active sites are too close, the ability of the substrate to react with the first enzyme was hindered, suggesting that even the most efficient orientations can be improved for a system that is allowed to rotate or change orientation to optimize the likelihood of reaction at both sites.

Translating access into utilization: lessons from the design and evaluation of a health insurance Web site to promote reproductive health care for young women in Massachusetts.

PubMed

Janiak, Elizabeth; Rhodes, Elizabeth; Foster, Angel M

2013-12-01

Following state-level health care reform in Massachusetts, young women reported confusion over coverage of contraception and other sexual and reproductive health services under newly available health insurance products. To address this gap, a plain-language Web site titled "My Little Black Book for Sexual Health" was developed by a statewide network of reproductive health stakeholders. The purpose of this evaluation was to assess the health literacy demands and usability of the site among its target audience, women ages 18-26 years. We performed an evaluation of the literacy demands of the Web site's written content and tested the Web site's usability in a health communications laboratory. Participants found the Web site visually appealing and its overall design concept accessible. However, the Web site's literacy demands were high, and all participants encountered problems navigating through the Web site. Following this evaluation, the Web site was modified to be more usable and more comprehensible to women of all health literacy levels. To avail themselves of sexual and reproductive health services newly available under expanded health insurance coverage, young women require customized educational resources that are rigorously evaluated to ensure accessibility. To maximize utilization of reproductive health services under expanded health insurance coverage, US women require customized educational resources commensurate with their literacy skills. The application of established research methods from the field of health communications will enable advocates to evaluate and adapt these resources to best serve their targeted audiences. © 2013.

Critical Access Hospitals and Retail Activity: An Empirical Analysis in Oklahoma

ERIC Educational Resources Information Center

Brooks, Lara; Whitacre, Brian E.

2011-01-01

Purpose: This paper takes an empirical approach to determining the effect that a critical access hospital (CAH) has on local retail activity. Previous research on the relationship between hospitals and economic development has primarily focused on single-case, multiplier-oriented analysis. However, as the efficacy of federal and state-level rural…

Cooperative activation of cardiac transcription through myocardin bridging of paired MEF2 sites

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

Anderson, Courtney M.; Hu, Jianxin; Thomas, Reuben

2017-03-28

Enhancers frequently contain multiple binding sites for the same transcription factor. These homotypic binding sites often exhibit synergy, whereby the transcriptional output from two or more binding sites is greater than the sum of the contributions of the individual binding sites alone. Although this phenomenon is frequently observed, the mechanistic basis for homotypic binding site synergy is poorly understood. Here in this paper, we identify a bona fide cardiac-specific Prkaa2 enhancer that is synergistically activated by homotypic MEF2 binding sites. We show that two MEF2 sites in the enhancer function cooperatively due to bridging of the MEF2C-bound sites by themore » SAP domain-containing co-activator protein myocardin, and we show that paired sites buffer the enhancer from integration site-dependent effects on transcription in vivo. Paired MEF2 sites are prevalent in cardiac enhancers, suggesting that this might be a common mechanism underlying synergy in the control of cardiac gene expression in vivo.« less

Maturation of the [Ni-4Fe-4S] active site of carbon monoxide dehydrogenases.

PubMed

Merrouch, Mériem; Benvenuti, Martino; Lorenzi, Marco; Léger, Christophe; Fourmond, Vincent; Dementin, Sébastien

2018-02-14

Nickel-containing enzymes are diverse in terms of function and active site structure. In many cases, the biosynthesis of the active site depends on accessory proteins which transport and insert the Ni ion. We review and discuss the literature related to the maturation of carbon monoxide dehydrogenases (CODH) which bear a nickel-containing active site consisting of a [Ni-4Fe-4S] center called the C-cluster. The maturation of this center has been much less studied than that of other nickel-containing enzymes such as urease and NiFe hydrogenase. Several proteins present in certain CODH operons, including the nickel-binding proteins CooT and CooJ, still have unclear functions. We question the conception that the maturation of all CODH depends on the accessory protein CooC described as essential for nickel insertion into the active site. The available literature reveals biological variations in CODH active site biosynthesis.

CASPASE-9 CARD:CORE DOMAIN INTERACTIONS REQUIRE A PROPERLY-FORMED ACTIVE SITE

PubMed Central

Huber, Kristen L.; Serrano, Banyuhay P.; Hardy, Jeanne A.

2018-01-01

Caspase-9 is a critical factor in the initiation of apoptosis, and as a result is tightly regulated by a number of mechanisms. Caspase-9 contains a Caspase Activation and Recruitment Domain (CARD), which enables caspase-9 to form a tight interaction with the apoptosome, a heptameric activating platform. The caspase-9 CARD has been thought to be principally involved in recruitment to the apoptosome, but its roles outside this interaction have yet to be uncovered. In this work we show that the CARD is involved in physical interactions with the catalytic core of caspase-9 in the absence of the apoptosome; this interaction requires a properly formed caspase-9 active site. The active sites of caspases are composed of four extremely mobile loops. When the active-site loops are not properly ordered, the CARD and core domains of caspase-9 do not interact and behave independently, like loosely tethered beads. When the active-site loop bundle is properly ordered, the CARD domain interacts with the catalytic core, forming a single folding unit. Together these findings provide mechanistic insight into a new level of caspase-9 regulation, prompting speculation that the CARD may also play a role in the recruitment or recognition of substrate. PMID:29500231

Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

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

Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

Active site densities, oxygen activation and adsorbed reactive oxygen in alcohol activation on npAu catalysts

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

Wang, Lu-Cun; Friend, C. M.; Fushimi, Rebecca

2016-01-01

The activation of molecular O 2as well as the reactivity of adsorbed oxygen species is of central importance in aerobic selective oxidation chemistry on Au-based catalysts. Herein, we address the issue of O 2activation on unsupported nanoporous gold (npAu) catalysts by applying a transient pressure technique, a temporal analysis of products (TAP) reactor, to measure the saturation coverage of atomic oxygen, its collisional dissociation probability, the activation barrier for O 2dissociation, and the facility with which adsorbed O species activate methanol, the initial step in the catalytic cycle of esterification. The results from these experiments indicate that molecular O 2dissociationmore » is associated with surface silver, that the density of reactive sites is quite low, that adsorbed oxygen atoms do not spill over from the sites of activation onto the surrounding surface, and that methanol reacts quite facilely with the adsorbed oxygen atoms. In addition, the O species from O 2dissociation exhibits reactivity for the selective oxidation of methanol but not for CO. The TAP experiments also revealed that the surface of the npAu catalyst is saturated with adsorbed O under steady state reaction conditions, at least for the pulse reaction.« less

Brownian aggregation rate of colloid particles with several active sites

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

Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V., E-mail: chern@ns.kinetics.nsc.ru

2014-08-14

We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shownmore » to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.« less

Stepwise Loop Insertion Strategy for Active Site Remodeling to Generate Novel Enzyme Functions.

PubMed

Hoque, Md Anarul; Zhang, Yong; Chen, Liuqing; Yang, Guangyu; Khatun, Mst Afroza; Chen, Haifeng; Hao, Liu; Feng, Yan

2017-05-19

The remodeling of active sites to generate novel biocatalysts is an attractive and challenging task. We developed a stepwise loop insertion strategy (StLois), in which randomized residue pairs are inserted into active site loops. The phosphotriesterase-like lactonase from Geobacillus kaustophilus (GkaP-PLL) was used to investigate StLois's potential for changing enzyme function. By inserting six residues into active site loop 7, the best variant ML7-B6 demonstrated a 16-fold further increase in catalytic efficiency toward ethyl-paraoxon compared with its initial template, that is a 609-fold higher, >10 7 fold substrate specificity shift relative to that of wild-type lactonase. The remodeled variants displayed 760-fold greater organophosphate hydrolysis activity toward the organophosphates parathion, diazinon, and chlorpyrifos. Structure and docking computations support the source of notably inverted enzyme specificity. Considering the fundamental importance of active site loops, the strategy has potential for the rapid generation of novel enzyme functions by loop remodeling.

Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality.

PubMed

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A; Schimmel, Paul; Yang, Xiang-Lei

2010-01-01

Protein multifunctionality is an emerging explanation for the complexity of higher organisms. In this regard, aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, but some also act in pathways for inflammation, angiogenesis and apoptosis. It is unclear how these multiple functions evolved and how they relate to the active site. Here structural modeling analysis, mutagenesis and cell-based functional studies show that the potent angiostatic, natural fragment of human tryptophanyl-tRNA synthetase (TrpRS) associates via tryptophan side chains that protrude from its cognate cellular receptor vascular endothelial cadherin (VE-cadherin). VE-cadherin's tryptophan side chains fit into the tryptophan-specific active site of the synthetase. Thus, specific side chains of the receptor mimic amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.

Identification of protein–protein interfaces by decreased amide proton solvent accessibility

PubMed Central

Mandell, Jeffrey G.; Falick, Arnold M.; Komives, Elizabeth A.

1998-01-01

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity. PMID:9843953

In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

PubMed

Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

2012-05-01

Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin

PubMed Central

Treuheit, Nicholas A.; Beach, Muneera A.; Komives, Elizabeth A.

2011-01-01

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethylketone to the active site serine, as well as non-covalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1, however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-L-arginine-(3-methyl-1,5-pantanediyl) amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause the same reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or to exosite 1. PMID:21526769

Thermodynamic compensation upon binding to exosite 1 and the active site of thrombin.

PubMed

Treuheit, Nicholas A; Beach, Muneera A; Komives, Elizabeth A

2011-05-31

Several lines of experimental evidence including amide exchange and NMR suggest that ligands binding to thrombin cause reduced backbone dynamics. Binding of the covalent inhibitor dPhe-Pro-Arg chloromethyl ketone to the active site serine, as well as noncovalent binding of a fragment of the regulatory protein, thrombomodulin, to exosite 1 on the back side of the thrombin molecule both cause reduced dynamics. However, the reduced dynamics do not appear to be accompanied by significant conformational changes. In addition, binding of ligands to the active site does not change the affinity of thrombomodulin fragments binding to exosite 1; however, the thermodynamic coupling between exosite 1 and the active site has not been fully explored. We present isothermal titration calorimetry experiments that probe changes in enthalpy and entropy upon formation of binary ligand complexes. The approach relies on stringent thrombin preparation methods and on the use of dansyl-l-arginine-(3-methyl-1,5-pantanediyl)amide and a DNA aptamer as ligands with ideal thermodynamic signatures for binding to the active site and to exosite 1. Using this approach, the binding thermodynamic signatures of each ligand alone as well as the binding signatures of each ligand when the other binding site was occupied were measured. Different exosite 1 ligands with widely varied thermodynamic signatures cause a similar reduction in ΔH and a concomitantly lower entropy cost upon DAPA binding at the active site. The results suggest a general phenomenon of enthalpy-entropy compensation consistent with reduction of dynamics/increased folding of thrombin upon ligand binding to either the active site or exosite 1.

Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

PubMed Central

Miao, Yinglong; Baudry, Jerome

2011-01-01

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431

Active Site Mutations Change the Cleavage Specificity of Neprilysin

PubMed Central

Sexton, Travis; Hitchcook, Lisa J.; Rodgers, David W.; Bradley, Luke H.; Hersh, Louis B.

2012-01-01

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential. PMID:22384224

Effectiveness of off-line and web-based promotion of health information web sites.

PubMed

Jones, Craig E; Pinnock, Carole B

2002-01-01

The relative effectiveness of off-line and web-based promotional activities in increasing the use of health information web sites by target audiences were compared. Visitor sessions were classified according to their method of arrival at the site (referral) as external web site, search engine, or "no referrer" (i.e., visitor arriving at the site by inputting URL or using bookmarks). The number of Australian visitor sessions correlated with no referrer referrals but not web site or search-engine referrals. Results showed that the targeted consumer group is more likely to access the web site as a result of off-line promotional activities. The properties of target audiences likely to influence the effectiveness of off-line versus on-line promotional strategies include the size of the Internet using population of the target audience, their proficiency in the use of the Internet, and the increase in effectiveness of off-line promotional activities when applied to locally defined target audiences.

Carbinolamine Formation and Dehydration in a DNA Repair Enzyme Active Site

PubMed Central

Dodson, M. L.; Walker, Ross C.; Lloyd, R. Stephen

2012-01-01

In order to suggest detailed mechanistic hypotheses for the formation and dehydration of a key carbinolamine intermediate in the T4 pyrimidine dimer glycosylase (T4PDG) reaction, we have investigated these reactions using steered molecular dynamics with a coupled quantum mechanics–molecular mechanics potential (QM/MM). We carried out simulations of DNA abasic site carbinolamine formation with and without a water molecule restrained to remain within the active site quantum region. We recovered potentials of mean force (PMF) from thirty replicate reaction trajectories using Jarzynski averaging. We demonstrated feasible pathways involving water, as well as those independent of water participation. The water–independent enzyme–catalyzed reaction had a bias–corrected Jarzynski–average barrier height of approximately for the carbinolamine formation reaction and ) for the reverse reaction at this level of representation. When the proton transfer was facilitated with an intrinsic quantum water, the barrier height was approximately in the forward (formation) reaction and for the reverse. In addition, two modes of unsteered (free dynamics) carbinolamine dehydration were observed: in one, the quantum water participated as an intermediate proton transfer species, and in the other, the active site protonated glutamate hydrogen was directly transferred to the carbinolamine oxygen. Water–independent unforced proton transfer from the protonated active site glutamate carboxyl to the unprotonated N–terminal amine was also observed. In summary, complex proton transfer events, some involving water intermediates, were studied in QM/MM simulations of T4PDG bound to a DNA abasic site. Imine carbinolamine formation was characterized using steered QM/MM molecular dynamics. Dehydration of the carbinolamine intermediate to form the final imine product was observed in free, unsteered, QM/MM dynamics simulations, as was unforced acid-base transfer between the active site

Immobilization of alkaline phosphatase on solid surface through self-assembled monolayer and by active-site protection.

PubMed

Gao, En-Feng; Kang, Kyung Lhi; Kim, Jeong Hee

2014-06-01

Retaining biological activity of a protein after immobilization is an important issue and many studies reported to enhance the activity of proteins after immobilization. We recently developed a new immobilization method of enzyme using active-site protection and minimization of the cross-links between enzyme and surface with a DNA polymerase as a model system. In this study, we extended the new method to an enzyme with a small mono-substrate using alkaline phosphatase (AP) as another model system. A condition to apply the new method is that masking agents, in this case its own substrate needs to stay at the active-site of the enzyme to be immobilized in order to protect the active-site during the harsh immobilization process. This could be achieved by removal of essential divalent ion, Zn2+ that is required for full enzyme activity of AP from the masking solution while active-site of AP was protected with p-nitrophenyl phosphate (pNPP). Approximately 40% of the solution-phase activity was acquired with active-site protected immobilized AP. In addition to protection active-site of AP, the number of immobilization links was kinetically controlled. When the mole fraction of the activated carboxyl group of the linker molecule in self-assembled monolayer (SAM) of 12-mercaptododecanoic acid and 6-mercapto-1-ethanol was varied, 10% of 12-mercaptododecanoic acid gave the maximum enzyme activity. Approximately 51% increase in enzyme activity of the active-site protected AP was observed compared to that of the unprotected group. It was shown that the concept of active-site protection and kinetic control of the number of covalent immobilization bonds can be extended to enzymes with small mono-substrates. It opens the possibility of further extension of the new methods of active-site protection and kinetic control of immobilization bond to important enzymes used in research and industrial fields.

Preorganization of molecular binding sites in designed diiron proteins.

PubMed

Maglio, Ornella; Nastri, Flavia; Pavone, Vincenzo; Lombardi, Angela; DeGrado, William F

2003-04-01

De novo protein design provides an attractive approach to critically test the features that are required for metalloprotein structure and function. Previously we designed and crystallographically characterized an idealized dimeric model for the four-helix bundle class of diiron and dimanganese proteins [Dueferri 1 (DF1)]. Although the protein bound metal ions in the expected manner, access to its active site was blocked by large bulky hydrophobic residues. Subsequently, a substrate-access channel was introduced proximal to the metal-binding center, resulting in a protein with properties more closely resembling those of natural enzymes. Here we delineate the energetic and structural consequences associated with the introduction of these binding sites. To determine the extent to which the binding site was preorganized in the absence of metal ions, the apo structure of DF1 in solution was solved by NMR and compared with the crystal structure of the di-Zn(II) derivative. The overall fold of the apo protein was highly similar to that of the di-Zn(II) derivative, although there was a rotation of one of the helices. We also examined the thermodynamic consequences associated with building a small molecule-binding site within the protein. The protein exists in an equilibrium between folded dimers and unfolded monomers. DF1 is a highly stable protein (K(diss) = 0.001 fM), but the dissociation constant increases to 0.6 nM (deltadeltaG = 5.4 kcalmol monomer) as the active-site cavity is increased to accommodate small molecules.

Effects of Home Access to Active Videogames on Child Self-Esteem, Enjoyment of Physical Activity, and Anxiety Related to Electronic Games: Results from a Randomized Controlled Trial.

PubMed

Abbott, Rebecca A; Smith, Anne J; Howie, Erin K; Pollock, Clare; Straker, Leon

2014-08-01

Active-input videogames could provide a useful conduit for increasing physical activity by improving a child's self-confidence, physical activity enjoyment, and reducing anxiety. Therefore this study evaluated the impact of (a) the removal of home access to traditional electronic games or (b) their replacement with active-input videogames, on child self-perception, enjoyment of physical activity, and electronic game use anxiety. This was a crossover, randomized controlled trial, conducted over a 6-month period in participants' family homes in metropolitan Perth, Australia, from 2007 to 2010. Children 10-12 years old were recruited through school and community media. Of 210 children who were eligible, 74 met inclusion criteria, and 8 withdrew, leaving 66 children (33 girls) for analysis. A counterbalanced randomized order of three conditions sustained for 8 weeks each: No home access to electronic games, home access to traditional electronic games, and home access to active-input electronic games. Perception of self-esteem (Harter's Self Perception Profile for Children), enjoyment of physical activity (Physical Activity Enjoyment Scale questionnaire), and anxiety toward electronic game use (modified Loyd and Gressard Computer Anxiety Subscale) were assessed. Compared with home access to traditional electronic games, neither removal of all electronic games nor replacement with active-input games resulted in any significant change to child self-esteem, enjoyment of physical activity, or anxiety related to electronic games. Although active-input videogames have been shown to be enjoyable in the short term, their ability to impact on psychological outcomes is yet to be established.

The Design, Synthesis, and Characterization of Open Sites on Metal Clusters

NASA Astrophysics Data System (ADS)

Nigra, Michael Mark

Coordinatively unsaturated corner and edge atoms have been hypothesized to have the highest activity of sites responsible for many catalytic reactions on a metal surface. Recent studies have validated this hypothesis in varied reaction systems. However, quantification of different types of coordinatively unsaturated sites, and elucidation of their individual catalytic rates has remained a largely unresolved challenge when understanding catalysis on metal surfaces. Yet such structure-function knowledge would be invaluable to the design of more active and selective metal-surface catalysts in the future. I investigated the catalytic contributions of undercoordinated sites such as corner and edge atoms are investigated in a model reaction system using organic ligands bound to the gold nanoparticle surface. The catalyst consisted of 4 nm gold nanoparticles on a metal oxide support, using resazurin to resorufin as a model reaction system. My results demonstrate that in this system, corner atom sites are the most undercoordinated sites, and are over an order of magnitude more active when compared to undercoordinated edge atom sites, while terrace sites remain catalytically inactive for the reduction reaction of resazurin to resorufin. Catalytic activity has been also demonstrated for calixarene-bound gold nanoparticles using the reduction of 4-nitrophenol. With the 4-nitrophenol reduction reaction, a comparative study was undertaken to compare calixarene phosphine and calixarene thiol bound 4 nm gold particles. The results of the study suggested that a leached site was responsible for catalysis and not sites on the original gold nanoparticles. Future experiments with calixarene bound gold clusters could investigate ligand effects in reactions where the active site is not a leached or aggregated gold species, possibly in oxidation reactions, where electron-rich gold is hypothesized to be a good catalyst. The results that emphasize the enhanced catalytic activity of

Dynamic Perturbation of the Active Site Determines Reversible Thermal Inactivation in Glycoside Hydrolase Family 12.

PubMed

Jiang, Xukai; Li, Wen; Chen, Guanjun; Wang, Lushan

2017-02-27

The temperature dependence of enzyme catalysis is highly debated. Specifically, how high temperatures induce enzyme inactivation has broad implications for both fundamental and applied science. Here, we explored the mechanism of the reversible thermal inactivation in glycoside hydrolase family 12 (GH12) using comparative molecular dynamics simulations. First, we investigated the distribution of structural flexibility over the enzyme and found that the active site was the general thermal-sensitive region in GH12 cellulases. The dynamic perturbation of the active site before enzyme denaturation was explored through principal-component analysis, which indicated that variations in the collective motion and conformational ensemble of the active site may precisely correspond to enzyme transition from its active form to the inactive form. Furthermore, the degree of dynamic perturbation of the active site was found to be negatively correlated with the melting temperatures of GH12 enzymes, further proving the importance of the dynamic stability of the active site. Additionally, analysis of the residue-interaction network revealed that the active site in thermophilic enzyme was capable of forming additional contacts with other amino acids than those observed in the mesophilic enzyme. These interactions are likely the key mechanisms underlying the differences in rigidity of the active site. These findings provide further biophysical insights into the reversible thermal inactivation of enzymes and potential applications in future protein engineering.

Elimination of a ligand gating site generates a supersensitive olfactory receptor.

PubMed

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

2016-06-21

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

Elimination of a ligand gating site generates a supersensitive olfactory receptor

PubMed Central

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

2016-01-01

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

Non-active site mutation (Q123A) in New Delhi metallo-β-lactamase (NDM-1) enhanced its enzyme activity.

PubMed

Ali, Abid; Azam, Mohd W; Khan, Asad U

2018-06-01

New Delhi metallo β-lactamase-1 is one of the carbapenemases, causing hydrolysis of almost all β-lactamase antibiotics. Seventeen different NDM variants have been reported so far, they varied in their sequences either by single or multiple amino acid substitutions. Hence, it is important to understand its structural and functional relation. In the earlier studies role of active site residues has been studied but non-active site residues has not studied in detail. Therefore, we have initiated to further comprehend its structure and function relation by mutating some of its non-active site residues. A laboratory mutant of NDM-1 was generated by PCR-based site-directed mutagenesis, replacing Q to A at 123 position. The MICs of imipenem and meropenem for NDM-1 Q123A were found increased by 2 fold as compare to wild type and so the hydrolytic activity was enhanced (Kcat/Km) as compared to NDM-1 wild type. GOLD fitness scores were also found in favour of kinetics data. Secondary structure for α-helical content was determined by Far-UV circular dichroism (CD), which showed significant conformational changes. We conclude a noteworthy role of non-active-site amino acid residues in the catalytic activity of NDM-1. This study also provides an insight of emergence of new variants through natural evolution. Copyright © 2018 Elsevier B.V. All rights reserved.

Secure web-based access to radiology: forms and databases for fast queries

NASA Astrophysics Data System (ADS)

McColl, Roderick W.; Lane, Thomas J.

2002-05-01

Currently, Web-based access to mini-PACS or similar databases commonly utilizes either JavaScript, Java applets or ActiveX controls. Many sites do not permit applets or controls or other binary objects for fear of viruses or worms sent by malicious users. In addition, the typical CGI query mechanism requires several parameters to be sent with the http GET/POST request, which may identify the patient in some way; this in unacceptable for privacy protection. Also unacceptable are pages produced by server-side scripts which can be cached by the browser, since these may also contain sensitive information. We propose a simple mechanism for access to patient information, including images, which guarantees security of information, makes it impossible to bookmark the page, or to return to the page after some defined length of time. In addition, this mechanism is simple, therefore permitting rapid access without the need to initially download an interface such as an applet or control. In addition to image display, the design of the site allows the user to view and save movies of multi-phasic data, or to construct multi-frame datasets from entire series. These capabilities make the site attractive for research purposes such as teaching file preparation.

Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

PubMed

Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

2016-08-01

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Heterogeneous Electrocatalyst with Molecular Cobalt Ions Serving as the Center of Active Sites.

PubMed

Wang, Jiong; Ge, Xiaoming; Liu, Zhaolin; Thia, Larissa; Yan, Ya; Xiao, Wei; Wang, Xin

2017-02-08

Molecular Co 2+ ions were grafted onto doped graphene in a coordination environment, resulting in the formation of molecularly well-defined, highly active electrocatalytic sites at a heterogeneous interface for the oxygen evolution reaction (OER). The S dopants of graphene are suggested to be one of the binding sites and to be responsible for improving the intrinsic activity of the Co sites. The turnover frequency of such Co sites is greater than that of many Co-based nanostructures and IrO 2 catalysts. Through a series of carefully designed experiments, the pathway for the evolution of the Co cation-based molecular catalyst for the OER was further demonstrated on such a single Co-ion site for the first time. The Co 2+ ions were successively oxidized to Co 3+ and Co 4+ states prior to the OER. The sequential oxidation was coupled with the transfer of different numbers of protons/hydroxides and generated an active Co 4+ ═O fragment. A side-on hydroperoxo ligand of the Co 4+ site is proposed as a key intermediate for the formation of dioxygen.

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

PubMed Central

Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

2015-01-01

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

Mars-Learning AN Open Access Educational Database

NASA Astrophysics Data System (ADS)

Kolankowski, S. M.; Fox, P. A.

2016-12-01

Schools across America have begun focusing more and more on science and technology, giving their students greater opportunities to learn about planetary science and engineering. With the development of rovers and advanced scientific instrumentation, we are learning about Mars' geologic history on a daily basis. These discoveries are crucial to our understanding of Earth and our solar system. By bringing these findings into the classroom, students can learn key concepts about Earth and Planetary sciences while focusing on a relevant current event. However, with an influx of readily accessible information, it is difficult for educators and students to find accurate and relevant material. Mars-Learning seeks to unify these discoveries and resources. This site will provide links to educational resources, software, and blogs with a focus on Mars. Activities will be grouped by grade for the middle and high school levels. Programs and software will be labeled, open access, free, or paid to ensure users have the proper tools to get the information they need. For new educators or those new to the subject, relevant blogs and pre-made lesson plans will be available so instructors can ensure their success. The expectation of Mars-Learning is to provide stress-free access to learning materials that falls within a wide range of curriculum. By providing a thorough and encompassing site, Mars-Learning hopes to further our understanding of the Red Planet and equip students with the knowledge and passion to continue this research.

Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.

PubMed

Spatzal, Thomas; Perez, Kathryn A; Howard, James B; Rees, Douglas C

2015-12-16

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.

Access to environmental resources and physical activity levels of adults in Hawaii.

PubMed

Geller, Karly S; Nigg, Claudio R; Ollberding, Nicholas J; Motl, Robert W; Horwath, Caroline; Dishman, Rodney K

2015-03-01

Examine associations between physical activity (PA) and spatial accessibility to environmental PA resources in Hawaii. Metabolic equivalents (METs) of mild, moderate, and strenuous PA were compared for accessibility with environmental PA resources within a population-based sample of Hawaiian adults (n = 381). Multiple linear regression estimated differences in PA levels for residing further from a PA resource or residing in an area with a greater number of resources. No associations were found in the total sample. Analyses within subsamples stratified by ethnicity revealed that greater spatial accessibility to a PA resource was positively associated with strenuous PA among Caucasians (P = .04) but negatively associated with moderate PA among Native Hawaiians (P = .00). The lack of association in the total sample may be a consequence of Hawaii's unique environment. Results of stratified sample analyses are unique, providing groundwork for future examinations within parallel environments and among similar ethnic groups. © 2012 APJPH.

28 CFR 16.41 - Requests for access to records.

Code of Federal Regulations, 2010 CFR

2010-07-01

... the Government Printing Office's World Wide Web site (which can be found at http://www.access.gpo.gov... accessed electronically at the Government Printing Office's World Wide Web site (which can be found at http... requested records, you may also, at your option, include your social security number. (e) Verification of...

28 CFR 16.41 - Requests for access to records.

Code of Federal Regulations, 2011 CFR

2011-07-01

... the Government Printing Office's World Wide Web site (which can be found at http://www.access.gpo.gov... accessed electronically at the Government Printing Office's World Wide Web site (which can be found at http... requested records, you may also, at your option, include your social security number. (e) Verification of...

Sociospatial distribution of access to facilities for moderate and vigorous intensity physical activity in Scotland by different modes of transport

PubMed Central

2012-01-01

Background People living in neighbourhoods of lower socioeconomic status have been shown to have higher rates of obesity and a lower likelihood of meeting physical activity recommendations than their more affluent counterparts. This study examines the sociospatial distribution of access to facilities for moderate or vigorous intensity physical activity in Scotland and whether such access differs by the mode of transport available and by Urban Rural Classification. Methods A database of all fixed physical activity facilities was obtained from the national agency for sport in Scotland. Facilities were categorised into light, moderate and vigorous intensity activity groupings before being mapped. Transport networks were created to assess the number of each type of facility accessible from the population weighted centroid of each small area in Scotland on foot, by bicycle, by car and by bus. Multilevel modelling was used to investigate the distribution of the number of accessible facilities by small area deprivation within urban, small town and rural areas separately, adjusting for population size and local authority. Results Prior to adjustment for Urban Rural Classification and local authority, the median number of accessible facilities for moderate or vigorous intensity activity increased with increasing deprivation from the most affluent or second most affluent quintile to the most deprived for all modes of transport. However, after adjustment, the modelling results suggest that those in more affluent areas have significantly higher access to moderate and vigorous intensity facilities by car than those living in more deprived areas. Conclusions The sociospatial distributions of access to facilities for both moderate intensity and vigorous intensity physical activity were similar. However, the results suggest that those living in the most affluent neighbourhoods have poorer access to facilities of either type that can be reached on foot, by bicycle or by bus than

Methylation of an alpha-foetoprotein gene intragenic site modulates gene activity.

PubMed Central

Opdecamp, K; Rivière, M; Molné, M; Szpirer, J; Szpirer, C

1992-01-01

By comparing the methylation pattern of Mspl/Hpall sites in the 5' region of the mouse alpha-foetoprotein (AFP) gene of different cells (hepatoma cells, foetal and adult liver, fibroblasts), we found a correlation between gene expression and unmethylation of a site located in the first intron of the gene. Other sites did not show this correlation. In transfection experiments of unmethylated and methylated AFP-CAT chimeric constructions, we then showed that methylation of the intronic site negatively modulates expression of CAT activity. We also found that a DNA segment centered on this site binds nuclear proteins; however methylation did not affect protein binding. Images PMID:1371343

6 CFR 5.21 - Requests for access to records.

Code of Federal Regulations, 2011 CFR

2011-01-01

... accessed electronically at the Government Printing Office's World Wide Web site (which can be found at http... Printing Office's World Wide Web site (which can be found at http://www.access.gpo.gov/su_docs). (c... requested records, you may also, at your option, include your social security number. (e) Verification of...

6 CFR 5.21 - Requests for access to records.

Code of Federal Regulations, 2010 CFR

2010-01-01

... accessed electronically at the Government Printing Office's World Wide Web site (which can be found at http... Printing Office's World Wide Web site (which can be found at http://www.access.gpo.gov/su_docs). (c... requested records, you may also, at your option, include your social security number. (e) Verification of...

Improving Web Accessibility in a University Setting

ERIC Educational Resources Information Center

Olive, Geoffrey C.

2010-01-01

Improving Web accessibility for disabled users visiting a university's Web site is explored following the World Wide Web Consortium (W3C) guidelines and Section 508 of the Rehabilitation Act rules for Web page designers to ensure accessibility. The literature supports the view that accessibility is sorely lacking, not only in the USA, but also…

Access NASA Satellite Global Precipitation Data Visualization on YouTube

NASA Technical Reports Server (NTRS)

Liu, Z.; Su, J.; Acker, J.; Huffman, G.; Vollmer, B.; Wei, J.; Meyer, D.

2017-01-01

Since the satellite era began, NASA has collected a large volume of Earth science observations for research and applications around the world. The collected and archived satellite data at 12 NASA data centers can also be used for STEM education and activities such as disaster events, climate change, etc. However, accessing satellite data can be a daunting task for non-professional users such as teachers and students because of unfamiliarity of terminology, disciplines, data formats, data structures, computing resources, processing software, programming languages, etc. Over the years, many efforts including tools, training classes, and tutorials have been developed to improve satellite data access for users, but barriers still exist for non-professionals. In this presentation, we will present our latest activity that uses a very popular online video sharing Web site, YouTube (https://www.youtube.com/), for accessing visualizations of our global precipitation datasets at the NASA Goddard Earth Sciences (GES) Data and Information Services Center (DISC). With YouTube, users can access and visualize a large volume of satellite data without the necessity to learn new software or download data. The dataset in this activity is a one-month animation for the GPM (Global Precipitation Measurement) Integrated Multi-satellite Retrievals for GPM (IMERG). IMERG provides precipitation on a near-global (60 deg. N-S) coverage at half-hourly time interval, providing more details on precipitation processes and development compared to the 3-hourly TRMM (Tropical Rainfall Measuring Mission) Multisatellite Precipitation Analysis (TMPA, 3B42) product. When the retro-processing of IMERG during the TRMM era is finished in 2018, the entire video will contain more than 330,000 files and will last 3.6 hours. Future plans include development of flyover videos for orbital data for an entire satellite mission or project. All videos, including the one-month animation, will be uploaded and

Methodology for Localized and Accessible Image Formation and Elucidation

ERIC Educational Resources Information Center

Patil, Sandeep R.; Katiyar, Manish

2009-01-01

Accessibility is one of the key checkpoints in all software products, applications, and Web sites. Accessibility with digital images has always been a major challenge for the industry. Images form an integral part of certain type of documents and most Web 2.0-compliant Web sites. Individuals challenged with blindness and many dyslexics only make…

Social Media Use and Access to Digital Technology in US Young Adults in 2016

PubMed Central

Johnson, Amanda L; Ilakkuvan, Vinu; Jacobs, Megan A; Graham, Amanda L; Rath, Jessica M

2017-01-01

Background In 2015, 90% of US young adults with Internet access used social media. Digital and social media are highly prevalent modalities through which young adults explore identity formation, and by extension, learn and transmit norms about health and risk behaviors during this developmental life stage. Objective The purpose of this study was to provide updated estimates of social media use from 2014 to 2016 and correlates of social media use and access to digital technology in data collected from a national sample of US young adults in 2016. Methods Young adult participants aged 18-24 years in Wave 7 (October 2014, N=1259) and Wave 9 (February 2016, N=989) of the Truth Initiative Young Adult Cohort Study were asked about use frequency for 11 social media sites and access to digital devices, in addition to sociodemographic characteristics. Regular use was defined as using a given social media site at least weekly. Weighted analyses estimated the prevalence of use of each social media site, overlap between regular use of specific sites, and correlates of using a greater number of social media sites regularly. Bivariate analyses identified sociodemographic correlates of access to specific digital devices. Results In 2014, 89.42% (weighted n, 1126/1298) of young adults reported regular use of at least one social media site. This increased to 97.5% (weighted n, 965/989) of young adults in 2016. Among regular users of social media sites in 2016, the top five sites were Tumblr (85.5%), Vine (84.7%), Snapchat (81.7%), Instagram (80.7%), and LinkedIn (78.9%). Respondents reported regularly using an average of 7.6 social media sites, with 85% using 6 or more sites regularly. Overall, 87% of young adults reported access or use of a smartphone with Internet access, 74% a desktop or laptop computer with Internet access, 41% a tablet with Internet access, 29% a smart TV or video game console with Internet access, 11% a cell phone without Internet access, and 3% none of these

Lunar base siting

NASA Technical Reports Server (NTRS)

Staehle, Robert L.; Dowling, Richard

1991-01-01

As with any planetary body, the lunar surface is quite heterogeneous. There are widely dispersed sites of particular interest for known and potential resource availability, selenology, and lunar observatories. Discriminating characteristics include solar illumination, view of earth, local topography, engineering properties of the regolith and certain geological features, and local mineralogy and petrology. Space vehicle arrival and departure trajectories constitute a minor consideration. Over time, a variety of base sites will be developed serving different purposes. Resource-driven sites may see the fastest growth during the first decades of lunar development, but selection of the most favorable sites is likely to be driven by suitability for a combination of activities. As on earth, later development may be driven by geographical advantages of surface transportation routes. With the availability of near-constant sunlight for power generation, as well as permanently shadowed areas at cryogenic temperatures, polar sites are attractive because they require substantially less earth-launched mass and lower equipment complexity for an initial permanent base. Discovery of accessible volatiles reservoirs, either in the form of polar permafrost or gas reservoirs at other locations, would dramatically increase the attractiveness of any site from a logistical support and selenological point of view. Amid such speculation, no reliable evidence of such volatiles exist. More reliable evidence exists for areas of certain mineral concentrations, such as ilmenite, which could form a feedstock for some proposed resource extraction schemes. While tentative selections of advantageous base sites are made, new data from lunar polar orbiters and the Galileo polar flybys would be very helpful.

Extrapulmonary sites of radiogallium accumulation in sarcoidosis

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

Sulavik, S.B.; Palestro, C.J.; Spencer, R.P.

1990-12-01

In an effort to detect extrapulmonary sites of radiogallium accumulation in cases of sarcoidosis, 145 separate Ga-67 citrate studies of 114 patients with biopsy-proven sarcoidosis were examined. The most characteristic extrapulmonary radiogallium uptake pattern was the panda sign in 47 patients (41%). The most common site of prominent extrapulmonary radiogallium uptake was the lacrimal glands in 101 patients (88%). Second most common was activity in one or more superficial lymph node regions such as the cervical, axillary, femoral, or inguinal in 19 patients (17%). Other extrapulmonary sites included breast uptake in 6 out of 80 women (8%), prominent splenic andmore » nasal uptake in 9 (8%) patients, periportal accumulation in 7 (6%), and cutaneous/subcutaneous activity in 4 (4%). Because many of these individuals were receiving corticosteroids, the natural (untreated) prevalence of extrapulmonary findings may be even higher. Although the sensitivity and specificity of extrapulmonary radiogallium accumulation has still to be determined, many of the sites may be accessible to biopsy both for diagnostic purposes and to follow the effects of medications. It is therefore suggested that whole-body imaging be performed when radiogallium is administered to patients with suspected or known sarcoidosis.« less

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity.

PubMed

Li, Jiang-Wei; Xia, Lijie; Su, Youhong; Liu, Hongchun; Xia, Xueqing; Lu, Qinxia; Yang, Chunjin; Reheman, Kalbinur

2012-04-20

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.

Synthesis of a molecularly defined single-active site heterogeneous catalyst for selective oxidation of N-heterocycles.

PubMed

Zhang, Yujing; Pang, Shaofeng; Wei, Zhihong; Jiao, Haijun; Dai, Xingchao; Wang, Hongli; Shi, Feng

2018-04-13

Generally, a homogeneous catalyst exhibits good activity and defined active sites but it is difficult to recycle. Meanwhile, a heterogeneous catalyst can easily be reused but its active site is difficult to reveal. It is interesting to bridge the gap between homogeneous and heterogeneous catalysis via controllable construction of a heterogeneous catalyst containing defined active sites. Here, we report that a molecularly defined, single-active site heterogeneous catalyst has been designed and prepared via the oxidative polymerization of maleimide derivatives. These polymaleimide derivatives can be active catalysts for the selective oxidation of heterocyclic compounds to quinoline and indole via the recycling of -C=O and -C-OH groups, which was confirmed by tracing the reaction with GC-MS using maleimide as the catalyst and by FT-IR analysis with polymaleimide as the catalyst. These results might promote the development of heterogeneous catalysts with molecularly defined single active sites exhibiting a comparable activity to homogeneous catalysts.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

PubMed

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Analysis of Substrate Access to Active Sites in Bacterial Multicomponent Monooxygenase Hydroxylases: X-ray Crystal Structure of Xenon-Pressurized Phenol Hydroxylase from Pseudomonas sp. OX1†,‡

PubMed Central

McCormick, Michael S.; Lippard, Stephen J.

2011-01-01

In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. Computational analyses of the hydrophobic cavities in the hydroxylase α-subunits of phenol hydroxylase (PHH), toluene/o-xylene monooxygenase (ToMOH), and soluble methane monooxygenase (sMMOH) are also presented. The results, together with previous findings from crystallographic studies of xenon-pressurized sMMO hydroxylase, clearly identify the propensity for these cavities to bind hydrophobic gas molecules in the protein interior. This proposed functional role is supported by recent stopped flow kinetic studies of ToMOH variants (Song, et al., 2011). In addition to information about the Xe sites, the structure determination revealed significantly reduced regulatory protein binding to the hydroxylase in comparison to the previously reported structure of PHH, as well as the presence of a newly identified metal binding site in the α-subunit that adopts a linear coordination environment consistent with Cu(I), and a glycerol molecule bound to Fe1 in a fashion that is unique among hydrocarbon-diiron site adducts reported to date in BMM hydroxylase structures. Finally, a comparative analysis of the α-subunit structures of MMOH, ToMOH, and PHH details proposed routes for the other three BMM substrates, the hydrocarbon, electrons, and protons, comprising cavities, channels, hydrogen-bonding networks, and pores in the structures of their α-subunits. PMID:22136180

Interactive flare sites within an active region complex

NASA Technical Reports Server (NTRS)

Poletto, G.; Gary, G. A.; Machado, M. E.

1993-01-01

We examine here a set of images of an active region complex, acquired on June 24-25, 1980, by the Hard X-ray Imaging Spectrometer on SMM, with the purpose of establishing whether there was any interplay between the frequent activity observed at different sites in the activity center and, in such a case, how the interaction was established. By analyzing both quiet and active orbits we show that, as a rule, activity originating in one region triggers the other region's activity. However, we find little unambiguous evidence for the presence of large-scale interconnecting loops. A comparison of X-ray images with magnetic field observations suggested that we interpret the active region behavior in terms of the interaction between different loop systems, in a scenario quite analogous to the interacting bipole representation of individual flares. We conclude that active region interplay provides an easily observable case to study the time-dependent topology and the mechanisms for the spreading of activity in transient events over all energy scales.

10 CFR 1046.14 - Access authorization.

Code of Federal Regulations, 2010 CFR

2010-01-01

... authorization for the highest level of classified matter to which they potentially have access. Security police... by the site security organization and approved by the Head of the Field Element. Security police officers shall possess a minimum of an “L” or DOE Secret access authorization. Security police officers...

Update on Virginia College Access Provider Activity

ERIC Educational Resources Information Center

Corning, Amy; Senechal, Jesse; Hutton, Amy

2015-01-01

In the fall of 2008, the State Council of Higher Education for Virginia (SCHEV) commissioned a research study comparing the college access provider resources in Virginia to the access and academic achievement needs of the state. That study--conducted by researchers from the College of William and Mary and West Carolina University--resulted in the…

Ultrafast infrared spectroscopy reveals water-mediated coherent dynamics in an enzyme active site.

PubMed

Adamczyk, Katrin; Simpson, Niall; Greetham, Gregory M; Gumiero, Andrea; Walsh, Martin A; Towrie, Michael; Parker, Anthony W; Hunt, Neil T

2015-01-01

Understanding the impact of fast dynamics upon the chemical processes occurring within the active sites of proteins and enzymes is a key challenge that continues to attract significant interest, though direct experimental insight in the solution phase remains sparse. Similar gaps in our knowledge exist in understanding the role played by water, either as a solvent or as a structural/dynamic component of the active site. In order to investigate further the potential biological roles of water, we have employed ultrafast multidimensional infrared spectroscopy experiments that directly probe the structural and vibrational dynamics of NO bound to the ferric haem of the catalase enzyme from Corynebacterium glutamicum in both H 2 O and D 2 O. Despite catalases having what is believed to be a solvent-inaccessible active site, an isotopic dependence of the spectral diffusion and vibrational lifetime parameters of the NO stretching vibration are observed, indicating that water molecules interact directly with the haem ligand. Furthermore, IR pump-probe data feature oscillations originating from the preparation of a coherent superposition of low-frequency vibrational modes in the active site of catalase that are coupled to the haem ligand stretching vibration. Comparisons with an exemplar of the closely-related peroxidase enzyme family shows that they too exhibit solvent-dependent active-site dynamics, supporting the presence of interactions between the haem ligand and water molecules in the active sites of both catalases and peroxidases that may be linked to proton transfer events leading to the formation of the ferryl intermediate Compound I. In addition, a strong, water-mediated, hydrogen bonding structure is suggested to occur in catalase that is not replicated in peroxidase; an observation that may shed light on the origins of the different functions of the two enzymes.

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

PubMed

Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

2003-05-13

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

Web Site Evaluation: How Would Your School's Web Site Measure Up?

ERIC Educational Resources Information Center

Riccardi, Megan; Easton, D'Anne; Small, Ruth

2004-01-01

One of the many responsibilities teacher-librarians have recently assumed is the development of their school library's web site. Such site provide an organic "window" to the library's programs, services and resources that can be accessed by students, teachers, administrators, parents and community members. As such, they represent another vital…

Engineering streptokinase for generation of active site-labeled plasminogen analogs*

PubMed Central

Laha, Malabika; Panizzi, Peter; Nahrendorf, Matthias; Bock, Paul E.

2011-01-01

We previously demonstrated that streptokinase (SK) can be used to generate active site-labeled fluorescent analogs of plasminogen (Pg) by virtue of its non-proteolytic activation of the zymogen. The method is versatile and allows for stoichiometric and active site-specific incorporation of any one of many molecular probes. The limitation of the labeling approach is that it is both time-consuming and low yield. Here we demonstrate an improved method for the preparation of labeled Pg analogs by the use of an engineered SK mutant fusion protein with both COOH- and NH2-terminal His6-tags. The NH2-terminal tag is followed by a tobacco etch virus proteinase cleavage site to ensure that the SK Ile1 residue, essential for conformational activation of Pg, is preserved. The SK COOH-terminal Lys414 residue and residues Arg253-Leu260 in the SK β-domain were deleted to prevent cleavage by plasmin (Pm), and to disable Pg substrate binding to the SK·Pg*/Pm catalytic complexes, respectively. Near-elimination of Pm generation with the SKΔ(R253-L260)ΔK414-His6 mutant increased the yield of labeled Pg 2.6-fold and reduced the time required >2-fold. The versatility of the labeling method was extended to the application of Pg labeled with a near-infrared probe to quantitate Pg receptors on immune cells by flow cytometry. PMID:21570944

Technology solutions to support supervisory activities and also to provide information access to the society

NASA Astrophysics Data System (ADS)

Paladini, D.; Mello, A. B.

2016-07-01

Inmetro's data about the conformity of certificated products, process and services are, usually, displayed at fragmented databases of difficult access for several reasons, for instance, the lack of computational solutions which allow this kind of access to its users. A discussion about some of the technological solutions to support supervisory activities by the appropriate regulatory bodies and also to provide information access to society in general is herein presented, along with a theoretical explanation of the pros and cons of such technologies to the conclusion that a mobile platform seems to be the best tool for the requirements of Inmetro.

Accessing Digital Libraries: A Study of ARL Members' Digital Projects

ERIC Educational Resources Information Center

Kahl, Chad M.; Williams, Sarah C.

2006-01-01

To ensure efficient access to and integrated searching capabilities for their institution's new digital library projects, the authors studied Web sites of the Association of Research Libraries' (ARL) 111 academic, English-language libraries. Data were gathered on 1117 digital projects, noting library Web site and project access, metadata, and…

U.S. EPAs Geospatial Data Access Project

EPA Pesticide Factsheets

To improve public health and the environment, the United States Environmental Protection Agency (EPA) collects information about facilities, sites, or places subject to environmental regulation or of environmental interest. Through the Geospatial Data Download Service, the public is now able to download the EPA Geodata Shapefile, Feature Class or extensible markup language (XML) file containing facility and site information from EPA's national program systems. The files are Internet accessible from the Envirofacts Web site (https://www3.epa.gov/enviro/). The data may be used with geospatial mapping applications. (Note: The files omit facilities without latitude/longitude coordinates.) The EPA Geospatial Data contains the name, location (latitude/longitude), and EPA program information about specific facilities and sites. In addition, the files contain a Uniform Resource Locator (URL), which allows mapping applications to present an option to users to access additional EPA data resources on a specific facility or site.

Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

PubMed Central

Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

2015-01-01

Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

Comparison of the active-site design of molybdenum oxo-transfer enzymes by quantum mechanical calculations.

PubMed

Li, Jilai; Ryde, Ulf

2014-11-17

There are three families of mononuclear molybdenum enzymes that catalyze oxygen atom transfer (OAT) reactions, named after a typical example from each family, viz., dimethyl sulfoxide reductase (DMSOR), sulfite oxidase (SO), and xanthine oxidase (XO). These families differ in the construction of their active sites, with two molybdopterin groups in the DMSOR family, two oxy groups in the SO family, and a sulfido group in the XO family. We have employed density functional theory calculations on cluster models of the active sites to understand the selection of molybdenum ligands in the three enzyme families. Our calculations show that the DMSOR active site has a much stronger oxidative power than the other two sites, owing to the extra molybdopterin ligand. However, the active sites do not seem to have been constructed to make the OAT reaction as exergonic as possible, but instead to keep the reaction free energy close to zero (to avoid excessive loss of energy), thereby making the reoxidation (SO and XO) or rereduction of the active sites (DMSOR) after the OAT reaction facile. We also show that active-site models of the three enzyme families can all catalyze the reduction of DMSO and that the DMSOR model does not give the lowest activation barrier. Likewise, all three models can catalyze the oxidation of sulfite, provided that the Coulombic repulsion between the substrate and the enzyme model can be overcome, but for this harder reaction, the SO model gives the lowest activation barrier, although the differences are not large. However, only the XO model can catalyze the oxidation of xanthine, owing to its sulfido ligand.

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.

Active Site Flexibility of Mycobacterium tuberculosis Isocitrate Lyase in Dimer Form.

PubMed

Lee, Yie-Vern; Choi, Sy Bing; Wahab, Habibah A; Choong, Yee Siew

2017-09-25

Tuberculosis (TB) still remains a global threat due to the emergence of a drug-resistant strain. Instead of focusing on the drug target of active stage TB, we are highlighting the isocitrate lyase (ICL) at the dormant stage TB. ICL is one of the persistent factors for Mycobacterium tuberculosis (MTB) to survive during the dormant phase. In addition, the absence of ICL in human has made ICL a potential drug target for TB therapy. However, the dynamic details of ICL which could give insights to the ICL-ligand interaction have yet to be solved. Therefore, a series of ICL dimer dynamics studies through molecular dynamics simulation were performed in this work. The ICL active site entrance gate closure is contributed to by hydrogen bonding and electrostatic interactions with the C-terminal. Analysis suggested that the open-closed behavior of the ICL active site entrance depends on the type of ligand present in the active site. We also observed four residues (Ser91, Asp108, Asp153, and Cys191) which could possibly be the nucleophiles for nucleophilic attack on the cleavage of isocitrate at the C 2 -C 3 bond. We hope that the elucidation of ICL dynamics can benefit future works such as lead identification or antibody design against ICL for TB therapeutics.

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

Nevada National Security Site Environmental Report Summary 2016

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

Wills, Cathy

This document is a summary of the full 2016 Nevada National Security Site Environmental Report (NNSSER) prepared by the U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office (NNSA/ NFO). This summary provides an abbreviated and more readable version of the full NNSSER. NNSA/NFO prepares the NNSSER to provide the public an understanding of the environmental monitoring and compliance activities that are conducted on the Nevada National Security Site (NNSS) to protect the public and the environment from radiation hazards and from potential nonradiological impacts. It is a comprehensive report of environmental activities performed at the NNSS andmore » offsite facilities over the previous calendar year. The NNSS is currently the nation’s unique site for ongoing national security–related missions and high-risk operations. The NNSS is located about 65 miles northwest of Las Vegas. The approximately 1,360-square-mile site is one of the largest restricted access areas in the United States. It is surrounded by federal installations with strictly controlled access as well as by lands that are open to public entry. In 2016, National Security Technologies, LLC (NSTec), was the NNSS Management and Operations Contractor accountable for ensuring work was performed in compliance with environmental regulations. NNSS activities in 2016 continued to be diverse, with the primary goal to ensure that the existing U.S. stockpile of nuclear weapons remains safe and reliable. Other activities included weapons of mass destruction first responder training; the controlled release of hazardous material at the Nonproliferation Test and Evaluation Complex (NPTEC); remediation of legacy contamination sites; characterization of waste destined for the Waste Isolation Pilot Plant in Carlsbad, New Mexico, or the Idaho National Laboratory in Idaho Falls, Idaho; disposal of low-level and mixed low-level radioactive waste; and environmental research. Facilities

Social Media Use and Access to Digital Technology in US Young Adults in 2016.

PubMed

Villanti, Andrea C; Johnson, Amanda L; Ilakkuvan, Vinu; Jacobs, Megan A; Graham, Amanda L; Rath, Jessica M

2017-06-07

In 2015, 90% of US young adults with Internet access used social media. Digital and social media are highly prevalent modalities through which young adults explore identity formation, and by extension, learn and transmit norms about health and risk behaviors during this developmental life stage. The purpose of this study was to provide updated estimates of social media use from 2014 to 2016 and correlates of social media use and access to digital technology in data collected from a national sample of US young adults in 2016. Young adult participants aged 18-24 years in Wave 7 (October 2014, N=1259) and Wave 9 (February 2016, N=989) of the Truth Initiative Young Adult Cohort Study were asked about use frequency for 11 social media sites and access to digital devices, in addition to sociodemographic characteristics. Regular use was defined as using a given social media site at least weekly. Weighted analyses estimated the prevalence of use of each social media site, overlap between regular use of specific sites, and correlates of using a greater number of social media sites regularly. Bivariate analyses identified sociodemographic correlates of access to specific digital devices. In 2014, 89.42% (weighted n, 1126/1298) of young adults reported regular use of at least one social media site. This increased to 97.5% (weighted n, 965/989) of young adults in 2016. Among regular users of social media sites in 2016, the top five sites were Tumblr (85.5%), Vine (84.7%), Snapchat (81.7%), Instagram (80.7%), and LinkedIn (78.9%). Respondents reported regularly using an average of 7.6 social media sites, with 85% using 6 or more sites regularly. Overall, 87% of young adults reported access or use of a smartphone with Internet access, 74% a desktop or laptop computer with Internet access, 41% a tablet with Internet access, 29% a smart TV or video game console with Internet access, 11% a cell phone without Internet access, and 3% none of these. Access to all digital devices with

Blogs and Social Network Sites as Activity Systems: Exploring Adult Informal Learning Process through Activity Theory Framework

ERIC Educational Resources Information Center

Heo, Gyeong Mi; Lee, Romee

2013-01-01

This paper uses an Activity Theory framework to explore adult user activities and informal learning processes as reflected in their blogs and social network sites (SNS). Using the assumption that a web-based space is an activity system in which learning occurs, typical features of the components were investigated and each activity system then…

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

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

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Identification and characterization of the sodium-binding site of activated protein C.

PubMed

He, X; Rezaie, A R

1999-02-19

Activated protein C (APC) requires both Ca2+ and Na+ for its optimal catalytic function. In contrast to the Ca2+-binding sites, the Na+-binding site(s) of APC has not been identified. Based on a recent study with thrombin, the 221-225 loop is predicted to be a potential Na+-binding site in APC. The sequence of this loop is not conserved in trypsin. We engineered a Gla domainless form of protein C (GDPC) in which the 221-225 loop was replaced with the corresponding loop of trypsin. We found that activated GDPC (aGDPC) required Na+ (or other alkali cations) for its amidolytic activity with dissociation constant (Kd(app)) = 44.1 +/- 8.6 mM. In the presence of Ca2+, however, the requirement for Na+ by aGDPC was eliminated, and Na+ stimulated the cleavage rate 5-6-fold with Kd(app) = 2.3 +/- 0.3 mM. Both cations were required for efficient factor Va inactivation by aGDPC. In the presence of Ca2+, the catalytic function of the mutant was independent of Na+. Unlike aGDPC, the mutant did not discriminate among monovalent cations. We conclude that the 221-225 loop is a Na+-binding site in APC and that an allosteric link between the Na+ and Ca2+ binding loops modulates the structure and function of this anticoagulant enzyme.

A prototype of Virtual Observatory access for planetary data in the framework of Europlanet-RI/IDIS

NASA Astrophysics Data System (ADS)

Gangloff, M.; Cecconi, B.; Bourrel, N.; Jacquey, C.; Le Sidaner, P.; Berthier, J.; André, N.; Pallier, E.; Erard, S.; Aboudarham, J.; Chanteur, G. M.; Capria, M. T.; Khodachenko, M.; Manaud, N.; Schmidt, W.; Schmitt, B.; Topf, F.; Trautan, F.; Sarkissian, A.

2011-12-01

Europlanet RI is a four-year project supported by the European Union under the Seventh Framework Programme. Launched in January 2009, it is an Integrated Infrastructure Initiative, ie. A combination of Networking Activities, Transnational Access Activities and Joint Research Activities. The Networking Activities aim at further fostering a culture of cooperation in the field of Planetary Sciences. The objective of the Transnational Access Activities is to provide transnational access to a range of laboratory and field site facilities tailored to the needs of planetary research and on-line access to the available planetary science data, information and software tools, through the IDIS e-service. The overall aim of the Joint Research Activities (JRA) is to improve the services provided by the ensemble of Transnational Access Activities. In EuroPlaNet-RI, JRA4 must prepare essential tools for IDIS (Integrated and Distributed Information Service) allowing the planetary science community to interrogate some selected data centres, access and process data and visualize the results. This is the first step towards a Planetary Virtual Observatory. The first requirement for different data centres to be able to operate together collectively is adequate standardization. In particular a common description of data and services is essential. This is why the major part of JRA4/Task2 activity is focussing on data models, associated dictionnaries, and protocols to exchange queries. A specific data model is being developed for IDIS, associated with the PDAP protocol, a standard defined by the IPDA (International Planetary Data Alliance) The scope of this prototype is to demonstrate the capabilities of the IDIS Data Model, and the PDAP protocol to search and retrieve data in the wide topical planetology context.

Orthogonal use of a human tRNA synthetase active site to achieve multi-functionality

PubMed Central

Zhou, Quansheng; Kapoor, Mili; Guo, Min; Belani, Rajesh; Xu, Xiaoling; Kiosses, William B.; Hanan, Melanie; Park, Chulho; Armour, Eva; Do, Minh-Ha; Nangle, Leslie A.; Schimmel, Paul; Yang, Xiang-Lei

2011-01-01

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multi-functionality of human tRNA synthetases and other proteins. PMID:20010843

When roads appear jaguars decline: Increased access to an Amazonian wilderness area reduces potential for jaguar conservation

PubMed Central

Celis, Gerardo; Branch, Lyn C.

2018-01-01

Roads are a main threat to biodiversity conservation in the Amazon, in part, because roads increase access for hunters. We examine how increased landscape access by hunters may lead to cascading effects that influence the prey community and abundance of the jaguar (Panthera onca), the top Amazonian terrestrial predator. Understanding such ecological effects originating from anthropogenic actions is essential for conservation and management of wildlife populations in areas undergoing infrastructure development. Our study was conducted in Yasuní Biosphere Reserve, the protected area with highest potential for jaguar conservation in Ecuador, and an area both threatened by road development and inhabited by indigenous groups dependent upon bushmeat. We surveyed prey and jaguar abundance with camera traps in four sites that differed in accessibility to hunters and used site occupancy and spatially explicit capture-recapture analyses to evaluate prey occurrence and estimate jaguar density, respectively. Higher landscape accessibility to hunters was linked with lower occurrence and biomass of game, particularly white-lipped peccary (Tayassu pecari) and collared peccary (Pecari tajacu), the primary game for hunters and prey for jaguars. Jaguar density was up to 18 times higher in the most remote site compared to the most accessible site. Our results provide a strong case for the need to: 1) consider conservation of large carnivores and other wildlife in policies about road construction in protected areas, 2) coordinate conservation initiatives with local governments so that development activities do not conflict with conservation objectives, and 3) promote development of community-based strategies for wildlife management that account for the needs of large carnivores. PMID:29298311

When roads appear jaguars decline: Increased access to an Amazonian wilderness area reduces potential for jaguar conservation.

PubMed

Espinosa, Santiago; Celis, Gerardo; Branch, Lyn C

2018-01-01

Roads are a main threat to biodiversity conservation in the Amazon, in part, because roads increase access for hunters. We examine how increased landscape access by hunters may lead to cascading effects that influence the prey community and abundance of the jaguar (Panthera onca), the top Amazonian terrestrial predator. Understanding such ecological effects originating from anthropogenic actions is essential for conservation and management of wildlife populations in areas undergoing infrastructure development. Our study was conducted in Yasuní Biosphere Reserve, the protected area with highest potential for jaguar conservation in Ecuador, and an area both threatened by road development and inhabited by indigenous groups dependent upon bushmeat. We surveyed prey and jaguar abundance with camera traps in four sites that differed in accessibility to hunters and used site occupancy and spatially explicit capture-recapture analyses to evaluate prey occurrence and estimate jaguar density, respectively. Higher landscape accessibility to hunters was linked with lower occurrence and biomass of game, particularly white-lipped peccary (Tayassu pecari) and collared peccary (Pecari tajacu), the primary game for hunters and prey for jaguars. Jaguar density was up to 18 times higher in the most remote site compared to the most accessible site. Our results provide a strong case for the need to: 1) consider conservation of large carnivores and other wildlife in policies about road construction in protected areas, 2) coordinate conservation initiatives with local governments so that development activities do not conflict with conservation objectives, and 3) promote development of community-based strategies for wildlife management that account for the needs of large carnivores.

Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

DOE Data Explorer

John Peterson

2015-04-17

These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

PubMed Central

Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

2015-01-01

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

Web site access statistics and delivery of research results

Treesearch

Daniel L. Schmoldt; Matthew F. Winn; Philip A. Araman

1998-01-01

For the past 2-1/2 years, our Research Work Unit (RWU) has been operating a Web site (http://www.se4702.forprod.vt.edu). The site became operational in October 1995. In the beginning, it was primarily designed to stake out a piece of the Internet with our name and our RWU’s organizational mission. Quickly, it became apparent to us, however, that our user community...

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

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

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Systematic Functional Analysis of Active-Site Residues in l-Threonine Dehydrogenase from Thermoplasma volcanium

DOE PAGES

Desjardins, Morgan; Mak, Wai Shun; O’Brien, Terrence E.; ...

2017-07-07

Enzymes have been through millions of years of evolution during which their active-site microenvironments are fine-tuned. Active-site residues are commonly conserved within protein families, indicating their importance for substrate recognition and catalysis. In this work, we systematically mutated active-site residues of l-threonine dehydrogenase from Thermoplasma volcanium and characterized the mutants against a panel of substrate analogs. Our results demonstrate that only a subset of these residues plays an essential role in substrate recognition and catalysis and that the native enzyme activity can be further enhanced roughly 4.6-fold by a single point mutation. Kinetic characterization of mutants on substrate analogs showsmore » that l-threonine dehydrogenase possesses promiscuous activities toward other chemically similar compounds not previously observed. Quantum chemical calculations on the hydride-donating ability of these substrates also reveal that this enzyme did not evolve to harness the intrinsic substrate reactivity for enzyme catalysis. Our analysis provides insights into connections between the details of enzyme active-site structure and specific function. Finally, these results are directly applicable to rational enzyme design and engineering.« less

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

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

Tomkinson, B.; Wernstedt, C.; Hellman, U.

1987-11-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residuesmore » 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.« less

Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

PubMed Central

Tomkinson, B; Wernstedt, C; Hellman, U; Zetterqvist, O

1987-01-01

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases. PMID:3313395

Calorimetric studies of the interactions of metalloenzyme active site mimetics with zinc-binding inhibitors.

PubMed

Robinson, Sophia G; Burns, Philip T; Miceli, Amanda M; Grice, Kyle A; Karver, Caitlin E; Jin, Lihua

2016-07-19

The binding of drugs to metalloenzymes is an intricate process that involves several interactions, including binding of the drug to the enzyme active site metal, as well as multiple interactions between the drug and the enzyme residues. In order to determine the free energy contribution of Zn(2+) binding by known metalloenzyme inhibitors without the other interactions, valid active site zinc structural mimetics must be formed and binding studies need to be performed in biologically relevant conditions. The potential of each of five ligands to form a structural mimetic with Zn(2+) was investigated in buffer using Isothermal Titration Calorimetry (ITC). All five ligands formed strong 1 : 1 (ligand : Zn(2+)) binary complexes. The complexes were used in further ITC experiments to study their interaction with 8-hydroxyquinoline (8-HQ) and/or acetohydroxamic acid (AHA), two bidentate anionic zinc-chelating enzyme inhibitors. It was found that tetradentate ligands were not suitable for creating zinc structural mimetics for inhibitor binding in solution due to insufficient coordination sites remaining on Zn(2+). A stable binary complex, [Zn(BPA)](2+), which was formed by a tridentate ligand, bis(2-pyridylmethyl)amine (BPA), was found to bind one AHA in buffer or a methanol : buffer mixture (60 : 40 by volume) at pH 7.25 or one 8-HQ in the methanol : buffer mixture at pH 6.80, making it an effective structural mimetic for the active site of zinc metalloenzymes. These results are consistent with the observation that metalloenzyme active site zinc ions have three residues coordinated to them, leaving one or two sites open for inhibitors to bind. Our findings indicate that Zn(BPA)X2 can be used as an active site structural mimetic for zinc metalloenzymes for estimating the free energy contribution of zinc binding to the overall inhibitor active site interactions. Such use will help aid in the rational design of inhibitors to a variety of zinc metalloenzymes.

Cost effective Internet access and video conferencing for a community cancer network.

PubMed Central

London, J. W.; Morton, D. E.; Marinucci, D.; Catalano, R.; Comis, R. L.

1995-01-01

Utilizing the ubiquitous personal computer as a platform, and Integrated Services Digital Network (ISDN) communications, cost effective medical information access and consultation can be provided for physicians at geographically remote sites. Two modes of access are provided: information retrieval via the Internet, and medical consultation video conferencing. Internet access provides general medical information such as current treatment options, literature citations, and active clinical trials. During video consultations, radiographic and pathology images, and medical text reports (e.g., history and physical, pathology, radiology, clinical laboratory reports), may be viewed and simultaneously annotated by either video conference participant. Both information access modes have been employed by physicians at community hospitals which are members of the Jefferson Cancer Network, and oncologists at Thomas Jefferson University Hospital. This project has demonstrated the potential cost effectiveness and benefits of this technology. Images Figure 1 Figure 2 Figure 3 PMID:8563397

Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

PubMed Central

Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

2009-01-01

Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

Creating Patient and Family Education Web Sites

PubMed Central

YADRICH, DONNA MACAN; FITZGERALD, SHARON A.; WERKOWITCH, MARILYN; SMITH, CAROL E.

2013-01-01

This article gives details about the methods and processes used to ensure that usability and accessibility were achieved during development of the Home Parenteral Nutrition Family Caregivers Web site, an evidence-based health education Web site for the family members and caregivers of chronically ill patients. This article addresses comprehensive definitions of usability and accessibility and illustrates Web site development according to Section 508 standards and the national Health and Human Services’ Research-Based Web Design and Usability Guidelines requirements. PMID:22024970

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

PubMed

Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

PubMed Central

Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

2016-01-01

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

NREL: International Activities - Energy Access

Science.gov Websites

experience with off-grid solutions to support mini and microgrid projects, policies, and programs that are prohibitively expensive. Investment interest in mini and microgrids for energy access has been growing among Quality Assurance Framework (QAF) for mini-grids was developed to address the root challenges to providing

DMSP SSJ4 Data Restoration, Classification, and On-Line Data Access

NASA Technical Reports Server (NTRS)

Wing, Simon; Bredekamp, Joseph H. (Technical Monitor)

2000-01-01

Compress and clean raw data file for permanent storage We have identified various error conditions/types and developed algorithms to get rid of these errors/noises, including the more complicated noise in the newer data sets. (status = 100% complete). Internet access of compacted raw data. It is now possible to access the raw data via our web site, http://www.jhuapl.edu/Aurora/index.html. The software to read and plot the compacted raw data is also available from the same web site. The users can now download the raw data, read, plot, or manipulate the data as they wish on their own computer. The users are able to access the cleaned data sets. Internet access of the color spectrograms. This task has also been completed. It is now possible to access the spectrograms from the web site mentioned above. Improve the particle precipitation region classification. The algorithm for doing this task has been developed and implemented. As a result, the accuracies improved. Now the web site routinely distributes the results of applying the new algorithm to the cleaned data set. Mark the classification region on the spectrograms. The software to mark the classification region in the spectrograms has been completed. This is also available from our web site.

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase

DOE PAGES

Murphy, Jesse R.; Donini, Stefano; Kappock, T. Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open'more » structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. In conclusion, as a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.« less

An active site-tail interaction in the structure of hexahistidine-tagged Thermoplasma acidophilum citrate synthase.

PubMed

Murphy, Jesse R; Donini, Stefano; Kappock, T Joseph

2015-10-01

Citrate synthase (CS) plays a central metabolic role in aerobes and many other organisms. The CS reaction comprises two half-reactions: a Claisen aldol condensation of acetyl-CoA (AcCoA) and oxaloacetate (OAA) that forms citryl-CoA (CitCoA), and CitCoA hydrolysis. Protein conformational changes that `close' the active site play an important role in the assembly of a catalytically competent condensation active site. CS from the thermoacidophile Thermoplasma acidophilum (TpCS) possesses an endogenous Trp fluorophore that can be used to monitor the condensation reaction. The 2.2 Å resolution crystal structure of TpCS fused to a C-terminal hexahistidine tag (TpCSH6) reported here is an `open' structure that, when compared with several liganded TpCS structures, helps to define a complete path for active-site closure. One active site in each dimer binds a neighboring His tag, the first nonsubstrate ligand known to occupy both the AcCoA and OAA binding sites. Solution data collectively suggest that this fortuitous interaction is stabilized by the crystalline lattice. As a polar but almost neutral ligand, the active site-tail interaction provides a new starting point for the design of bisubstrate-analog inhibitors of CS.

A simplified method for active-site titration of lipases immobilised on hydrophobic supports.

PubMed

Nalder, Tim D; Kurtovic, Ivan; Barrow, Colin J; Marshall, Susan N

2018-06-01

The aim of this work was to develop a simple and accurate protocol to measure the functional active site concentration of lipases immobilised on highly hydrophobic supports. We used the potent lipase inhibitor methyl 4-methylumbelliferyl hexylphosphonate to titrate the active sites of Candida rugosa lipase (CrL) bound to three highly hydrophobic supports: octadecyl methacrylate (C18), divinylbenzene crosslinked methacrylate (DVB) and styrene. The method uses correction curves to take into account the binding of the fluorophore (4-methylumbelliferone, 4-MU) by the support materials. We showed that the uptake of the detection agent by the three supports is not linear relative to the weight of the resin, and that the uptake occurs in an equilibrium that is independent of the total fluorophore concentration. Furthermore, the percentage of bound fluorophore varied among the supports, with 50 mg of C18 and styrene resins binding approximately 64 and 94%, respectively. When the uptake of 4-MU was calculated and corrected for, the total 4-MU released via inhibition (i.e. the concentration of functional lipase active sites) could be determined via a linear relationship between immobilised lipase weight and total inhibition. It was found that the functional active site concentration of immobilised CrL varied greatly among different hydrophobic supports, with 56% for C18, compared with 14% for DVB. The described method is a simple and robust approach to measuring functional active site concentration in immobilised lipase samples. Copyright © 2018 Elsevier Inc. All rights reserved.

Macroscopic characterisations of Web accessibility

NASA Astrophysics Data System (ADS)

Lopes, Rui; Carriço, Luis

2010-12-01

The Web Science framework poses fundamental questions on the analysis of the Web, by focusing on how microscopic properties (e.g. at the level of a Web page or Web site) emerge into macroscopic properties and phenomena. One research topic on the analysis of the Web is Web accessibility evaluation, which centres on understanding how accessible a Web page is for people with disabilities. However, when framing Web accessibility evaluation on Web Science, we have found that existing research stays at the microscopic level. This article presents an experimental study on framing Web accessibility evaluation into Web Science's goals. This study resulted in novel accessibility properties of the Web not found at microscopic levels, as well as of Web accessibility evaluation processes themselves. We observed at large scale some of the empirical knowledge on how accessibility is perceived by designers and developers, such as the disparity of interpretations of accessibility evaluation tools warnings. We also found a direct relation between accessibility quality and Web page complexity. We provide a set of guidelines for designing Web pages, education on Web accessibility, as well as on the computational limits of large-scale Web accessibility evaluations.

Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase.

PubMed

Fenwick, Michael K; Mehta, Angad P; Zhang, Yang; Abdelwahed, Sameh H; Begley, Tadhg P; Ealick, Steven E

2015-03-27

Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

Making Physical Activity Accessible to Older Adults with Memory Loss: A Feasibility Study

ERIC Educational Resources Information Center

Logsdon, Rebecca G.; McCurry, Susan M.; Pike, Kenneth C.; Teri, Linda

2009-01-01

Purpose: For individuals with mild cognitive impairment (MCI), memory loss may prevent successful engagement in exercise, a key factor in preventing additional disability. The Resources and Activities for Life Long Independence (RALLI) program uses behavioral principles to make exercise more accessible for these individuals. Exercises are broken…

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Insights into the glycyl radical enzyme active site of benzylsuccinate synthase: a computational study.

PubMed

Bharadwaj, Vivek S; Dean, Anthony M; Maupin, C Mark

2013-08-21

The fumarate addition reaction, catalyzed by the enzyme benzylsuccinate synthase (BSS), is considered to be one of the most intriguing and energetically challenging reactions in biology. BSS belongs to the glycyl radical enzyme family and catalyzes the fumarate addition reaction, which enables microorganisms to utilize hydrocarbons as an energy source under anaerobic conditions. Unfortunately, the extreme sensitivity of the glycyl radical to oxygen has hampered the structural and kinetic characterization of BSS, thereby limiting our knowledge on this enzyme. To enhance our molecular-level understanding of BSS, a computational approach involving homology modeling, docking studies, and molecular dynamics (MD) simulations has been used to deduce the structure of BSS's catalytic subunit (BSSα) and illuminate the molecular basis for the fumarate addition reaction. We have identified two conserved and distinct binding pockets at the BSSα active site: a hydrophobic pocket for toluene binding and a polar pocket for fumaric acid binding. Subsequent dynamical and energetic evaluations have identified Glu509, Ser827, Leu390, and Phe384 as active site residues critical for substrate binding. The orientation of substrates at the active site observed in MD simulations is consistent with experimental observations of the syn addition of toluene to fumaric acid. It is also found that substrate binding tightens the active site and restricts the conformational flexibility of the thiyl radical, leading to hydrogen transfer distances conducive to the proposed reaction mechanism. The stability of substrates at the active site and the occurrence of feasible radical transfer distances between the thiyl radical, substrates, and the active site glycine indicate a substrate-assisted radical transfer pathway governing fumarate addition.