Flexible Precision: Air Force’s Answer to Army Transformation and Intratheater Airlift on the 21st Century Battlefield

DTIC Science & Technology

2007-06-01

humanitarian relief operations that result from natural disasters —both abroad and at home—illustrates the continued importance of intratheater airlift...Light Cargo Aircraft ( LCA ). The two services were pursuing separate acquisition programs until late 2005 when the Office of the Secretary of Defense...adopting procedures currently used by the Close Air Support (CAS) community. On-call airdrop will result in a “time-sensitive” employment capability

Close Air Support Allocation for Extended Counterinsurgency: Is Our Doctrine Lacking

DTIC Science & Technology

2010-04-01

timely and accurate deconfliction of those aircraft. They act as the primary command and control for all aircraft in the theater and are extremely...only merge the real time CAS control fuctions into a single entity. It is not to eliminate the capacity of the ASOC to participate in land

The Best Aircraft for Close Air Support in the Twenty First Century

DTIC Science & Technology

2016-09-01

movement of those forces.”8 In addition to CAS, the Air Force em - ploys its aircraft to perform a myriad of roles during combat operations, such as offen...distance to bombing ranges, and so forth.14 Once these figures are accumulated for each major com- mand’s fleet, the second step is developing a CPFH...the GBU-39 small diameter bomb and the AGM-176 Griffin missile, it is still in operational test and development.26 The new squadron (replacement

Digital Avionics Information System (DAIS): Impact of DAIS Concept on Life Cycle Cost. Final Report.

ERIC Educational Resources Information Center

Goclowski, John C.; And Others

Designed to identify and quantify the potential impacts of the Digital Avionics Information System (DAIS) on weapon system personnel requirements and life cycle cost (LCC), this study postulated a typical close-air-support (CAS) mission avionics suite to serve as a basis for comparing present day and DAIS configuration specifications. The purpose…

Clean Air Slots Amid Dense Atmospheric Pollution in Southern Africa

NASA Technical Reports Server (NTRS)

Hobbs, Peter V.

2003-01-01

During the flights of the University of Washington's Convair-580 in the Southern African Regional Science Initiative (SAFARI 2000) in southern Africa, a phenomenon was observed that has not been reported previously. This was the occurrence of thin layers of remarkably clean air, sandwiched between heavily polluted air, which persisted for many hours during the day. Photographs are shown of these clean air slots (CAS), and particle concentrations and light scattering coefficients in and around such slot are presented. An explanation is proposed for the propensity of CAS to form in southern Africa during the dry season.

NASA Controller Acceptability Study 1(CAS-1) Experiment Description and Initial Observations

NASA Technical Reports Server (NTRS)

Chamberlain, James P.; Consiglio, Maria C.; Comstock, James R., Jr.; Ghatas, Rania W.; Munoz, Cesar

2015-01-01

This paper describes the Controller Acceptability Study 1 (CAS-1) experiment that was conducted by NASA Langley Research Center personnel from January through March 2014 and presents partial CAS-1 results. CAS-1 employed 14 air traffic controller volunteers as research subjects to assess the viability of simulated future unmanned aircraft systems (UAS) operating alongside manned aircraft in moderate-density, moderate-complexity Class E airspace. These simulated UAS were equipped with a prototype pilot-in-the-loop (PITL) Detect and Avoid (DAA) system, specifically the Self-Separation (SS) function of such a system based on Stratway+ software to replace the see-and-avoid capabilities of manned aircraft pilots. A quantitative CAS-1 objective was to determine horizontal miss distance (HMD) values for SS encounters that were most acceptable to air traffic controllers, specifically HMD values that were assessed as neither unsafely small nor disruptively large. HMD values between 0.5 and 3.0 nautical miles (nmi) were assessed for a wide array of encounter geometries between UAS and manned aircraft. The paper includes brief introductory material about DAA systems and their SS functions, followed by descriptions of the CAS-1 simulation environment, prototype PITL SS capability, and experiment design, and concludes with presentation and discussion of partial CAS-1 data and results.

Scorpion: Close Air Support (CAS) aircraft

NASA Technical Reports Server (NTRS)

Allen, Chris; Cheng, Rendy; Koehler, Grant; Lyon, Sean; Paguio, Cecilia

1991-01-01

The objective is to outline the results of the preliminary design of the Scorpion, a proposed close air support aircraft. The results obtained include complete preliminary analysis of the aircraft in the areas of aerodynamics, structures, avionics and electronics, stability and control, weight and balance, propulsion systems, and costs. A conventional wing, twin jet, twin-tail aircraft was chosen to maximize the desirable characteristics. The Scorpion will feature low speed maneuverability, high survivability, low cost, and low maintenance. The life cycle cost per aircraft will be 17.5 million dollars. The maximum takeoff weight will be 52,760 pounds. Wing loading will be 90 psf. The thrust to weight will be 0.6 lbs/lb. This aircraft meets the specified mission requirements. Some modifications have been suggested to further optimize the design.

Special Operations Forces Aviation on a Shoestring Budget: An Effectiveness Analysis of Light and Medium Fixed Wing Aircraft

DTIC Science & Technology

2012-12-01

C2 Command and Control CAIG Cost Analysis Improvement Group CAS Close Air Support CASA Construcciones Aeronáuticas SA CLS Contract...Commercially Available Off-the-Shelf Commercially available off-the shelf (COTS) is defined as any item of supply (including construction material...uses are found for old weapons that were constructed for use in industrial war against soldiers and heavy armament. 6. The sides are mostly non

Rapid, Value-based, Evolutionary Acquisition and Its Application to a USMC Tactical Service Oriented Architecture

DTIC Science & Technology

2009-06-01

Availability C2PC Command and Control Personal Computer CAS Close Air Support CCA Clinger-Cohen Act CDR Critical Design Review CJCSI Chairman of the Joint... kids , Jackie and Anna and my future boy whose name is TBD, I think my time at NPS has made me a better person and hopefully a better father. Thank... can the USMC apply the essential principles of rapid, value-based, evolutionary acquisition to the development and procurement of a TSOA? 4 THIS

F-16 Low Altitude Navigation and Targeting Infrared System for Night (LANTIRN) and the Night Close Air Support (CAS) Mission

DTIC Science & Technology

1989-06-02

physical reference can be a natural feature, river bend, distinctly shaped wooded area, or lake. Artificial references such as colored smoke or...fluorescent ground panels can also be placed instead of, or as an aid to recognition of the natural feature.ដ> But, artificial references that are effective...Government Printin-gOffice, 1959. Central Intelligence Agenc7. National Intellignece Survey, East Germany, Section 23, Weather and Climate. Washington

A Diagnostic Marker to Discriminate Childhood Apraxia of Speech From Speech Delay: III. Theoretical Coherence of the Pause Marker with Speech Processing Deficits in Childhood Apraxia of Speech.

PubMed

Shriberg, Lawrence D; Strand, Edythe A; Fourakis, Marios; Jakielski, Kathy J; Hall, Sheryl D; Karlsson, Heather B; Mabie, Heather L; McSweeny, Jane L; Tilkens, Christie M; Wilson, David L

2017-04-14

Previous articles in this supplement described rationale for and development of the pause marker (PM), a diagnostic marker of childhood apraxia of speech (CAS), and studies supporting its validity and reliability. The present article assesses the theoretical coherence of the PM with speech processing deficits in CAS. PM and other scores were obtained for 264 participants in 6 groups: CAS in idiopathic, neurogenetic, and complex neurodevelopmental disorders; adult-onset apraxia of speech (AAS) consequent to stroke and primary progressive apraxia of speech; and idiopathic speech delay. Participants with CAS and AAS had significantly lower scores than typically speaking reference participants and speech delay controls on measures posited to assess representational and transcoding processes. Representational deficits differed between CAS and AAS groups, with support for both underspecified linguistic representations and memory/access deficits in CAS, but for only the latter in AAS. CAS-AAS similarities in the age-sex standardized percentages of occurrence of the most frequent type of inappropriate pauses (abrupt) and significant differences in the standardized occurrence of appropriate pauses were consistent with speech processing findings. Results support the hypotheses of core representational and transcoding speech processing deficits in CAS and theoretical coherence of the PM's pause-speech elements with these deficits.

Development Process of a Praxeology for Supporting the Teaching of Proofs in a CAS Environment Based on Teachers' Experience in a Professional Development Course

ERIC Educational Resources Information Center

Zehavi, Nurit; Mann, Giora

2011-01-01

This paper presents the development process of a "praxeology" (theory-of-practice) for supporting the teaching of proofs in a CAS environment. The characteristics of the praxeology were elaborated within the frame of a professional development course for teaching analytic geometry with CAS. The theoretical framework draws on Chevallard's…

Reliance on auditory feedback in children with childhood apraxia of speech.

PubMed

Iuzzini-Seigel, Jenya; Hogan, Tiffany P; Guarino, Anthony J; Green, Jordan R

2015-01-01

Children with childhood apraxia of speech (CAS) have been hypothesized to continuously monitor their speech through auditory feedback to minimize speech errors. We used an auditory masking paradigm to determine the effect of attenuating auditory feedback on speech in 30 children: 9 with CAS, 10 with speech delay, and 11 with typical development. The masking only affected the speech of children with CAS as measured by voice onset time and vowel space area. These findings provide preliminary support for greater reliance on auditory feedback among children with CAS. Readers of this article should be able to (i) describe the motivation for investigating the role of auditory feedback in children with CAS; (ii) report the effects of feedback attenuation on speech production in children with CAS, speech delay, and typical development, and (iii) understand how the current findings may support a feedforward program deficit in children with CAS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

21 CFR 177.2910 - Ultra-filtration membranes.

Code of Federal Regulations, 2010 CFR

2010-04-01

... of a sintered carbon support that is coated with zirconium oxide (CAS Reg. No. 1314-23-4) containing... of an aluminum oxide support that is coated with zirconium oxide (CAS Reg. No. 1314-23-4) containing...

40 CFR Appendix IV to Part 266 - Reference Air Concentrations*

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 28 2013-07-01 2013-07-01 false Reference Air Concentrations* IV Appendix IV to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... MANAGEMENT FACILITIES Pt. 266, App. IV Appendix IV to Part 266—Reference Air Concentrations* Constituent CAS...

40 CFR Appendix IV to Part 266 - Reference Air Concentrations*

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 27 2014-07-01 2014-07-01 false Reference Air Concentrations* IV Appendix IV to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... MANAGEMENT FACILITIES Pt. 266, App. IV Appendix IV to Part 266—Reference Air Concentrations* Constituent CAS...

40 CFR Appendix IV to Part 266 - Reference Air Concentrations*

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 27 2011-07-01 2011-07-01 false Reference Air Concentrations* IV Appendix IV to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... MANAGEMENT FACILITIES Pt. 266, App. IV Appendix IV to Part 266—Reference Air Concentrations* Constituent CAS...

40 CFR Appendix IV to Part 266 - Reference Air Concentrations*

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 26 2010-07-01 2010-07-01 false Reference Air Concentrations* IV Appendix IV to Part 266 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID WASTES... MANAGEMENT FACILITIES Pt. 266, App. IV Appendix IV to Part 266—Reference Air Concentrations* Constituent CAS...

A Diagnostic Marker to Discriminate Childhood Apraxia of Speech From Speech Delay: III. Theoretical Coherence of the Pause Marker with Speech Processing Deficits in Childhood Apraxia of Speech

PubMed Central

Strand, Edythe A.; Fourakis, Marios; Jakielski, Kathy J.; Hall, Sheryl D.; Karlsson, Heather B.; Mabie, Heather L.; McSweeny, Jane L.; Tilkens, Christie M.; Wilson, David L.

2017-01-01

Purpose Previous articles in this supplement described rationale for and development of the pause marker (PM), a diagnostic marker of childhood apraxia of speech (CAS), and studies supporting its validity and reliability. The present article assesses the theoretical coherence of the PM with speech processing deficits in CAS. Method PM and other scores were obtained for 264 participants in 6 groups: CAS in idiopathic, neurogenetic, and complex neurodevelopmental disorders; adult-onset apraxia of speech (AAS) consequent to stroke and primary progressive apraxia of speech; and idiopathic speech delay. Results Participants with CAS and AAS had significantly lower scores than typically speaking reference participants and speech delay controls on measures posited to assess representational and transcoding processes. Representational deficits differed between CAS and AAS groups, with support for both underspecified linguistic representations and memory/access deficits in CAS, but for only the latter in AAS. CAS–AAS similarities in the age–sex standardized percentages of occurrence of the most frequent type of inappropriate pauses (abrupt) and significant differences in the standardized occurrence of appropriate pauses were consistent with speech processing findings. Conclusions Results support the hypotheses of core representational and transcoding speech processing deficits in CAS and theoretical coherence of the PM's pause-speech elements with these deficits. PMID:28384751

32 CFR 37.570 - What must I do if a CAS-covered participant accounts differently for its own and the Federal...

Code of Federal Regulations, 2010 CFR

2010-07-01

... Accounting Standard (CAS) 402. Noncompliance with CAS 402 is a potential issue only for a participant that... contractors with their ACOs (currently on the World Wide Web at http://alerts.dcmdw.dcma.mil/support, a site...

The Evolutionary History of Daphniid α-Carbonic Anhydrase within Animalia

PubMed Central

Culver, Billy W.; Morton, Philip K.

2015-01-01

Understanding the mechanisms that drive acid-base regulation in organisms is important, especially for organisms in aquatic habitats that experience rapidly fluctuating pH conditions. Previous studies have shown that carbonic anhydrases (CAs), a family of zinc metalloenzymes, are responsible for acid-base regulation in many organisms. Through the use of phylogenetic tools, this present study attempts to elucidate the evolutionary history of the α-CA superfamily, with particular interest in the emerging model aquatic organism Daphnia pulex. We provide one of the most extensive phylogenies of the evolution of α-CAs, with the inclusion of 261 amino acid sequences across taxa ranging from Cnidarians to Homo sapiens. While the phylogeny supports most of our previous understanding on the relationship of how α-CAs have evolved, we find that, contrary to expectations, amino acid conservation with bacterial α-CAs supports the supposition that extracellular α-CAs are the ancestral state of animal α-CAs. Furthermore, we show that two cytosolic and one GPI-anchored α-CA in Daphnia genus have homologs in sister taxa that are possible candidate genes to study for acid-base regulation. In addition, we provide further support for previous findings of a high rate of gene duplication within Daphnia genus, as compared with other organisms. PMID:25893130

What Does the Cognitive Assessment System (CAS) Measure? Joint Confirmatory Factor Analysis of the CAS and the Woodcock-Johnson Tests of Cognitive Ability (3rd Edition).

ERIC Educational Resources Information Center

Keith, Timothy Z.; Kranzler, John H.; Flanagan, Dawn P.

2001-01-01

Reports the results of the first joint confirmatory factor analysis (CFA) of the Cognitive Assessment System (CAS) and the Woodcock-Johnson Tests of Cognitive Abilities-3rd Edition (WJ III). Results of these analyses do not support the construct validity of the CAS as a measure of the PASS (planning, attention, simultaneous, and sequential)…

Influence of anisotropy on the electrical conductivity and diffusion coefficient of dry K-feldspar: Implications of the mechanism of conduction

NASA Astrophysics Data System (ADS)

Dai, Li-Dong; Hu, Hai-Ying; Li, He-Ping; Sun, Wen-Qing; Jiang, Jian-Jun

2018-02-01

Not Available Project supported by the Strategic Priority Research Program (B) of the Chinese Academy of Sciences (CAS) (Grant No. XDB 18010401), the Key Research Program of Frontier Sciences of CAS (Grant No. QYZDB-SSW-DQC009), the “135” Program of the Institute of Geochemistry of CAS, the Hundred-Talent Program of CAS, and the National Natural Science Foundation of China (Grant Nos. 41474078, 41774099, and 41772042).

Air Quality Study Using Satellites - Current Capability and Future Plans

NASA Technical Reports Server (NTRS)

Bhartia, Pawan K.; Joiner, Joanna; Gleason, James; Liu, Xiong; Torres, Omar; Krotkov, Nickolay; Ziemke, Jerry; Chandra, Sushil

2008-01-01

Satellite instruments have had great success in monitoring the stratospheric ozone and in understanding the processes that control its daily to decadal scale variations. This field is now reaching its zenith with a number of satellite instruments from the US, Europe and Canada capping several decades of active research in this field. The primary public policy imperative of this research was to make reliable prediction of increases in biologically active surface UV radiation due to human activity. By contrast retrieval from satellite data of atmospheric constituents and photo-chemically active radiation that affect air quality is a new and growing field that is presenting us with unique challenges in measurement and data interpretation. A key distinction compared to stratospheric sensors is the greatly enhanced role of clouds, aerosols, and surfaces (CAS) in determining the quality and quantity of useful data that is available for air quality research. In our presentation we will use data from several sensors that are currently flying on the A-train satellite constellation, including OMI, MODIS, CLOUDSAT, and CALIPSO, to highlight that CAS can have both positive and negative effects on the information content of satellite measurements. This is in sharp contrast to other fields of remote sensing where CAS are usually considered an interference except in those cases when they are the primary subject of study. Our analysis has revealed that in the reflected wavelengths one often sees much further down into the atmosphere, through most cirrus, than one does in the emitted wavelengths. The lower level clouds provide a nice background against which one can track long-range transport of trace gases and aerosols. In addition, differences in trace gas columns estimated over cloudy and adjacent clear pixels can be used to measure boundary layer trace gases. However, in order to take full advantage of these features it will be necessary to greatly advance our understanding of how CAS affect the radiation at wavelengths that are used to derive the atmospheric constituents that affect air quality as well as the radiation that controls the photolysis of chemically active trace gases. We will discuss how we are using these new insights to design future satellite missions to study air quality.

40 CFR Appendix: Table 1 to... - List of Hazardous Air Pollutants (HAP) for Subpart HHH

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 11 2010-07-01 2010-07-01 true List of Hazardous Air Pollutants (HAP) for Subpart HHH Table Appendix: Table 1 to Subpart HHH of Part 63 Protection of Environment... HHH of Part 63—List of Hazardous Air Pollutants (HAP) for Subpart HHH CAS Number a Chemical name 75070...

Closure Report for Corrective Action Unit 562: Waste Systems, Nevada National Security Site, Nevada

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

NSTec Environmental Restoration

2012-08-15

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 562, Waste Systems, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 562 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management (FFACO, 1996 as amended). CAU 562 consists of the following 13 Corrective Action Sites (CASs), located in Areas 2, 23, and 25 of the Nevadamore » National Security Site: · CAS 02-26-11, Lead Shot · CAS 02-44-02, Paint Spills and French Drain · CAS 02-59-01, Septic System · CAS 02-60-01, Concrete Drain · CAS 02-60-02, French Drain · CAS 02-60-03, Steam Cleaning Drain · CAS 02-60-04, French Drain · CAS 02-60-05, French Drain · CAS 02-60-06, French Drain · CAS 02-60-07, French Drain · CAS 23-60-01, Mud Trap Drain and Outfall · CAS 23-99-06, Grease Trap · CAS 25-60-04, Building 3123 Outfalls Closure activities began in October 2011 and were completed in April 2012. Activities were conducted according to the Corrective Action Plan for CAU 562 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste and hazardous waste. Some wastes exceeded land disposal limits and required offsite treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite or offsite landfills. NNSA/NSO requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 562 · The transfer of CAU 562 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

40 CFR 721.3 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... concentration in air at 0.5 milligrams or less per liter of air (LC50). CAS Number means Chemical Abstracts..., trade name, brand name, or generic chemical name used to identify a chemical substance other than by its... Office of Pollution Prevention and Toxics means the Director of the EPA Office of Pollution Prevention...

40 CFR 721.3 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... concentration in air at 0.5 milligrams or less per liter of air (LC50). CAS Number means Chemical Abstracts..., trade name, brand name, or generic chemical name used to identify a chemical substance other than by its... Office of Pollution Prevention and Toxics means the Director of the EPA Office of Pollution Prevention...

40 CFR 721.3 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... concentration in air at 0.5 milligrams or less per liter of air (LC50). CAS Number means Chemical Abstracts..., trade name, brand name, or generic chemical name used to identify a chemical substance other than by its... Office of Pollution Prevention and Toxics means the Director of the EPA Office of Pollution Prevention...

40 CFR 721.3 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... concentration in air at 0.5 milligrams or less per liter of air (LC50). CAS Number means Chemical Abstracts..., trade name, brand name, or generic chemical name used to identify a chemical substance other than by its... Office of Pollution Prevention and Toxics means the Director of the EPA Office of Pollution Prevention...

Transformation of OODT CAS to Perform Larger Tasks

NASA Technical Reports Server (NTRS)

Mattmann, Chris; Freeborn, Dana; Crichton, Daniel; Hughes, John; Ramirez, Paul; Hardman, Sean; Woollard, David; Kelly, Sean

2008-01-01

A computer program denoted OODT CAS has been transformed to enable performance of larger tasks that involve greatly increased data volumes and increasingly intensive processing of data on heterogeneous, geographically dispersed computers. Prior to the transformation, OODT CAS (also alternatively denoted, simply, 'CAS') [wherein 'OODT' signifies 'Object-Oriented Data Technology' and 'CAS' signifies 'Catalog and Archive Service'] was a proven software component used to manage scientific data from spaceflight missions. In the transformation, CAS was split into two separate components representing its canonical capabilities: file management and workflow management. In addition, CAS was augmented by addition of a resource-management component. This third component enables CAS to manage heterogeneous computing by use of diverse resources, including high-performance clusters of computers, commodity computing hardware, and grid computing infrastructures. CAS is now more easily maintainable, evolvable, and reusable. These components can be used separately or, taking advantage of synergies, can be used together. Other elements of the transformation included addition of a separate Web presentation layer that supports distribution of data products via Really Simple Syndication (RSS) feeds, and provision for full Resource Description Framework (RDF) exports of metadata.

Is It Worth Using CAS for Symbolic Algebra Manipulation in the Middle Secondary Years? Some Teachers' Views

ERIC Educational Resources Information Center

Pierce, Robyn; Ball, Lynda; Stacey, Kaye

2009-01-01

The use of Computer Algebra Systems (CAS) in years 9 and 10 classrooms as a tool to support learning or in preparation for senior secondary mathematics is controversial. This paper presents an analysis of the positive and negative aspects of using CAS identified in the literature related to these year levels, along with the perceptions of 12…

Walking the Walk/Talking the Talk: Mission Planning with Speech-Interactive Agents

NASA Technical Reports Server (NTRS)

Bell, Benjamin; Short, Philip; Webb, Stewart

2010-01-01

The application of simulation technology to mission planning and rehearsal has enabled realistic overhead 2-D and immersive 3-D "fly-through" capabilities that can help better prepare tactical teams for conducting missions in unfamiliar locales. For aircrews, detailed terrain data can offer a preview of the relevant landmarks and hazards, and threat models can provide a comprehensive glimpse of potential hot zones and safety corridors. A further extension of the utility of such planning and rehearsal techniques would allow users to perform the radio communications planned for a mission; that is, the air-ground coordination that is critical to the success of missions such as close air support (CAS). Such practice opportunities, while valuable, are limited by the inescapable scarcity of complete mission teams to gather in space and time during planning and rehearsal cycles. Moreoever, using simulated comms with synthetic entities, despite the substantial training and cost benefits, remains an elusive objective. In this paper we report on a solution to this gap that incorporates "synthetic teammates" - intelligent software agents that can role-play entities in a mission scenario and that can communicate in spoken language with users. We employ a fielded mission planning and rehearsal tool so that our focus remains on the experimental objectives of the research rather than on developing a testbed from scratch. Use of this planning tool also helps to validate the approach in an operational system. The result is a demonstration of a mission rehearsal tool that allows aircrew users to not only fly the mission but also practice the verbal communications with air control agencies and tactical controllers on the ground. This work will be presented in a CAS mission planning example but has broad applicability across weapons systems, missions and tactical force compositions.

An evaluation of descent strategies for TNAV-equipped aircraft in an advanced metering environment

NASA Technical Reports Server (NTRS)

Izumi, K. H.; Schwab, R. W.; Groce, J. L.; Coote, M. A.

1986-01-01

Investigated were the effects on system throughput and fleet fuel usage of arrival aircraft utilizing three 4D RNAV descent strategies (cost optimal, clean-idle Mach/CAS and constant descent angle Mach/CAS), both individually and in combination, in an advanced air traffic control metering environment. Results are presented for all mixtures of arrival traffic consisting of three Boeing commercial jet types and for all combinations of the three descent strategies for a typical en route metering airport arrival distribution.

40 CFR Table 8 to Subpart Ffff of... - Partially Soluble Hazardous Air Pollutants

Code of Federal Regulations, 2012 CFR

2012-07-01

... CATEGORIES (CONTINUED) National Emission Standards for Hazardous Air Pollutants: Miscellaneous Organic Chemical Manufacturing Pt. 63, Subpt. FFFF, Table 8 Table 8 to Subpart FFFF of Part 63—Partially Soluble...: Chemical name . . . CAS No. 1. 1,1,1-Trichloroethane (methyl chloroform) 71556 2. 1,1,2,2-Tetrachloroethane...

Pipeline Corrosion and Friction Reduction Coatings.

DTIC Science & Technology

1986-06-01

surface energy, i.e., a lower friction surface. Due to the toxic nature of fluorine cas we elected to have Air Products and Chemicals , Inc . perform...Research and Chemical Corp. PR-319 A.P. 8717-21 (1) - 6.9 +19.0 +32.5 +14.9 (1) Indicates Air Products and Chemicals , Inc . proprietary exposure method

Closure Report for Corrective Action Unit 104: Area 7 Yucca Flat Atmospheric Test Sites, Nevada National Security Site, Nevada

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

None

2013-06-27

This Closure Report (CR) presents information supporting closure of Corrective Action Unit (CAU) 104, Area 7 Yucca Flat Atmospheric Test Sites, and provides documentation supporting the completed corrective actions and confirmation that closure objectives for CAU 104 were met. This CR complies with the requirements of the Federal Facility Agreement and Consent Order (FFACO) that was agreed to by the State of Nevada; the U.S. Department of Energy (DOE), Environmental Management; the U.S. Department of Defense; and DOE, Legacy Management. CAU 104 consists of the following 15 Corrective Action Sites (CASs), located in Area 7 of the Nevada National Securitymore » Site: · CAS 07-23-03, Atmospheric Test Site T-7C · CAS 07-23-04, Atmospheric Test Site T7-1 · CAS 07-23-05, Atmospheric Test Site · CAS 07-23-06, Atmospheric Test Site T7-5a · CAS 07-23-07, Atmospheric Test Site - Dog (T-S) · CAS 07-23-08, Atmospheric Test Site - Baker (T-S) · CAS 07-23-09, Atmospheric Test Site - Charlie (T-S) · CAS 07-23-10, Atmospheric Test Site - Dixie · CAS 07-23-11, Atmospheric Test Site - Dixie · CAS 07-23-12, Atmospheric Test Site - Charlie (Bus) · CAS 07-23-13, Atmospheric Test Site - Baker (Buster) · CAS 07-23-14, Atmospheric Test Site - Ruth · CAS 07-23-15, Atmospheric Test Site T7-4 · CAS 07-23-16, Atmospheric Test Site B7-b · CAS 07-23-17, Atmospheric Test Site - Climax Closure activities began in October 2012 and were completed in April 2013. Activities were conducted according to the Corrective Action Decision Document/Corrective Action Plan for CAU 104. The corrective actions included No Further Action and Clean Closure. Closure activities generated sanitary waste, mixed waste, and recyclable material. Some wastes exceeded land disposal limits and required treatment prior to disposal. Other wastes met land disposal restrictions and were disposed in appropriate onsite landfills. The U.S. Department of Energy, National Nuclear Security Administration Nevada Field Office (NNSA/NFO) requests the following: · A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NFO for closure of CAU 104 · The transfer of CAU 104 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

CRISPR/Cas9 in Genome Editing and Beyond.

PubMed

Wang, Haifeng; La Russa, Marie; Qi, Lei S

2016-06-02

The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.

RIFM fragrance ingredient safety assessment, linalyl benzoate, CAS Registry Number 126-64-7.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dkant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Penning, T M; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic. Data from the suitable read across analog linalyl phenylacetate (CAS # 7143-69-3) show that this material does not have skin sensitization potential. The repeated dose toxicity endpoint was completed using linalyl cinnamate (CAS # 78-37-5) as a suitable read across analog, which provided a MOE > 100. The developmental and reproductive toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8), benzoic acid (CAS # 65-85-0) and sodium benzoate (CAS # 532-32-1) as suitable read across analogs, which provided a MOE > 100. The local respiratory toxicity endpoint was completed using linalool (CAS # 78-70-6) and benzoic acid (CAS # 65-85-0) as suitable read across analogs, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework along with data from the suitable read across analog linalyl cinnamate (CAS # 78-375). Copyright © 2016 Elsevier Ltd. All rights reserved.

Projectile Motion with Mathematica.

ERIC Educational Resources Information Center

de Alwis, Tilak

2000-01-01

Describes how to use the computer algebra system (CAS) Mathematica to analyze projectile motion with and without air resistance. These experiments result in several conjectures leading to theorems. (Contains 17 references.) (Author/ASK)

Growth of Aeromonas hydrophila on fresh vegetables stored under a controlled atmosphere.

PubMed Central

Berrang, M E; Brackett, R E; Beuchat, L R

1989-01-01

The effects of controlled-atmosphere storage (CAS) on the survival and growth of Aeromonas hydrophila on fresh asparagus, broccoli, and cauliflower were examined. Two lots of each vegetable were inoculated with A. hydrophila 1653 or K144. A third lot served as an uninoculated control. Following inoculation, vegetables were stored at 4 or 15 degrees C under a CAS system previously shown to extend the shelf life of each commodity or under ambient air. Populations of A. hydrophila were enumerated on the initial day of inoculation and at various intervals for 10 days (15 degrees C) or 21 days (4 degrees C) of storage. Direct plating of samples with selective media was used to enumerate A. hydrophila. The organism was detected on most lots of vegetables as they were received from a commercial produce supplier. Without exception, the CAS system lengthened the time vegetables were subjectively considered acceptable for consumption. However, CAS did not significantly affect populations of A. hydrophila which survived or grew on inoculated vegetables. PMID:2802601

Analysis of Low Appropriateness Score Exam Trends in Decision Support-based Radiology Order Entry System.

PubMed

Gupta, Supriya; Klein, Kandace; Singh, Anand H; Thrall, James H

2017-05-01

Awareness of imaging utilization increased after implementation of Radiology Order Entry with decision support systems (ROE-DS). Our hypothesis is few exams with low Clinical Appropriateness Score (CAS) on ROE-DS are performed. Clinical indications of exams with CAS less than 3 (9-point scale) were re-reviewed and reports analyzed. Structured Query Language-based query retrieved exams with CAS less than 3 in ROE-DS from January 2007 to December 2011. Reasons provided by physicians for ordering these exams and reports of exams performed were analyzed. For each indication, number of exams ordered and performed was calculated. Statistical significance was assessed using Student's t test and χ 2 analysis (P < .05). From 445,984 exams, 12,615 exams (2.8%) had CAS less than 3, and 7,956 exams (63%) were performed. Reasons for ordering of 12,615 low CAS exams were as follows: Requests by physician specialists without further explanation (4,516 = 35.8%), notation of special clinical circumstances (2,877 = 22.8%), requests by nonphysician staff without further explanation (1,383 = 10.9%), absence of suspected finding on previous modality (1,099 = 8.7%), patient preference (737 = 5.8%), and requests based on radiologists' recommendations (706 = 5.6%). Difference between male and female (male < female) preferences for low CAS exams was statistically significant (P < .01). Imaging outcome was highest for extremity MRI cases (66.7%; P < .01). Less than 3% of exams ordered had low CAS and about two-thirds of these were performed. Most common indication for ordering these exams was physician specialist request based on opinion of medical necessity without specification. Extremity MRI constituted the highest positive findings for low CAS exams performed. Published by Elsevier Inc.

Involvement of the CasK/R two-component system in optimal unsaturation of the Bacillus cereus fatty acids during low-temperature growth.

PubMed

Diomandé, Sara Esther; Nguyen-the, Christophe; Abee, Tjakko; Tempelaars, Marcel H; Broussolle, Véronique; Brillard, Julien

2015-11-20

Bacillus cereus sensu lato is composed of a set of ubiquitous strains including human pathogens that can survive a range of food processing conditions, grow in refrigerated food, and sometimes cause food poisoning. We previously identified the two-component system CasK/R that plays a key role in cold adaptation. To better understand the CasK/R-controlled mechanisms that support low-temperature adaptation, we performed a transcriptomic analysis on the ATCC 14579 strain and its isogenic ∆casK/R mutant grown at 12°C. Several genes involved in fatty acid (FA) metabolism were downregulated in the mutant, including desA and desB encoding FA acyl-lipid desaturases that catalyze the formation of a double-bond on the FA chain in positions ∆5 and ∆10, respectively. A lower proportion of FAs presumably unsaturated by DesA was observed in the ΔcasK/R strain compared to the parental strain while no difference was found for FAs presumably unsaturated by DesB. Addition of phospholipids from egg yolk lecithin rich in unsaturated FAs, to growth medium, abolished the cold-growth impairment of ΔcasK/R suggesting that exogenous unsaturated FAs can support membrane-level modifications and thus compensate for the decreased production of these FAs in the B. cereus ∆casK/R mutant during growth at low temperature. Our findings indicate that CasK/R is involved in the regulation of FA metabolism, and is necessary for cold adaptation of B. cereus unless an exogenous source of unsaturated FAs is available. Copyright © 2015 Elsevier B.V. All rights reserved.

On the Origin of Reverse Transcriptase-Using CRISPR-Cas Systems and Their Hyperdiverse, Enigmatic Spacer Repertoires.

PubMed

Silas, Sukrit; Makarova, Kira S; Shmakov, Sergey; Páez-Espino, David; Mohr, Georg; Liu, Yi; Davison, Michelle; Roux, Simon; Krishnamurthy, Siddharth R; Fu, Becky Xu Hua; Hansen, Loren L; Wang, David; Sullivan, Matthew B; Millard, Andrew; Clokie, Martha R; Bhaya, Devaki; Lambowitz, Alan M; Kyrpides, Nikos C; Koonin, Eugene V; Fire, Andrew Z

2017-07-11

Cas1 integrase is the key enzyme of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas adaptation module that mediates acquisition of spacers derived from foreign DNA by CRISPR arrays. In diverse bacteria, the cas1 gene is fused (or adjacent) to a gene encoding a reverse transcriptase (RT) related to group II intron RTs. An RT-Cas1 fusion protein has been recently shown to enable acquisition of CRISPR spacers from RNA. Phylogenetic analysis of the CRISPR-associated RTs demonstrates monophyly of the RT-Cas1 fusion, and coevolution of the RT and Cas1 domains. Nearly all such RTs are present within type III CRISPR-Cas loci, but their phylogeny does not parallel the CRISPR-Cas type classification, indicating that RT-Cas1 is an autonomous functional module that is disseminated by horizontal gene transfer and can function with diverse type III systems. To compare the sequence pools sampled by RT-Cas1-associated and RT-lacking CRISPR-Cas systems, we obtained samples of a commercially grown cyanobacterium- Arthrospira platensis Sequencing of the CRISPR arrays uncovered a highly diverse population of spacers. Spacer diversity was particularly striking for the RT-Cas1-containing type III-B system, where no saturation was evident even with millions of sequences analyzed. In contrast, analysis of the RT-lacking type III-D system yielded a highly diverse pool but reached a point where fewer novel spacers were recovered as sequencing depth was increased. Matches could be identified for a small fraction of the non-RT-Cas1-associated spacers, and for only a single RT-Cas1-associated spacer. Thus, the principal source(s) of the spacers, particularly the hypervariable spacer repertoire of the RT-associated arrays, remains unknown. IMPORTANCE While the majority of CRISPR-Cas immune systems adapt to foreign genetic elements by capturing segments of invasive DNA, some systems carry reverse transcriptases (RTs) that enable adaptation to RNA molecules. From analysis of available bacterial sequence data, we find evidence that RT-based RNA adaptation machinery has been able to join with CRISPR-Cas immune systems in many, diverse bacterial species. To investigate whether the abilities to adapt to DNA and RNA molecules are utilized for defense against distinct classes of invaders in nature, we sequenced CRISPR arrays from samples of commercial-scale open-air cultures of Arthrospira platensis , a cyanobacterium that contains both RT-lacking and RT-containing CRISPR-Cas systems. We uncovered a diverse pool of naturally occurring immune memories, with the RT-lacking locus acquiring a number of segments matching known viral or bacterial genes, while the RT-containing locus has acquired spacers from a distinct sequence pool for which the source remains enigmatic. Copyright © 2017 Silas et al.

La prise en charge du pneumothorax spontané: à propos de 138 cas

PubMed Central

Habibi, Bouchra; Achachi, Leila; Hayoun, Sohaib; Raoufi, Mohammed; Herrak, Laila; Ftouh, Mustapha El

2017-01-01

Le pneumothorax est définit par la présence d’air dans la cavité pleurale. L’objectif de notre étude rétrospective du pneumothorax spontanés au servie de pneumologie à l’hôpital Ibn Sina rabat (2009-2011) est de déterminer le profil épidémiologique, clinique, radiologique, thérapeutique et évolutif. Il s’agit de 138 patients: 128 hommes et 10 femmes (17 à 83 ans), un âge moyen de 44,5 +/- 17,4 ans; sexe ratio 12/8. Le tabagisme est noté chez 81,2%. La symptomatologie clinique est la douleur thoracique (92%), la dyspnée (60%). Et sur la radiographie thoracique: on trouve un PNO (pneumothorax) unilatéral total (110 cas); partiel (10 cas); localisé (6 cas); bilatéral (4 cas); à droite dans 51,4% et à gauche dans 45,7%. On a recensé 70% de pneumothorax spontanés primitifs et 30% de PNO secondaire à (BPCO 44%, et tuberculose pulmonaire 39%). La prise en charge initiale est l’hospitalisation de tous les patients : le drainage thoracique (95%), l’exsufflation à l’aiguille (1%). Le repos et l’O2 (4%). Le retour du poumon à la paroi a été obtenu avant 10 jours chez 63%. L’évolution est favorable chez 89%. Et les complications immédiates: l’emphysème sous cutané (5 cas); une infection (6 cas) et 3 décès (arrêt cardio-respiratoire); les complications à distance sont les récidives dans 11,6%; une 1ère récidive chez 13 cas (drainage thoracique chez 11 cas et oxygénothérapie chez 2 cas) et une 2ème récidive chez 3 cas (recours à la chirurgie). Ce travail montre l’intérêt du drainage thoracique et la surveillance dans la prise en charge du pneumothorax pour éviter les complications et surtout pour éviter les récidives avec un éventuel recours à la chirurgie. PMID:28533875

Psychometric evaluation of the PainCAS Interference with Daily Activities, Psychological/Emotional Distress, and Pain scales.

PubMed

McCaffrey, Stacey A; Black, Ryan A; Butler, Stephen F

2018-03-01

The PainCAS is a web-based clinical tool for assessing and tracking pain and opioid risk in chronic pain patients. Despite evidence for its utility within the clinical setting, the PainCAS scales have never been subject to psychometric evaluation. The current study is the first to evaluate the psychometric properties of the PainCAS Interference with Daily Activities, Psychological/Emotional Distress, and Pain scales. Patients (N = 4797) from treatment centers and hospitals in 16 different states completed the PainCAS as part of routine clinical assessment. A subsample (n = 73) from two hospital-based treatment centers also completed comparator measures. Rasch Rating Scale Models were employed to evaluate the Interference with Daily Activities and Psychological/Emotional Distress scales, and empirical evaluation included assessment of dimensionality, discrimination, item fit, reliability, information, and person-to-item targeting. Additionally, convergent and discriminant validity were evaluated through classical test theory approaches. Convergent validity of the Pain scales was evaluated through correlations with corresponding comparator items. One Interference with Daily Activities item was removed due to poor functioning and discrimination. The retained items from the Interference with Daily Activities and Psychological/Emotional Distress scales conformed to unidimensional Rasch measurement models, yielding satisfactory item fit, reliability, precision, and coverage. Further, results provided support for the convergent and discriminant validity of these two scales. Convergent validity between the PainCAS Pain and BPI Pain items was also strong. Taken together, results provide strong psychometric support for these PainCAS Pain scales. Strengths and limitations of the current study are discussed.

A Diagnostic Marker to Discriminate Childhood Apraxia of Speech From Speech Delay: II. Validity Studies of the Pause Marker.

PubMed

Shriberg, Lawrence D; Strand, Edythe A; Fourakis, Marios; Jakielski, Kathy J; Hall, Sheryl D; Karlsson, Heather B; Mabie, Heather L; McSweeny, Jane L; Tilkens, Christie M; Wilson, David L

2017-04-14

The purpose of this 2nd article in this supplement is to report validity support findings for the Pause Marker (PM), a proposed single-sign diagnostic marker of childhood apraxia of speech (CAS). PM scores and additional perceptual and acoustic measures were obtained from 296 participants in cohorts with idiopathic and neurogenetic CAS, adult-onset apraxia of speech and primary progressive apraxia of speech, and idiopathic speech delay. Adjusted for questionable specificity disagreements with a pediatric Mayo Clinic diagnostic standard, the estimated sensitivity and specificity, respectively, of the PM were 86.8% and 100% for the CAS cohort, yielding positive and negative likelihood ratios of 56.45 (95% confidence interval [CI]: [1.15, 2763.31]) and 0.13 (95% CI [0.06, 0.30]). Specificity of the PM for 4 cohorts totaling 205 participants with speech delay was 98.5%. These findings are interpreted as providing support for the PM as a near-conclusive diagnostic marker of CAS.

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

NASA Astrophysics Data System (ADS)

Cooper, Edwin L.; Overstreet, Nicola

2014-03-01

Recent evidence supports that prokaryotes exhibit adaptive immunity in the form of CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) and Cas (CRISPR associated proteins). The CRISPR-Cas system confers resistance to exogenous genetic elements such as phages and plasmids by allowing for the recognition and silencing of these genetic elements. Moreover, CRISPR-Cas serves as a memory of past exposures. This suggests that the evolution of the immune system has counterparts among the prokaryotes, not exclusively among eukaryotes. Mathematical models have been proposed which simulate the evolutionary patterns of CRISPR, however large gaps in our understanding of CRISPR-Cas function and evolution still exist. The CRISPR-Cas system is analogous to small RNAs involved in resistance mechanisms throughout the tree of life, and a deeper understanding of the evolution of small RNA pathways is necessary before the relationship between these convergent systems is to be determined. Presented in this review are novel RNAi therapies based on CRISPR-Cas analogs and the potential for future therapies based on CRISPR-Cas system components.

QuickStrike ASOC Battlefield Simulation: Preparing the War Fighter to Win

NASA Technical Reports Server (NTRS)

Jones, Richard L.

2010-01-01

The QuickStrike ASOC (Air Support Operations Center) Battlefield Simulation fills a crucial gap in USAF and United Kingdom Close Air Support (CAS) and airspace manager training. The system now provides six squadrons with the capability to conduct total-mission training events whenever the personnel and time are available. When the 111th ASOC returned from their first deployment to Afghanistan they realized the training available prior to deployment was inadequate. They sought an organic training capability focused on the ASOC mission that was low cost, simple to use, adaptable, and available now. Using a commercial off-the-shelf simulation, they developed a complete training system by adapting the simulation to their training needs. Through more than two years of spiral development, incorporating lessons learned, the system has matured, and can now realistically replicate the Tactical Operations Center (TOC) in Kabul, Afghanistan, the TOC supporting the mission in Iraq, or can expand to support a major conflict scenario. The training system provides a collaborative workspace for the training audience and exercise control group via integrated software and workstations that can easily adapt to new mission reqUirements and TOC configurations. The system continues to mature. Based on inputs from the war fighter, new capabilities have been incorporated to add realism and simplify the scenario development process. The QuickStrike simulation can now import TBMCS Air Tasking Order air mission data and can provide air and ground tracks to a common operating picture; presented through either C2PC or JADOCS. This oranic capability to practice team processes and tasks and to conduct mission rehearsals proved its value in the 111 h ASOS's next deployment. The ease of scenario development and the simple to learn and intuitive gamelike interface enables the squadrons to develop and share scenarios incorporating lessons learned from every deployment. These war fighters have now filled the training gap and have the capability they need to train to win.

40 CFR 63.61 - Deletion of methyl ethyl ketone from the list of hazardous air pollutants.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 9 2011-07-01 2011-07-01 false Deletion of methyl ethyl ketone from... Designations, Source Category List § 63.61 Deletion of methyl ethyl ketone from the list of hazardous air pollutants. The substance methyl ethyl ketone (MEK, 2-Butanone) (CAS Number 78-93-3) is deleted from the list...

40 CFR 63.61 - Deletion of methyl ethyl ketone from the list of hazardous air pollutants.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Deletion of methyl ethyl ketone from... Designations, Source Category List § 63.61 Deletion of methyl ethyl ketone from the list of hazardous air pollutants. The substance methyl ethyl ketone (MEK, 2-Butanone) (CAS Number 78-93-3) is deleted from the list...

Source apportionment and dynamic changes of carbonaceous aerosols during the haze bloom-decay process in China based on radiocarbon and organic molecular tracers

NASA Astrophysics Data System (ADS)

Liu, Junwen; Li, Jun; Liu, Di; Ding, Ping; Shen, Chengde; Mo, Yangzhi; Wang, Xinming; Luo, Chunling; Cheng, Zhineng; Szidat, Sönke; Zhang, Yanlin; Chen, Yingjun; Zhang, Gan

2016-03-01

Fine carbonaceous aerosols (CAs) is the key factor influencing the currently filthy air in megacities in China, yet few studies simultaneously focus on the origins of different CAs species using specific and powerful source tracers. Here, we present a detailed source apportionment for various CAs fractions, including organic carbon (OC), water-soluble OC (WSOC), water-insoluble OC (WIOC), elemental carbon (EC) and secondary OC (SOC) in the largest cities of North (Beijing, BJ) and South China (Guangzhou, GZ), using the measurements of radiocarbon and anhydrosugars. Results show that non-fossil fuel sources such as biomass burning and biogenic emission make a significant contribution to the total CAs in Chinese megacities: 56 ± 4 in BJ and 46 ± 5 % in GZ, respectively. The relative contributions of primary fossil carbon from coal and liquid petroleum combustions, primary non-fossil carbon and secondary organic carbon (SOC) to total carbon are 19, 28 and 54 % in BJ, and 40, 15 and 46 % in GZ, respectively. Non-fossil fuel sources account for 52 in BJ and 71 % in GZ of SOC, respectively. These results suggest that biomass burning has a greater influence on regional particulate air pollution in North China than in South China. We observed an unabridged haze bloom-decay process in South China, which illustrates that both primary and secondary matter from fossil sources played a key role in the blooming phase of the pollution episode, while haze phase is predominantly driven by fossil-derived secondary organic matter and nitrate.

Source apportionment and dynamic changes of carbonaceous aerosols during the haze bloom-decay process in China based on radiocarbon and organic molecular tracers

NASA Astrophysics Data System (ADS)

Liu, J.; Li, J.; Liu, D.; Ding, P.; Shen, C.; Mo, Y.; Wang, X.; Luo, C.; Cheng, Z.; Szidat, S.; Zhang, Y.; Chen, Y.; Zhang, G.

2015-12-01

Fine carbonaceous aerosols (CAs) is the key factor influencing the currently filthy air in megacities of China, yet seldom study simultaneously focuses on the origins of different CAs species using specific and powerful source tracers. Here, we present a detailed source apportionment for various CAs fractions, including organic carbon (OC), water-soluble OC (WSOC), water-insoluble OC (WIOC), elemental carbon (EC) and secondary OC (SOC) in the largest cities of North (Beijing, BJ) and South China (Guangzhou, GZ), respectively, using the measurements of radiocarbon and anhydrosugars. Results show that non-fossil fuel sources such as biomass burning and biogenic emission make a significant contribution to the total CAs in Chinese megacities: 56 ± 4 % in BJ and 46 ± 5 % in GZ, respectively. The relative contributions of primary fossil carbon from coal and liquid petroleum combustions, primary non-fossil carbon and secondary organic carbon (SOC) to total carbon are 19, 28 and 54 % in BJ, and 40, 15 and 46 % in GZ, respectively. Non-fossil fuel sources account for 52 % in BJ and 71 % in GZ of SOC, respectively. These results suggest that biomass burning has a greater influence on regional particulate air pollution in North China than in South China. We observed an unabridged haze bloom-decay process in South China, which illustrates that both primary and secondary matter from fossil sources played a key role in the blooming phase of the pollution episode, while haze phase is predominantly driven by fossil-derived secondary organic matter and nitrate.

Guidelines for patient selection and performance of carotid artery stenting.

PubMed

2009-12-01

The endovascular treatment of carotid atherosclerosis with carotid artery stenting (CAS) remains controversial. Carotid endarterectomy (CEA) remains the benchmark in terms of procedural mortality and morbidity. Consensus Australasian guidelines for the safe performance of CAS were developed using the modified Delphi consensus method of iterative consultation. Selection of patients suitable for CAS needs careful consideration of clinical and patho-anatomical criteria. Randomised controlled trials and pooled analyses have demonstrated that CAS is more hazardous than CEA. The CGSC therefore recommends that CAS should not be performed in the majority of patients requiring carotid revascularisation. There is currently no evidence to support CAS as a treatment for asymptomatic carotid stenosis. The use of distal protection devices during CAS remains controversial with increased risk of clinically silent stroke. The knowledge requirements for the safe performance of CAS include an understanding of the evidence base from randomised controlled trials, carotid and aortic arch anatomy and pathology, and recognition and management of periprocedural complications. It is critical that all patients being considered for a carotid intervention have adequate pre-procedural neuroimaging and peri-procedural, independent, neurological assessment. Maintenance of proficiency in CAS requires active involvement in surgical/endovascular audit and continuing medical education programmes. These standards should apply in the public and private health-care settings. These guidelines represent the consensus of an intercollegiate committee in order to direct appropriate patient selection to perform CAS. Advances in endovascular technologies and the results of randomised controlled trials will guide future revisions of this document.

Cas IIgly Induces Apoptosis in Glioma C6 Cells In Vitro and In Vivo through Caspase-Dependent and Caspase-Independent Mechanisms1

PubMed Central

Trejo-Solís, Cristina; Palencia, Guadalupe; Zúñiga, Sergio; Rodríguez-Ropon, Andrea; Osorio-Rico, Laura; Torres Luvia, Sanchez; Gracia-Mora, Isabel; Marquez-Rosado, Lucrecia; Sánchez, Aurora; Moreno-García, Miguel E; Cruz, Arturo; Bravo-Gómez, María Elena; Ruiz-Ramírez, Lena; Rodríguez-Enriquez, Sara; Sotelo, Julio

2005-01-01

Abstract In this work, we investigated the effects of Casiopeina II-gly (Cas IIgly)—a new copper compound exhibiting antineoplastic activity—on glioma C6 cells under both in vitro and in vivo conditions, as an approach to identify potential therapeutic agents against malignant glioma. The exposure of C6 cells to Cas IIgly significantly inhibited cell proliferation, increased reactive oxygen species (ROS) formation, and induced apoptosis in a dose-dependent manner. In cultured C6 cells, Cas IIgly caused mitochondrio-nuclear translocation of apoptosis induction factor (AIF) and endonuclease G at all concentrations tested; in contrast, fragmentation of nucleosomal DNA, cytochrome c release, and caspase-3 activation were observed at high concentrations. Administration of N-acetyl-l-cystein, an antioxidant, resulted in significant inhibition of AIF translocation, nucleosomal DNA fragmentation, and caspase-3 activation induced by Cas IIgly. These results suggest that caspase-dependent and caspase-independent pathways both participate in apoptotic events elicited by Cas IIgly. ROS formation induced by Cas IIgly might also be involved in the mitochondrio-nuclear translocation of AIF and apoptosis. In addition, treatment of glioma C6-positive rats with Cas IIgly reduced tumor volume and mitotic and cell proliferation indexes, and increased apoptotic index. Our findings support the use of Cas IIgly for the treatment of malignant gliomas. PMID:16036107

Behavioral and neurobiological correlates of childhood apraxia of speech in Italian children.

PubMed

Chilosi, Anna Maria; Lorenzini, Irene; Fiori, Simona; Graziosi, Valentina; Rossi, Giuseppe; Pasquariello, Rosa; Cipriani, Paola; Cioni, Giovanni

2015-11-01

Childhood apraxia of speech (CAS) is a neurogenic Speech Sound Disorder whose etiology and neurobiological correlates are still unclear. In the present study, 32 Italian children with idiopathic CAS underwent a comprehensive speech and language, genetic and neuroradiological investigation aimed to gather information on the possible behavioral and neurobiological markers of the disorder. The results revealed four main aggregations of behavioral symptoms that indicate a multi-deficit disorder involving both motor-speech and language competence. Six children presented with chromosomal alterations. The familial aggregation rate for speech and language difficulties and the male to female ratio were both very high in the whole sample, supporting the hypothesis that genetic factors make substantial contribution to the risk of CAS. As expected in accordance with the diagnosis of idiopathic CAS, conventional MRI did not reveal macrostructural pathogenic neuroanatomical abnormalities, suggesting that CAS may be due to brain microstructural alterations. Copyright © 2015 Elsevier Inc. All rights reserved.

Survival of Listeria monocytogenes on fresh blueberries (Vaccinium corymbosum) stored under controlled atmosphere and ozone.

PubMed

Concha-Meyer, Anibal; Eifert, Joseph; Williams, Robert; Marcy, Joseph; Welbaum, Gregory

2014-05-01

Listeria monocytogenes is a foodborne pathogen that represents a high risk for consumers because it can grow under refrigeration conditions and can also develop acid tolerance. Fresh blueberries are hand-picked, packed, and transported under refrigeration without receiving a microbial inactivation treatment. The aim of this work was to study the survival of L. monocytogenes in fresh highbush blueberries stored at 4 or 12 °C under different controlled atmosphere conditions, including air (control); 5% O2, 15% CO2, 80% N2 (controlled atmosphere storage [CAS]); or ozone gas (O3), 4 ppm at 4 °C or 2.5 ppm at 12 °C, at high relative humidity (90 to 95%) for a total of 10 days. Fresh blueberries inside a plastic clamshell were spot inoculated with the bacteria and were stored at 4 or 12 °C in isolated cabinets under air, CAS, and O3 atmospheric conditions. Samples were evaluated on days 0, 1, 4, 7, and 10 for microbial growth using modified Oxford agar. CAS did not delay or inhibit L. monocytogenes growth in fresh blueberries after 10 days. O3 achieved 3- and 2-log reductions when compared with air treatment at 4 and 12 °C, respectively. Low concentrations of O3 together with proper refrigeration temperature can ensure product safety throughout transportation. O3 is a strong antimicrobial that safely decomposes to oxygen and water without leaving residues and can be used as an alternative method to prevent bacterial growth during a long transport period.

Prise en charge diagnostique et thérapeutique de la tuberculose ganglionnaire en Tunisie

PubMed Central

Ben Brahim, Hajer; Kooli, Ikbel; Aouam, Abir; Toumi, Adnene; Loussaief, Chawki; Koubaa, Jamel; Chakroun, Mohamed

2014-01-01

La tuberculose ganglionnaire est la localisation extra-pulmonaire la plus fréquente de la tuberculose. Nous nous proposons dans ce travail d’étudier les modalités diagnostiques, thérapeutiques et évolutives de cette localisation. Il s'agit d'une étude rétrospective portant sur 100 cas de tuberculose ganglionnaire. L’âge moyen était de 35 ± 15 ans (15-85 ans). Aucun malade n’était VIH positif. L'aire cervicale était la plus touchée (93 cas). L'intradermo-réaction à la tuberculine était positive dans 76/91 cas (83,5%). L'examen bactériologique des prélèvements au niveau des ganglions atteints avait mis en évidence des bacilles acido-alcoolo-résistants à l'examen direct dans 2/31 cas (6,4%) et la culture avait isolé Mycobacteruim tuberculosis dans 1/31 cas (3,2%). La cytoponction ganglionnaire (FNAC) était évocatrice de tuberculose dans 35/42 cas (83,3%). La biopsie ganglionnaire était réalisée dans 69 cas et avait permis de retenir le diagnostic de tuberculose dans tous les cas. La FNAC, comparativement à la biopsie, avait permis de raccourcir significativement le délai de la prise en charge (15,1 vs 22,8 jours; p=0,001) et la durée d'hospitalisation (17,3 vs 24,6; p=0,004). La durée moyenne du traitement antituberculeux était de 9,8 ± 4,6 mois (7 à 44 mois). Le traitement chirurgical initial avait raccourci significativement la durée du traitement médical. Il n'avait pas d'impact sur le taux de guérison. Nous avons noté 10 cas de réponse paradoxale aux antituberculeux, quatre cas de résistance clinique et une rechute dans deux cas. La tuberculose ganglionnaire pose un problème diagnostique et thérapeutique. La microbiologie est d'un faible apport. La FNAC est un moyen diagnostique très utiles dans les pays endémiques et à faibles ressources. Un traitement médical seul permet d’éviter les inconvénients de la chirurgie. PMID:25829976

Study of interactions between anionic exopolysaccharides produced by newly isolated probiotic bacteria and sodium caseinate.

PubMed

Abid, Yousra; Joulak, Ichrak; Ben Amara, Chedia; Casillo, Angela; Attia, Hamadi; Gharsallaoui, Adem; Azabou, Samia

2018-07-01

The present study aims to evaluate the interactions between four exopolysaccharides (EPS) produced by probiotic bacteria and sodium caseinate (Cas) in order to simulate their behavior in dairy products. Complexation between the produced EPS samples and Cas was investigated as a function of polysaccharide to protein ratio. The highest turbidity and average size of complexes were formed at an EPS/Cas ratio of 3 (corresponding to 1 g/L of EPS and 0.33 g/L of Cas) as a result of the combination of individual complexes to form aggregates. Zeta potential measurements and Cas surface hydrophobicity results suggested that complex formation occurred essentially through electrostatic attractions with a possible contribution of hydrophobic interaction for EPS-GM which was produced by Bacillus tequilensis-GM. Afterwards, the effect of pH on the complexation between biopolymers was studied when EPS and Cas concentrations were maintained constant at 1 and 0.33 g/L, respectively. pH was adjusted to 3.0 and 3.5, respectively. Results showed that the highest amount and sizes of EPS/Cas complexes were formed at pH 3.5 and that EPS-GM enabled to obtain the biggest and highest amount of aggregates. Therefore, the obtained results support the fact that the simultaneous presence of EPS and Cas in dairy products results in complexes formation via electrostatic interactions depending on EPS/Cas ratio and pH of the medium. Copyright © 2018 Elsevier B.V. All rights reserved.

Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes.

PubMed

Chu, Van Trung; Weber, Timm; Graf, Robin; Sommermann, Thomas; Petsch, Kerstin; Sack, Ulrike; Volchkov, Pavel; Rajewsky, Klaus; Kühn, Ralf

2016-01-16

The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes. We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.

Independent Confirmatory Factor Analysis of the Cognitive Assessment System (CAS): What Does CAS Measure?

ERIC Educational Resources Information Center

Kranzler, John H.; Keith, Timothy Z.

1999-01-01

Uses confirmatory factor analysis (CFA) to address unresolved issues concerning the structure of the Cognitive Assessment System, a test of intelligence based upon the planning, attention, and simultaneous-successive (PASS) processes theory of human cognition. Results reveal that the CFA of the standardization data do not support use of the CAS…

The Future of Online Services to Faculty: A Pilot Project with CAS Online.

ERIC Educational Resources Information Center

Culotta, Wendy A.

Lottery money in California, part of which is required by law to be distributed to educational institutions, provides avenues for innovative services. A pilot program was supported by the administration of California State University, Long Beach, to provide faculty access to CAS (Chemical Abstracts Service) ONLINE (a remote database), which could…

Prevalence and Phenotype of Childhood Apraxia of Speech In Youth with Galactosemia

PubMed Central

Shriberg, Lawrence D.; Potter, Nancy L.; Strand, Edythe A.

2010-01-01

Purpose We address the hypothesis that the severe and persistent speech disorder reported in persons with galactosemia meets contemporary diagnostic criteria for Childhood Apraxia of Speech (CAS). A positive finding for CAS in this rare metabolic disorder has the potential to impact treatment of persons with galactosemia and inform explanatory perspectives on CAS in neurological, neurodevelopmental, and idiopathic contexts. Method Thirty-three youth with galactosemia and significant prior or persistent speech sound disorder were assessed in their homes in 17 states. Participants completed a protocol yielding information on their cognitive, structural, sensorimotor, language, speech, prosody, and voice status and function. Results Eight of the 33 participants (24%) met contemporary diagnostic criteria for CAS. Two participants, one of whom was among the 8 with CAS, met criteria for ataxic or hyperkinetic dysarthria. Group-wise findings for the remaining 24 participants are consistent with a classification category termed Motor Speech Disorder-Not Otherwise Specified (MSD-NOS; Shriberg, Fourakis, et al., in press-a). Conclusion We estimate the prevalence of CAS in galactosemia at 18 per hundred, 180 times the estimated risk for idiopathic CAS. Findings support the need to study risk factors for the high occurrence of motor speech disorders in galactosemia, despite early compliant dietary management. PMID:20966389

[Anesthetic management for surgery of giant coronary aneurysms complicated with Churg-Strauss syndrome].

PubMed

Kido, Koji; Tokuda, Rui; Suzuki, Tomofumi; Hanashiro, Ako; Kobashigawa, Teruyo; Mayama, Takashi; Kamikawa, Michie

2014-04-01

Few cases of Churg-Strauss syndrome (CSS) complicated by giant coronary aneurysms (CAs)have been reported thus far. We report a case of CSS in a 60-year-old man who underwent surgery for giant CAs, and was managed with anesthetics. The patient developed acute myocardial infarction, and was diagnosed with giant CAs in the right coronary artery (RCA, 11 cm) and circumflex artery (3 cm). The CA in RCA was communicating with the right ventricle. He had a history of pericardiectomy for pericarditis caused by the CSS and developed thrombocytopenia due to consumptive coagulopathy within the CAs. An operation, including ligation and excision of the CAs, and coronary artery bypass grafting was performed under general anesthesia and cardiopulmonary bypass. There was massive hemorrhage followed by hemodynamic instability while detaching the tight pericardial adhesion and fragile surface of the CAs. Massive transfusion was required along with inotropes administration and intraaortic balloon support. In this case, determination of the appropriate surgical timing was difficult because symptoms of the CSS became worse followed by rapid enlargement of the CAs, myocardial infarction, and thrombocytopenia. Steroids were administered for treating CSS, and the blood transfusion was sufficient. However, it was difficult to control the hemorrhage and maintain hemodynamic stability.

RIFM fragrance ingredient safety assessment, 2-ethyl-1-butanol, CAS Registry Number 97-95-0.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Miyachi, Y; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data from the suitable read across analog 2-ethylhexanol (CAS # 104-76-7) show that this material is not genotoxic. Data from the suitable read across analog isopropyl alcohol (CAS # 67-63-0) show that this material does not have skin sensitization potential. The local respiratory toxicity endpoint was completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (1.4 mg/day). The repeated dose toxicity endpoint was completed using 2-ethylhexanol (CAS # 104-76-7) and 1-heptanol, 2-propyl (CAS # 10042-59-8) as suitable read across analogs, which provided a MOE > 100. The developmental and reproductive toxicity endpoint was completed using 2-ethyl-hexanol (CAS # 104-76-7) and isobutyl alcohol (CAS # 78-83-1) as suitable read across analogs, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

Advanced Propulsion for the XXIst Century

NASA Technical Reports Server (NTRS)

Frisbee, Robert H.

2003-01-01

This document represents a poster presentation offered at the AIAA/CAS International Air & Space Symposium and Exposition from July 14-17, 2003 in Dayton Ohio. This presentation outlines advanced space propulsion concepts as well as associated research and industry activities during the 21st century.

TRENDS IN RURAL SULFUR CONCENTRATIONS

EPA Science Inventory

This paper presents an analysis of regional trends in atmospheric concentrations in sulfur dioxide (502) and particulate sulfate (50~- ) at rural monitoring sites in the Clean Air Act Status and Trends Monitoring Network (CAsTNet) from 1990 to 1999. A two-stage approach is used t...

CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

PubMed Central

Garrett, Roger A.; Shah, Shiraz A.; Erdmann, Susanne; Liu, Guannan; Mousaei, Marzieh; León-Sobrino, Carlos; Peng, Wenfang; Gudbergsdottir, Soley; Deng, Ling; Vestergaard, Gisle; Peng, Xu; She, Qunxin

2015-01-01

The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. PMID:25764276

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli

PubMed Central

Díez-Villaseñor, César; Guzmán, Noemí M.; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J.M.

2013-01-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism. PMID:23445770

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

PubMed

Díez-Villaseñor, César; Guzmán, Noemí M; Almendros, Cristóbal; García-Martínez, Jesús; Mojica, Francisco J M

2013-05-01

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

Contralateral occlusion is not a clinically important reason for choosing carotid artery stenting for patients with significant carotid artery stenosis.

PubMed

Brewster, Luke P; Beaulieu, Robert; Kasirajan, Karthik; Corriere, Matthew A; Ricotta, Joseph J; Patel, Siddharth; Dodson, Thomas F

2012-11-01

Contralateral carotid artery occlusion by itself carries an increased risk of stroke. Carotid endarterectomy (CEA) in the presence of contralateral carotid artery occlusion has high reported rates of perioperative morbidity and mortality. Our objective was to determine if there is a clinical benefit to patients who receive carotid artery stenting (CAS) compared to CEA in the presence of contralateral carotid artery occlusion. We conducted a retrospective medical chart review over a 4.5-year institutional experience of persons with contralateral carotid artery occlusion and ipsilateral carotid artery stenosis who underwent CAS or CEA. The main outcome measures were 30-day cardiac, stroke, and mortality rate, and midterm mortality. Of a total of 713 patients treated for carotid artery stenosis during this time period, 57 had contralateral occlusion (~8%). Thirty-nine of these patients were treated with CAS, and 18 with CEA. The most common indications for CAS were prior neck surgery (18), contralateral internal carotid occlusion (nine), and prior neck radiation (seven). The average age was 70 ± 8.5 for CEA and 66.7 ± 9.3 for CAS (P = .20). Both groups were predominantly men (CEA 12 of 18; CAS 28 of 39; P = .76), with similar prevalence of symptomatic lesions (CEA 8 of 18, CAS 20 of 39; P = .77). Two patients died within 30 days in the CAS group (5%). No deaths occurred within 30 days in the CEA group (P = .50); the mortality rate for CAS and CEA combined was 3.5%. No perioperative strokes or myocardial infarction occurred in either group. Two transient ischemic attacks occurred after CAS. At mean follow-up of 29.4 ± 16 months (CEA) and 28 ± 14.4 months (CAS; range, 1.5-48.5 months), seven deaths occurred in the CAS group and one in the CEA group (17.9% vs 5.5%; P = .40). There were two reinterventions in the CAS group for in-stent restenosis and there were no reoperations in the CEA group. Although CEA and CAS can both be performed with good perioperative results and acceptable midterm mortality, the observed outcomes do not support use of contralateral carotid artery occlusion as a selection criterion for CAS over CEA in the absence of other indications. Copyright © 2012 Society for Vascular Surgery. All rights reserved.

Summary Report on the Navy Emergency Escape Breathing Device

DTIC Science & Technology

1983-08-08

recirculated cas. The device produces pure oxygen (02) and uses a venturi to recirculate unused and expired gases through the purifier. A rubber...passing the recirculated air through a lithium hydroxide (LiOH) scrubber . The scrubber reduces the CO. level by forming either lithium bicarbonate (LiSCO...transparent visor and an elastromeric neck seal; a chlorate candle-based 02 generator; an air purification filter or scrubberl and a venturi arrangement to

National trends in carotid artery revascularization surgery.

PubMed

Dumont, Travis M; Rughani, Anand I

2012-06-01

Several randomized trials have emerged with conflicting data on the overall safety of carotid artery stenting (CAS) in comparison with carotid endarterectomy (CEA). The authors hypothesize that changes in national trends correspond to publication of randomized trials, including an increase in utilization of CAS after publication of trials favorable to CAS (for example, Carotid and Vertebral Artery Transluminal Angioplasty Study [CAVATAS] and Stenting and Angioplasty with Protection in Patients at High Risk for Endarterectomy [SAPPHIRE]) and decrease in utilization of CAS after publication of trials favorable to CEA (for example, Endarterectomy versus Stenting in Patients with Symptomatic Severe Carotid Stenosis [EVA3-S] and Stent-Supported Percutaneous Angioplasty of the Carotid Artery versus Endarterectomy [SPACE]). The Nationwide Inpatient Sample was obtained for the years 1998-2008. Individual cases were isolated for principal diagnosis of unilateral or bilateral carotid artery stenosis or occlusion undergoing CEA or CAS. The percentage of CAS for all carotid revascularization procedures was calculated for each year. Perioperative inpatient morbidity, including stroke or death, were calculated and compared. The percentage of patients undergoing CAS increased yearly from the start of the observed period to the end, with the exception of a decrease in 2007. The peak utilization of CAS for carotid artery revascularization procedures was 15% of all cases in 2006. The stroke or death rate was consistent at 5% among all patients undergoing CEA for all years, while the incidence of stroke or death decreased among patients undergoing CAS from 9% in 1998 to 5% in 2008. The practice of CAS in the US is expanding, from less than 3% of all carotid artery revascularization procedures to 13% in 2008. The utilization of CAS was seen to correlate with publication of randomized trials. Utilization nearly doubled in 2005 after publication of the CAS-favorable SAPPHIRE in 2004, and decreased by 22% after publication of the CEA-favorable EVA-3S and SPACE in 2007. With the publication of Carotid Revascularization Endarterectomy Versus Stenting Trial (CREST), the authors predict a resultant increase in the rate of CAS for carotid artery disease in the upcoming years.

Using Common Assignments to Strengthen Teaching and Learning: Research on the Second Year of Implementation

ERIC Educational Resources Information Center

Reumann-Moore, Rebecca; Duffy, Mark

2015-01-01

Initiated for the 2013-14 school year, the Common Assignment Study (CAS) is a three-year effort being led by the Colorado Education Initiative (CEI) and The Fund for Transforming Education in Kentucky (The Fund) with support from the Bill & Melinda Gates Foundation. Conceptually, CAS builds on previous efforts to improve instruction through…

CAS -- A Turboprop Solution for the COIN Fight

DTIC Science & Technology

2009-04-14

Civilian Tiltrotor. http://www.bellagusta.com/air ba main.cfm. Accessed 13 Jan 2009. Blake, Brett R , AT-6-The Best Investmentfor the Long War, Air...Counterinsurgency, CRS for Congress RL32737, Washington DC: Congressional Research Service, 24 Jan. 2005. Blake, Brett R Major USAF. AT-6-The Best...www.aviationweek.com/aw/generic/channel .jsp?channel= awst # (accessed 15 Jan 2009). 7 Bolkcom and Katzman, 24. 8 U.S. Congress, Office ofTechnology Assessment, Global

Guidelines for patient selection and performance of carotid artery stenting.

PubMed

Bladin, Christopher; Chambers, Brian; New, Gishel; Denton, Michael; Lawrence-Brown, Michael

2010-06-01

The endovascular treatment of carotid atherosclerosis with carotid artery stenting (CAS) remains controversial. Carotid endarterectomy remains the benchmark in terms of procedural mortality and morbidity. At present, there are no consensus Australasian guidelines for the safe performance of CAS. We applied a modified Delphi consensus method of iterative consultation between the College representatives on the Carotid Stenting Guidelines Committee (CSGC). Selection of patients suitable for CAS needs careful consideration of clinical and patho-anatomical criteria and cannot be directly extrapolated from clinical indicators for carotid endarterectomy (CEA). Randomized controlled trials (including pooled analyses of results) comparing CAS with CEA for treatment of symptomatic stenosis have demonstrated that CAS is more hazardous than CEA. On current evidence, the CGSC therefore recommends that CAS should not be performed in the majority of patients requiring carotid revascularisation. The evidence for CAS in patients with symptomatic severe carotid stenosis who are considered medically high risk is weak, and there is currently no evidence to support CAS as a treatment for asymptomatic carotid stenosis. The use of distal protection devices during CAS remains controversial with increased risk of clinically silent stroke. The knowledge requirements for the safe performance of CAS include an understanding of the evidence base from randomized controlled trials, carotid and aortic arch anatomy and pathology, clinical stroke syndromes, the differing treatment options for stroke and carotid atherosclerosis, and recognition and management of periprocedural complications. It is critical that all patients being considered for a carotid intervention have adequate pre-procedural neuro-imaging and an independent, standardized neurological assessment before and after the procedure. Maintenance of proficiency in CAS requires active involvement in surgical/endovascular audit and continuing medical education programs. These standards should apply in the public and private health care settings. These guidelines represent the consensus of an inter-collegiate committee in order to direct appropriate patient selection and the range of cognitive and technical requirements to perform CAS. Advances in endovascular technologies and the results of randomized controlled trials will guide future revisions of these guidelines.

Synergistic Effects of Temperature, Oxidation and Stress Level on Fatigue Damage Evolution and Lifetime Prediction of Cross-Ply SiC/CAS Ceramic-Matrix Composites Through Hysteresis-Based Parameters

NASA Astrophysics Data System (ADS)

Li, Longbiao

2017-12-01

The damage development and cyclic fatigue lifetime of cross-ply SiC/CAS ceramic-matrix composites have been investigated at different testing temperatures in air atmosphere. The relationships between the fatigue hysteresis-based damage parameters, i.e., fatigue hysteresis dissipated energy, fatigue hysteresis modulus and fatigue peak strain and the damage mechanisms of matrix multicracking, fiber/matrix interface debonding, interface sliding and fibers failure, have been established. With the increase in the cycle number, the evolution of the fatigue hysteresis modulus, fatigue peak strain and fatigue hysteresis dissipated energy depends upon the fatigue peak stress levels, interface and fibers oxidation and testing temperature. The fatigue life S-N curves of cross-ply SiC/CAS composite at room and elevated temperatures have been predicted, and the fatigue limit stresses at room temperature, 750 and 850 °C, are 50, 36 and 30% of the tensile strength, respectively.

A Diagnostic Marker to Discriminate Childhood Apraxia of Speech from Speech Delay: IV. the Pause Marker Index

ERIC Educational Resources Information Center

Shriberg, Lawrence D.; Strand, Edythe A.; Fourakis, Marios; Jakielski, Kathy J.; Hall, Sheryl D.; Karlsson, Heather B.; Mabie, Heather L.; McSweeny, Jane L.; Tilkens, Christie M.; Wilson, David L.

2017-01-01

Purpose: Three previous articles provided rationale, methods, and several forms of validity support for a diagnostic marker of childhood apraxia of speech (CAS), termed the pause marker (PM). Goals of the present article were to assess the validity and stability of the PM Index (PMI) to scale CAS severity. Method: PM scores and speech, prosody,…

A facile and efficient dry transfer technique for two-dimensional Van derWaals heterostructure

NASA Astrophysics Data System (ADS)

Xie, Li; Du, Luojun; Lu, Xiaobo; Yang, Rong; Shi, Dongxia; Zhang, Guangyu

2017-08-01

Not Available Project supported by the National Basic Research Program of China (Grant Nos. 2013CB934500 and 2013CBA01602), the National Natural Science Foundation of China (Grant Nos. 61325021, 11574361, and 51572289), the Key Research Program of Frontier Sciences, CAS, (Grant No. QYZDB-SSW-SLH004), and the Strategic Priority Research Program (B), CAS (Grant No. XDB07010100).

Unique Sensor Plane Maps Invisible Toxins for First Responders

ScienceCinema

Kroutil, Robert; Thomas, Mark; Aten, Keith

2017-12-09

A unique airborne emergency response tool, ASPECT is a Los Alamos/U.S. Environmental Protection Agency project that can put chemical and radiological mapping tools in the air over an accident scene. The name ASPECT is an acronym for Airborne Spectral Photometric Environmental Collection Technology. Update, Sept. 19, 2008: Flying over storm-damaged refineries and chemical factories, a twin-engine plane carrying the ASPECT (Airborne Spectral Photometric Environmental Collection Technology) system has been on duty throughout the recent hurricanes that have swept the Florida and Gulf Coast areas. ASPECT is a project of the U.S. U.S. Environmental Protection Agencys National Decontamination Team. Los Alamos National Laboratory leads a science and technology program supporting the EPA and the ASPECT aircraft. Casting about with a combination of airborne photography and infrared spectroscopy, the highly instrumented plane provides emergency responders on the ground with a clear concept of where danger lies, and the nature of the sometimes-invisible plumes that could otherwise kill them. ASPECT is the nations only 24/7 emergency response aircraft with chemical plume mapping capability. Bob Kroutil of Bioscience Division is the project leader, and while he said the team has put in long hours, both on the ground and in the air, its a worthwhile effort. The plane flew over 320 targeted sites in four days, he noted. Prior to the deployment to the Gulf Coast, the plane had been monitoring the Democratic National Convention in Denver, Colorado. Los Alamos National Laboratory Divisions that are supporting ASPECT include, in addition to B-Division, CTN-5: Networking Engineering and IRM-CAS: Communication, Arts, and Services. Leslie Mansell, CTN-5, and Marilyn Pruitt, IRM-CAS, were recognized the the U.S. EPA for their outstanding support to the hurricane response of Gustav in Louisiana and Ike in Texas. The information from the data collected in the most recent event, Hurricane Ike, was sent to the EPA Region 6 Rapid Needs Assessment and the State of Texas Joint Field Office in Austin, Texas. It appears that though there is considerable damage in Galveston and Texas City, there are fewer chemical leaks than during either hurricanes Katrina or Rita. Specific information gathered from the data was reported out to the U.S. Environmental Protection Agency Headquarters, the Federal Emergency Management Agency, the Department of Homeland Security, and the State of Texas Emergency Management Agency.

40 CFR 60.617 - Chemicals affected by subpart III.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 6 2010-07-01 2010-07-01 false Chemicals affected by subpart III. 60... Compound (VOC) Emissions From the Synthetic Organic Chemical Manufacturing Industry (SOCMI) Air Oxidation Unit Processes § 60.617 Chemicals affected by subpart III. Chemical name CAS No.* Acetaldehyde 75-07-0...

40 CFR 60.617 - Chemicals affected by subpart III.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 6 2011-07-01 2011-07-01 false Chemicals affected by subpart III. 60... Compound (VOC) Emissions From the Synthetic Organic Chemical Manufacturing Industry (SOCMI) Air Oxidation Unit Processes § 60.617 Chemicals affected by subpart III. Chemical name CAS No.* Acetaldehyde 75-07-0...

Convergent Aeronautics Solutions (CAS) Showcase Presentation on Mission Adaptive Digital Composite Aerostructure Technologies (MADCAT)

NASA Technical Reports Server (NTRS)

Swei, Sean; Cheung, Kenneth

2016-01-01

This project is to develop a novel aerostructure concept that takes advantage of emerging digital composite materials and manufacturing methods to build high stiffness-to-density ratio, ultra-light structures that can provide mission adaptive and aerodynamically efficient future N+3N+4 air vehicles.

40 CFR 60.617 - Chemicals affected by subpart III.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 7 2014-07-01 2014-07-01 false Chemicals affected by subpart III. 60... Compound (VOC) Emissions From the Synthetic Organic Chemical Manufacturing Industry (SOCMI) Air Oxidation Unit Processes § 60.617 Chemicals affected by subpart III. Chemical name CAS No.* Acetaldehyde 75-07-0...

40 CFR 60.617 - Chemicals affected by subpart III.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 7 2012-07-01 2012-07-01 false Chemicals affected by subpart III. 60... Compound (VOC) Emissions From the Synthetic Organic Chemical Manufacturing Industry (SOCMI) Air Oxidation Unit Processes § 60.617 Chemicals affected by subpart III. Chemical name CAS No.* Acetaldehyde 75-07-0...

40 CFR 60.617 - Chemicals affected by subpart III.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 7 2013-07-01 2013-07-01 false Chemicals affected by subpart III. 60... Compound (VOC) Emissions From the Synthetic Organic Chemical Manufacturing Industry (SOCMI) Air Oxidation Unit Processes § 60.617 Chemicals affected by subpart III. Chemical name CAS No.* Acetaldehyde 75-07-0...

Childhood apraxia of speech: A survey of praxis and typical speech characteristics.

PubMed

Malmenholt, Ann; Lohmander, Anette; McAllister, Anita

2017-07-01

The purpose of this study was to investigate current knowledge of the diagnosis childhood apraxia of speech (CAS) in Sweden and compare speech characteristics and symptoms to those of earlier survey findings in mainly English-speakers. In a web-based questionnaire 178 Swedish speech-language pathologists (SLPs) anonymously answered questions about their perception of typical speech characteristics for CAS. They graded own assessment skills and estimated clinical occurrence. The seven top speech characteristics reported as typical for children with CAS were: inconsistent speech production (85%), sequencing difficulties (71%), oro-motor deficits (63%), vowel errors (62%), voicing errors (61%), consonant cluster deletions (54%), and prosodic disturbance (53%). Motor-programming deficits described as lack of automatization of speech movements were perceived by 82%. All listed characteristics were consistent with the American Speech-Language-Hearing Association (ASHA) consensus-based features, Strand's 10-point checklist, and the diagnostic model proposed by Ozanne. The mode for clinical occurrence was 5%. Number of suspected cases of CAS in the clinical caseload was approximately one new patient/year and SLP. The results support and add to findings from studies of CAS in English-speaking children with similar speech characteristics regarded as typical. Possibly, these findings could contribute to cross-linguistic consensus on CAS characteristics.

Near-Infrared Emission Lines of Nova Cassiopeiae 1995

NASA Astrophysics Data System (ADS)

Rudy, R. J.; Lynch, D. K.; Mazuk, S. M.; Venturini, C. C.; Puetter, R. C.

2000-12-01

The slow nova V 723 Cas (Nova Cas 1995) exhibits comparatively narrow emission features (FWHM 500 km sec-1) that make it ideal for classifying weak lines and lines blended with stronger features. We present spectra from 0.8-2.5 microns that track the gradual incrase in excitation of Nova Cas and discuss the emission lines that were present. During the period encompassed by these observations Nova Cas reached only moderate excitation-the most energetic coronal lines were [S VIII] 9913 and [Al IX] 20444; lines such as [S IX] 12523 that are prominent in some novae were not detected. Additional coronal lines present include [Si VI] 19641, [Ca VIII] 23205, and [Si VII] 24807. New lines identified include features of [Fe V], [Fe VI]. These iron features are not coronal lines, arising from transitions among low-lying terms rather than within the ground term itself. Also detected was [Ti VI] 17151 that was first identified in V1974 Cygni (Nova Cyg 1992), and possibly [Ti VII] 22050. Accurate wavelengths for a number of unidentified lines are also presented. These unidentified features are discussed with regard to their likely level of excitation and their presence in other novae. This work was supported by the IR&D program of the Aerospace Corporation. RCP acknowledges support from NASA.

Project CHECO Southeast Asia Report. Air Operations in Northern Laos, 1 November 1970 - 1 April 1971

DTIC Science & Technology

1971-05-03

to be of sufficient size and scope to warrant a significant increase in the daily tacair sortie rate provided by Seventh Air Force. The execution of...weather, however, delayed the operation. A last-minute change of HLZs was made by CAS, but weather and faulty execution precluded proper zone...Artillery Airborne Battlefield Command and Control Center Attacks by Fire Jtq 1£4+, .. Auto Defense du Choc . Technically an obsolete tenn now. Refers

Novel Roles for Kv7 Channels in Shaping Histamine-Induced Contractions and Bradykinin-Dependent Relaxations in Pig Coronary Arteries

PubMed Central

Chen, Xingjuan; Li, Wennan; Hiett, S. Christopher; Obukhov, Alexander G.

2016-01-01

Voltage-gated Kv7 channels are inhibited by agonists of Gq-protein-coupled receptors, such as histamine. Recent works have provided evidence that inhibition of vascular Kv7 channels may trigger vessel contractions. In this study, we investigated how Kv7 activity modulates the histamine-induced contractions in “healthy” and metabolic syndrome (MetS) pig right coronary arteries (CAs). We performed isometric tension and immunohistochemical studies with domestic, lean Ossabaw, and MetS Ossabaw pig CAs. We found that neither the Kv7.2/Kv7.4/Kv7.5 activator ML213 nor the general Kv7 inhibitor XE991 altered the tension of CA rings under preload, indicating that vascular Kv7 channels are likely inactive in the preloaded rings. Conversely, ML213 potently dilated histamine-pre-contracted CAs, suggesting that Kv7 channels are activated during histamine applications and yet partially inhibited by histamine. Immunohistochemistry analysis revealed strong Kv7.4 immunostaining in the medial and intimal layers of the CA wall, whereas Kv7.5 immunostaining intensity was strong in the intimal but weak in the medial layers. The medial Kv7 immunostaining was significantly weaker in MetS Ossabaw CAs as compared to lean Ossabaw or domestic CAs. Consistently, histamine-pre-contracted MetS Ossabaw CAs exhibited attenuated ML213-dependent dilations. In domestic pig CAs, where medial Kv7 immunostaining intensity was stronger, histamine-induced contractions spontaneously decayed to ~31% of the peak amplitude within 4 minutes. Oppositely, in Ossabaw CAs, where Kv7 immunostaining intensity was weaker, the histamine-induced contractions were more sustained. XE991 pretreatment significantly slowed the decay rate of histamine-induced contractions in domestic CAs, supporting the hypothesis that increased Kv7 activity correlates with a faster rate of histamine-induced contraction decay. Alternatively, XE991 significantly decreased the amplitude of bradykinin-dependent dilations in pre-contracted CAs. We propose that in CAs, a decreased expression or a loss of function of Kv7 channels may lead to sustained histamine-induced contractions and reduced endothelium-dependent relaxation, both risk factors for coronary spasm. PMID:26844882

Whole-exome sequencing supports genetic heterogeneity in childhood apraxia of speech.

PubMed

Worthey, Elizabeth A; Raca, Gordana; Laffin, Jennifer J; Wilk, Brandon M; Harris, Jeremy M; Jakielski, Kathy J; Dimmock, David P; Strand, Edythe A; Shriberg, Lawrence D

2013-10-02

Childhood apraxia of speech (CAS) is a rare, severe, persistent pediatric motor speech disorder with associated deficits in sensorimotor, cognitive, language, learning and affective processes. Among other neurogenetic origins, CAS is the disorder segregating with a mutation in FOXP2 in a widely studied, multigenerational London family. We report the first whole-exome sequencing (WES) findings from a cohort of 10 unrelated participants, ages 3 to 19 years, with well-characterized CAS. As part of a larger study of children and youth with motor speech sound disorders, 32 participants were classified as positive for CAS on the basis of a behavioral classification marker using auditory-perceptual and acoustic methods that quantify the competence, precision and stability of a speaker's speech, prosody and voice. WES of 10 randomly selected participants was completed using the Illumina Genome Analyzer IIx Sequencing System. Image analysis, base calling, demultiplexing, read mapping, and variant calling were performed using Illumina software. Software developed in-house was used for variant annotation, prioritization and interpretation to identify those variants likely to be deleterious to neurodevelopmental substrates of speech-language development. Among potentially deleterious variants, clinically reportable findings of interest occurred on a total of five chromosomes (Chr3, Chr6, Chr7, Chr9 and Chr17), which included six genes either strongly associated with CAS (FOXP1 and CNTNAP2) or associated with disorders with phenotypes overlapping CAS (ATP13A4, CNTNAP1, KIAA0319 and SETX). A total of 8 (80%) of the 10 participants had clinically reportable variants in one or two of the six genes, with variants in ATP13A4, KIAA0319 and CNTNAP2 being the most prevalent. Similar to the results reported in emerging WES studies of other complex neurodevelopmental disorders, our findings from this first WES study of CAS are interpreted as support for heterogeneous genetic origins of this pediatric motor speech disorder with multiple genes, pathways and complex interactions. We also submit that our findings illustrate the potential use of WES for both gene identification and case-by-case clinical diagnostics in pediatric motor speech disorders.

Design and verification by nonlinear simulation of a Mach/CAS control law for the NASA TCV B737 aircraft

NASA Technical Reports Server (NTRS)

Bruce, Kevin R.

1986-01-01

A Mach/CAS control system using an elevator was designed and developed for use on the NASA TCV B737 aircraft to support research in profile descent procedures and approach energy management. The system was designed using linear analysis techniques primarily. The results were confirmed and the system validated at additional flight conditions using a nonlinear 737 aircraft simulation. All design requirements were satisfied.

A Diagnostic Marker to Discriminate Childhood Apraxia of Speech from Speech Delay: III. Theoretical Coherence of the Pause Marker with Speech Processing Deficits in Childhood Apraxia of Speech

ERIC Educational Resources Information Center

Shriberg, Lawrence D.; Strand, Edythe A.; Fourakis, Marios; Jakielski, Kathy J.; Hall, Sheryl D.; Karlsson, Heather B.; Mabie, Heather L.; McSweeny, Jane L.; Tilkens, Christie M.; Wilson, David L.

2017-01-01

Purpose: Previous articles in this supplement described rationale for and development of the pause marker (PM), a diagnostic marker of childhood apraxia of speech (CAS), and studies supporting its validity and reliability. The present article assesses the theoretical coherence of the PM with speech processing deficits in CAS. Method: PM and other…

Embracing uncertainty, managing complexity: applying complexity thinking principles to transformation efforts in healthcare systems.

PubMed

Khan, Sobia; Vandermorris, Ashley; Shepherd, John; Begun, James W; Lanham, Holly Jordan; Uhl-Bien, Mary; Berta, Whitney

2018-03-21

Complexity thinking is increasingly being embraced in healthcare, which is often described as a complex adaptive system (CAS). Applying CAS to healthcare as an explanatory model for understanding the nature of the system, and to stimulate changes and transformations within the system, is valuable. A seminar series on systems and complexity thinking hosted at the University of Toronto in 2016 offered a number of insights on applications of CAS perspectives to healthcare that we explore here. We synthesized topics from this series into a set of six insights on how complexity thinking fosters a deeper understanding of accepted ideas in healthcare, applications of CAS to actors within the system, and paradoxes in applications of complexity thinking that may require further debate: 1) a complexity lens helps us better understand the nebulous term "context"; 2) concepts of CAS may be applied differently when actors are cognizant of the system in which they operate; 3) actor responses to uncertainty within a CAS is a mechanism for emergent and intentional adaptation; 4) acknowledging complexity supports patient-centred intersectional approaches to patient care; 5) complexity perspectives can support ways that leaders manage change (and transformation) in healthcare; and 6) complexity demands different ways of implementing ideas and assessing the system. To enhance our exploration of key insights, we augmented the knowledge gleaned from the series with key articles on complexity in the literature. Ultimately, complexity thinking acknowledges the "messiness" that we seek to control in healthcare and encourages us to embrace it. This means seeing challenges as opportunities for adaptation, stimulating innovative solutions to ensure positive adaptation, leveraging the social system to enable ideas to emerge and spread across the system, and even more important, acknowledging that these adaptive actions are part of system behaviour just as much as periods of stability are. By embracing uncertainty and adapting innovatively, complexity thinking enables system actors to engage meaningfully and comfortably in healthcare system transformation.

Army Fixed-Wing Ground Attack Aircraft: A Historical Precedent and Contemporary Rationale

DTIC Science & Technology

2015-06-12

platforms at the tactical level. Fielding such aircraft would free the Air Force to focus on its broader missions while enhancing the capabilities of...ground forces. In fact, an Army attack aircraft would reduce, but not eliminate, the requirement for USAF CAS, freeing the USAF to focus on its

40 CFR Table 6 to Subpart Jj of... - VHAP of Potential Concern

Code of Federal Regulations, 2014 CFR

2014-07-01

... Subpart JJ of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS...) National Emission Standards for Wood Furniture Manufacturing Operations Pt. 63, Subpt. JJ, Table 6 Table 6 to Subpart JJ of Part 63—VHAP of Potential Concern CAS No. Chemical name EPA de minimis, tons/yr...

40 CFR Table 6 to Subpart Jj of... - VHAP of Potential Concern

Code of Federal Regulations, 2012 CFR

2012-07-01

... Subpart JJ of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS...) National Emission Standards for Wood Furniture Manufacturing Operations Pt. 63, Subpt. JJ, Table 6 Table 6 to Subpart JJ of Part 63—VHAP of Potential Concern CAS No. Chemical name EPA de minimis, tons/yr...

40 CFR Table 6 to Subpart Jj of... - VHAP of Potential Concern

Code of Federal Regulations, 2013 CFR

2013-07-01

... Subpart JJ of Part 63 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS...) National Emission Standards for Wood Furniture Manufacturing Operations Pt. 63, Subpt. JJ, Table 6 Table 6 to Subpart JJ of Part 63—VHAP of Potential Concern CAS No. Chemical name EPA de minimis, tons/yr...

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

Rodgers, A J; Petersson, N A; Morency, C E

The California Academy of Sciences (CAS) Morrison Planetarium is producing a 'full-dome' planetarium show on earthquakes and asked LLNL to produce content for the show. Specifically the show features numerical ground motion simulations of the M 7.9 1906 San Francisco and a possible future M 7.05 Hayward fault scenario earthquake. The show also features concepts of plate tectonics and mantle convection using images from LLNL's G3D global seismic tomography. This document describes the data that was provided to the CAS in support of production of the 'Earthquake' show. The CAS is located in Golden Gate Park, San Francisco and hostsmore » over 1.6 million visitors. The Morrison Planetarium, within the CAS, is the largest all digital planetarium in the world. It features a 75-foot diameter spherical section projection screen tilted at a 30-degree angle. Six projectors cover the entire field of view and give a three-dimensional immersive experience. CAS shows strive to use scientifically accurate digital data in their productions. The show, entitled simply 'Earthquake', will debut on 26 May 2012. They are working on graphics and animations based on the same data sets for display on LLNL powerwalls and flat-screens as well as for public release.« less

CRISPR-Cas9 Mediated Telomere Removal Leads to Mitochondrial Stress and Protein Aggregation.

PubMed

Kim, Hyojung; Ham, Sangwoo; Jo, Minkyung; Lee, Gum Hwa; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

2017-10-03

Aging is considered the major risk factor for neurodegenerative diseases including Parkinson's disease (PD). Telomere shortening is associated with cellular senescence. In this regard, pharmacological or genetic inhibition of telomerase activity has been used to model cellular aging. Here, we employed CRISPR-Cas9 technology to instantly remove the telomere to induce aging in a neuroblastoma cell line. Expression of both Cas9 and guide RNA targeting telomere repeats ablated the telomere, leading to retardation of cell proliferation. Instant deletion of telomere in SH-SY5Y cells impaired mitochondrial function with diminished mitochondrial respiration and cell viability. Supporting the pathological relevance of cell aging by CRISPR-Cas9 mediated telomere removal, alterations were observed in the levels of PD-associated proteins including PTEN-induced putative kinase 1, peroxisome proliferator-activated receptor γ coactivator 1-α, nuclear respiratory factor 1, parkin, and aminoacyl tRNA synthetase complex interacting multifunctional protein 2. Significantly, α-synuclein expression in the background of telomere removal led to the enhancement of protein aggregation, suggesting positive feed-forward interaction between aging and PD pathogenesis. Collectively, our results demonstrate that CRISPR-Cas9 can be used to efficiently model cellular aging and PD.

Effets de la pollution de l’air sur la santé

PubMed Central

Abelsohn, Alan; Stieb, Dave M.

2011-01-01

Résumé Objectif Faire connaître aux médecins de famille les effets de la pollution atmosphérique sur la santé et indiquer quels conseils donner aux patients vulnérables pour qu’ils soient moins exposés. Sources de l’information On a consulté MEDLINE à l’aide des termes relatifs à la pollution atmosphérique et à ses effets indésirables. On a révisé les articles en anglais publiés entre janvier 2008 et décembre 2009. La plupart des études contenaient des preuves de niveau II. Principal message Au Canada, la pollution de l’air extérieur cause une morbidité et une mortalité importantes. Elle peut affecter le système respiratoire (exacerbation de l’asthme et de la maladie pulmonaire obstructive chronique) et le système cardiovasculaire (déclencher l’arythmie, l’insuffisance cardiaque et les AVC). La cote air santé (CAS) est un nouvel outil de communication mis au point par Santé Canada et Environnement Canada qui indique sur une échelle de 1 à 10, le risque pour la santé causé par la pollution atmosphérique. La CAS est largement diffusée dans les médias et cet outil pourrait être utile au médecin de famille pour inciter les patients à haut risque (comme ceux qui souffrent d’asthme, de maladie pulmonaire obstructive chronique ou d’insuffisance cardiaque) à réduire leur exposition à la pollution atmosphérique. Conclusion Le médecin de famille peut se servir de la CAS et de ses messages sur la santé pour enseigner aux asthmatiques et aux autres patients à risque élevé la façon de réduire les risques pour la santé causés par la pollution atmosphérique.

ESnet authentication services and trust federations

NASA Astrophysics Data System (ADS)

Muruganantham, Dhivakaran; Helm, Mike; Genovese, Tony

2005-01-01

ESnet provides authentication services and trust federation support for SciDAC projects, collaboratories, and other distributed computing applications. The ESnet ATF team operates the DOEGrids Certificate Authority, available to all DOE Office of Science programs, plus several custom CAs, including one for the National Fusion Collaboratory and one for NERSC. The secure hardware and software environment developed to support CAs is suitable for supporting additional custom authentication and authorization applications that your program might require. Seamless, secure interoperation across organizational and international boundaries is vital to collaborative science. We are fostering the development of international PKI federations by founding the TAGPMA, the American regional PMA, and the worldwide IGTF Policy Management Authority (PMA), as well as participating in European and Asian regional PMAs. We are investigating and prototyping distributed authentication technology that will allow us to support the "roaming scientist" (distributed wireless via eduroam), as well as more secure authentication methods (one-time password tokens).

Comprehensive protocols for CRISPR/Cas9-based gene editing in human pluripotent stem cells

PubMed Central

Santos, David P.; Kiskinis, Evangelos; Eggan, Kevin; Merkle, Florian T.

2016-01-01

Application of the CRISPR/Cas9 system to edit the genomes of human pluripotent stem cells (hPSCs) has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. PMID:27532820

RIFM fragrance ingredient safety assessment, linalyl cinnamate, CAS Registry Number 78-37-5.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Penning, T M; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic nor does it have skin sensitization potential. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (0.03 and 1.4 mg/day, respectively). The developmental toxicity endpoint was completed using linalool (CAS # 78-70-6), dehydrolinalool (CAS # 29171-20-8) and cinnamic acid (CAS # 621-82-9) as suitable read across analogs, which provided a MOE > 100. The repeated dose toxicity endpoint was completed using data on the target material which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

Direct measurements of conductivity and mobility in millimeter-sized single-crystalline graphene via van der Pauw geometry

NASA Astrophysics Data System (ADS)

Ma, Rui-Song; Huan, Qing; Wu, Liang-Mei; Yan, Jia-Hao; Zhang, Yu-Yang; Bao, Li-Hong; Liu, Yun-Qi; Du, Shi-Xuan; Gao, Hong-Jun

2017-06-01

Not Available Project supported by the Science Fund from the Ministry of Science and Technology of China (Grant No. 2013CBA01600), the National Key Research & Development Project of China (Grant No. 2016YFA0202300), the National Natural Science Foundation of China (Grant Nos. 61474141, 61674170, 61335006, 61390501, 51325204, and 51210003), the Chinese Academy of Sciences (CAS), and Youth Innovation Promotion Association of CAS (Grant No. 20150005).

[Stent and surgery for symptomatic carotid stenosis. SPACE study results].

PubMed

Ringleb, P A; Hacke, W

2007-10-01

The SPACE trial compared risk and effectiveness of stent-supported angioplasty (CAS) vs carotid endarterectomy (CEA) using a noninferiority design in patients with symptomatic stenoses. Intention-to-treat analysis of the entire study population of 1,214 patients showed that primary endpoint events (ipsilateral stroke or death between randomisation and day 30) occurred in 6.92% of the CAS group and 6.45% of the CEA group. The 95% confidence interval (CI) of the absolute risk difference ranged from -1.94% to +2.87%, therefore the noninferiority was not proven. The same was true for the analysis of protocols. No significant differences between the two treatment methods were found in primary or any of the secondary endpoints. There were also no differences in short-term prevention. The endpoint 'ipsilateral ischemic stroke or vascular death between randomisation and 6 months' occurred in 7.4% of the CAS and 6.5% of the CEA patients (odds ratio 1.16, 95% confidence interval 0.74-1.82). Instent restenoses were significantly more common in the CAS group (4.6% vs 2.2%, odds ratio 2.14, 95% CI 1.10-4.18). Surgery remains the gold standard in treatment of patients with symptomatic carotid artery stenosis. Stent-supported angioplasty can be an alternative only in the hands of an experienced interventionalist with proven low periprocedural complication rate.

AFTI/F-16

NASA Technical Reports Server (NTRS)

1991-01-01

The AFTI F-16 flying at high angle of attack, shown in the final configuration and paint finish. Dummy Sidewinder air-to-air missles are attached to the wing tips. The white objects visible on the wing racks represent practice bomb dispensers, used in weapon tests. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

AFTI/F-16 in banked flight

NASA Technical Reports Server (NTRS)

1989-01-01

This photo depicts the AFTI F-16 in the configuration used midway through the program. The sensor pods were added to the fuselage, but the chin canards remained in place. Painted in non-standard gray tones, it carried Sidewinder air-to-air missles on its wingtips. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

AFTI/F-16

NASA Technical Reports Server (NTRS)

1992-01-01

The AFTI F-16 in its final configuration, flying in the vicinity of Edwards Air Force Base, California. During this phase, the two forward infrared turrets were added ahead of the cockpit, the chin canards were removed, and the aircraft was repainted in a standard Air Force scheme. A fuel drop tank is visible below the wing. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

Regulation of expression and biochemical characterization of a beta-class carbonic anhydrase from the plant growth-promoting rhizobacterium, Azospirillum brasilense Sp7.

PubMed

Kaur, Simarjot; Mishra, Mukti Nath; Tripathi, Anil K

2009-10-01

Carbonic anhydrase (CA; [EC 4.2.1.1]) is a ubiquitous enzyme catalysing the reversible hydration of CO(2) to bicarbonate, a reaction that supports various biochemical and physiological functions. Genome analysis of Azospirillum brasilense, a nonphotosynthetic, nitrogen-fixing, rhizobacterium, revealed an ORF with homology to beta-class carbonic anhydrases (CAs). Biochemical characteristics of the beta-class CA of A. brasilense, analysed after cloning the gene (designated as bca), overexpressing in Escherichia coli and purifying the protein by affinity purification, revealed that the native recombinant enzyme is a homotetramer, inhibited by the known CA inhibitors. CA activity in A. brasilense cell extracts, reverse transcriptase (RT)-PCR and Western blot analyses showed that bca was constitutively expressed under aerobic conditions. Lower beta-galactosidase activity in A. brasilense cells harbouring bca promoter: lacZ fusion during the stationary phase or during growth on 3% CO(2) enriched air or at acidic pH indicated that the transcription of bca was downregulated by the stationary phase, elevated CO(2) levels and acidic pH conditions. These observations were also supported by RT-PCR analysis. Thus, beta-CA in A. brasilense seems to be required for scavenging CO(2) from the ambient air and the requirement of CO(2) hydration seems to be higher for the cultures growing exponentially at neutral to alkaline pH.

Defense Logistics: Enhanced Policy and Procedures Needed to Improve Management of Sensitive Conventional Ammunition

DTIC Science & Technology

2016-02-01

Information System-Retail OIS-MC Ordnance Information System-Marine Corps SAAS -MOD Standard Army Ammunition System-Modernization SRC Security Risk...automated information systems. For the Army we used LMP, SAAS , and WARS-NT; for the Navy and Marine Corps we used OIS-W, OIS-R, and OIS-MC; and for the Air...Army we used LMP, SAAS , and WARS-NT; for the Navy and Marine Corps we used OIS-W, OIS-R, and OIS-MC; and for the Air Force we used CAS. Military

40 CFR Appendix to Subpart Cc of... - Tables

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 11 2013-07-01 2013-07-01 false Tables Appendix to Subpart CC of Part... to Subpart CC of Part 63—Tables Table 1—Hazardous Air Pollutants Chemical name CAS No.a Benzene 71432... salts, esters, or derivatives. Table 2—Leak Definitions for Pumps and Valves Standard a Phase Leak...

40 CFR Appendix to Subpart Cc of... - Tables

Code of Federal Regulations, 2012 CFR

2012-07-01

... 40 Protection of Environment 11 2012-07-01 2012-07-01 false Tables Appendix to Subpart CC of Part... to Subpart CC of Part 63—Tables Table 1—Hazardous Air Pollutants Chemical name CAS No.a Benzene 71432... salts, esters, or derivatives. Table 2—Leak Definitions for Pumps and Valves Standard a Phase Leak...

40 CFR Appendix to Subpart Cc of... - Tables

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 11 2014-07-01 2014-07-01 false Tables Appendix to Subpart CC of Part... to Subpart CC of Part 63—Tables Table 1—Hazardous Air Pollutants Chemical name CAS No.a Benzene 71432... salts, esters, or derivatives. Table 2—Leak Definitions for Pumps and Valves Standard a Phase Leak...

40 CFR 721.10057 - Dodecanedioic acid, 1, 12-dihydrazide.

Code of Federal Regulations, 2010 CFR

2010-07-01

... significant new uses subject to reporting. (1) The chemical substance identified as dodecanedioic acid, 1, 12-dihydrazide (PMNs P-01-759 and P-05-555; CAS No. 4080-98-2) is subject to reporting under this section for the... respirators meet the minimum requirement for § 721.63(a)(4): Air-purifying, tight-fitting full-face respirator...

Comprehensive Protocols for CRISPR/Cas9-based Gene Editing in Human Pluripotent Stem Cells.

PubMed

Santos, David P; Kiskinis, Evangelos; Eggan, Kevin; Merkle, Florian T

2016-08-17

Genome editing of human pluripotent stem cells (hPSCs) with the CRISPR/Cas9 system has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Developing an index to measure the voluntariness of consent to research.

PubMed

Dugosh, Karen L; Festinger, David S; Marlowe, Douglas B; Clements, Nicolle T

2014-10-01

The goals of the current study were to expand the content domain and further validate the Coercion Assessment Scale (CAS), a measure of perceived coercion for criminally involved substance abusers being recruited into research. Unlike the few existing measures of this construct, the CAS identifies specific external sources of pressure that may influence one's decision to participate. In Phase 1, we conducted focus groups with criminal justice clients and stakeholders to expand the instrument by identifying additional sources of pressure. In Phase 2, we evaluated the expanded measure (i.e., endorsement rates, reliability, validity) in an ongoing research trial. Results identified new sources of pressure and provided evidence supporting the CAS's utility and reliability over time as well as convergent and discriminative validity. © The Author(s) 2014.

RIFM fragrance ingredient safety assessment, p-Isopropylbenzyl acetate, CAS Registry Number 59230-57-8.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current use conditions is supported by the existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental toxicity, reproductive toxicity, local respiratory toxicity, phototoxicity, skin sensitization, as well as environmental safety. Data from the suitable read across analog, benzyl acetate (CAS # 140-11-4), show that this material is not genotoxic nor does it have skin sensitization potential. The repeated dose, developmental and reproductive, and local respiratory toxicity endpoints were completed using benzyl acetate (CAS # 140-11-4) as a suitable read across analog, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System

PubMed Central

Cooper, Lauren A.; Stringer, Anne M.

2018-01-01

ABSTRACT In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo, for the type I-E system of Escherichia coli. Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5′ end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. PMID:29666291

Active and adaptive Legionella CRISPR-Cas reveals a recurrent challenge to the pathogen.

PubMed

Rao, Chitong; Guyard, Cyril; Pelaz, Carmen; Wasserscheid, Jessica; Bondy-Denomy, Joseph; Dewar, Ken; Ensminger, Alexander W

2016-10-01

Clustered regularly interspaced short palindromic repeats with CRISPR-associated gene (CRISPR-Cas) systems are widely recognized as critical genome defense systems that protect microbes from external threats such as bacteriophage infection. Several isolates of the intracellular pathogen Legionella pneumophila possess multiple CRISPR-Cas systems (type I-C, type I-F and type II-B), yet the targets of these systems remain unknown. With the recent observation that at least one of these systems (II-B) plays a non-canonical role in supporting intracellular replication, the possibility remained that these systems are vestigial genome defense systems co-opted for other purposes. Our data indicate that this is not the case. Using an established plasmid transformation assay, we demonstrate that type I-C, I-F and II-B CRISPR-Cas provide protection against spacer targets. We observe efficient laboratory acquisition of new spacers under 'priming' conditions, in which initially incomplete target elimination leads to the generation of new spacers and ultimate loss of the invasive DNA. Critically, we identify the first known target of L. pneumophila CRISPR-Cas: a 30 kb episome of unknown function whose interbacterial transfer is guarded against by CRISPR-Cas. We provide evidence that the element can subvert CRISPR-Cas by mutating its targeted sequences - but that primed spacer acquisition may limit this mechanism of escape. Rather than generally impinging on bacterial fitness, this element drives a host specialization event - with improved fitness in Acanthamoeba but a reduced ability to replicate in other hosts and conditions. These observations add to a growing body of evidence that host range restriction can serve as an existential threat to L. pneumophila in the wild. © 2016 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

PubMed

Yiu, Glenn; Tieu, Eric; Nguyen, Anthony T; Wong, Brittany; Smit-McBride, Zeljka

2016-10-01

To employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells. CRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay. Five gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected. The CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

CRISPR/Cas9-mediated genome editing of Epstein-Barr virus in human cells.

PubMed

Yuen, Kit-San; Chan, Chi-Ping; Wong, Nok-Hei Mickey; Ho, Chau-Ha; Ho, Ting-Hin; Lei, Ting; Deng, Wen; Tsao, Sai Wah; Chen, Honglin; Kok, Kin-Hang; Jin, Dong-Yan

2015-03-01

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a highly efficient and powerful tool for RNA-guided editing of the cellular genome. Whether CRISPR/Cas9 can also cleave the genome of DNA viruses such as Epstein-Barr virus (EBV), which undergo episomal replication in human cells, remains to be established. Here, we reported on CRISPR/Cas9-mediated editing of the EBV genome in human cells. Two guide RNAs (gRNAs) were used to direct a targeted deletion of 558 bp in the promoter region of BART (BamHI A rightward transcript) which encodes viral microRNAs (miRNAs). Targeted editing was achieved in several human epithelial cell lines latently infected with EBV, including nasopharyngeal carcinoma C666-1 cells. CRISPR/Cas9-mediated editing of the EBV genome was efficient. A recombinant virus with the desired deletion was obtained after puromycin selection of cells expressing Cas9 and gRNAs. No off-target cleavage was found by deep sequencing. The loss of BART miRNA expression and activity was verified, supporting the BART promoter as the major promoter of BART RNA. Although CRISPR/Cas9-mediated editing of the multicopy episome of EBV in infected HEK293 cells was mostly incomplete, viruses could be recovered and introduced into other cells at low m.o.i. Recombinant viruses with an edited genome could be further isolated through single-cell sorting. Finally, a DsRed selectable marker was successfully introduced into the EBV genome during the course of CRISPR/Cas9-mediated editing. Taken together, our work provided not only the first genetic evidence that the BART promoter drives the expression of the BART transcript, but also a new and efficient method for targeted editing of EBV genome in human cells. © 2015 The Authors.

A motor speech assessment for children with severe speech disorders: reliability and validity evidence.

PubMed

Strand, Edythe A; McCauley, Rebecca J; Weigand, Stephen D; Stoeckel, Ruth E; Baas, Becky S

2013-04-01

In this article, the authors report reliability and validity evidence for the Dynamic Evaluation of Motor Speech Skill (DEMSS), a new test that uses dynamic assessment to aid in the differential diagnosis of childhood apraxia of speech (CAS). Participants were 81 children between 36 and 79 months of age who were referred to the Mayo Clinic for diagnosis of speech sound disorders. Children were given the DEMSS and a standard speech and language test battery as part of routine evaluations. Subsequently, intrajudge, interjudge, and test-retest reliability were evaluated for a subset of participants. Construct validity was explored for all 81 participants through the use of agglomerative cluster analysis, sensitivity measures, and likelihood ratios. The mean percentage of agreement for 171 judgments was 89% for test-retest reliability, 89% for intrajudge reliability, and 91% for interjudge reliability. Agglomerative hierarchical cluster analysis showed that total DEMSS scores largely differentiated clusters of children with CAS vs. mild CAS vs. other speech disorders. Positive and negative likelihood ratios and measures of sensitivity and specificity suggested that the DEMSS does not overdiagnose CAS but sometimes fails to identify children with CAS. The value of the DEMSS in differential diagnosis of severe speech impairments was supported on the basis of evidence of reliability and validity.

Spectra of Cas A's Highest Velocity Ejecta

NASA Astrophysics Data System (ADS)

Fesen, Robert A.; Milisavljevic, Dan

2010-08-01

The young age and close distance of the Galactic supernova remnant Cassiopeia A (Cas A) make it perhaps our best case study and clearest look at the explosion dynamics of a core-collapse supernova (CCSN). Interestingly, Cas A exhibits two nearly opposing streams of high velocity ejecta or `jets' in its NE and SW regions racing outward at speeds more than twice that of the main shell. The nature of these jets, however, and their possible association with an aspherical supernova explosion mechanism is controversial. A handful of existing low-resolution spectra of outer knots in the NE jet display chemical abundances hinting at an origin from the S-Si-Ca- Ar rich layer deep inside the progenitor. If these abundances could be firmly established in both the NE and SW jets, it would be very strong evidence in support of a highly asymmetrical explosion engine for Cas A's progenitor and, in turn, for CCSNe in general. We request KPNO 4m telescope + MARS time to obtain high quality multi-object spectroscopy of Cas A's highest velocity ejecta to measure their nitrogen, sulfur, oxygen, calcium, and argon abundances. These spectra will be analyzed with the metal-rich shock models of J. Raymond and then compared to current sets of CCSN models paying particular attention to knot composition vs. ejection velocity and ejecta mixing.

Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.

PubMed

Gao, Hong; Prasad, G L; Zacharias, Wolfgang

2014-05-01

The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Serum levels of chemerin, apelin, vaspin, and omentin-1 in obese type 2 diabetic Egyptian patients with coronary artery stenosis.

PubMed

Motawi, Tarek M K; Mahdy, Soliman G; El-Sawalhi, Maha M; Ali, Eman N; El-Telbany, Rania Farag A

2018-01-01

Cardiovascular diseases (CVD) are the leading cause of death in the diabetic population. Obesity is a serious problem that has been linked with CVD and diabetes via a variety of adipokines. The aims of this study were to evaluate and correlate circulating chemerin, apelin, vaspin, and omentin-1 levels in obese type 2 diabetic Egyptian patients with coronary artery stenosis (CAS), and to assess their usefulness as noninvasive diagnostic biomarkers. Chemerin, apelin, vaspin, and omentin-1 levels were determined by enzyme immunoassay in coronary artery disease (CAD) I patients (45 non-obese, nondiabetic with CAS), CAD II patients (45 obese, diabetic with CAS), and 30 controls. Patients in CAD I and CAD II groups exhibited higher levels of chemerin and apelin together with lower levels of vaspin and omentin-1 than in controls. These alterations were more significant in CAD II than in CAD I patients. Additionally, adipokine levels were individually correlated with each other and with certain biochemical variables. Moreover, chemerin and vaspin levels could differentiate CAD II patients from CAD I and controls. Alterations of these adipokines may play a crucial role in the pathogenesis of CAS in obese type 2 diabetic Egyptian patients. Chemerin and vaspin could be used as markers to support diagnosis of CAS.

Dietary Whole Egg Consumption Attenuates Body Weight Gain and Is More Effective than Supplemental Cholecalciferol in Maintaining Vitamin D Balance in Type 2 Diabetic Rats.

PubMed

Saande, Cassondra J; Jones, Samantha K; Hahn, Kaylee E; Reed, Carter H; Rowling, Matthew J; Schalinske, Kevin L

2017-09-01

Background: Type 2 diabetes (T2D) is characterized by vitamin D insufficiency owing to excessive urinary loss of 25-hydroxycholecalciferol [25(OH)D]. We previously reported that a diet containing dried whole egg, a rich source of vitamin D, was effective at maintaining circulating 25(OH)D concentrations in rats with T2D. Furthermore, whole egg consumption reduced body weight gain in rats with T2D. Objective: This study was conducted to compare whole egg consumption with supplemental cholecalciferol with respect to vitamin D balance, weight gain, and body composition in rats with T2D. Methods: Male Zucker diabetic fatty (ZDF) rats ( n = 24) and their lean controls ( n = 24) were obtained at 5 wk of age and randomly assigned to 3 treatment groups: a casein-based diet (CAS), a dried whole egg-based diet (WE), or a casein-based diet containing supplemental cholecalciferol (CAS+D) at the same amount of cholecalciferol provided by WE (37.6 μg/kg diet). Rats were fed their respective diets for 8 wk. Weight gain and food intake were measured daily, circulating 25(OH)D concentrations were measured by ELISA, and body composition was analyzed by dual X-ray absorptiometry. Results: Weight gain and percentage of body fat were reduced by ∼20% and 11%, respectively, in ZDF rats fed WE compared with ZDF rats fed CAS or CAS+D. ZDF rats fed CAS had 21% lower serum 25(OH)D concentrations than lean rats fed CAS. In ZDF rats, WE consumption increased serum 25(OH)D concentrations 130% compared with CAS, whereas consumption of CAS+D increased serum 25(OH)D concentrations 35% compared with CAS. Conclusions: Our data suggest that dietary consumption of whole eggs is more effective than supplemental cholecalciferol in maintaining circulating 25(OH)D concentrations in rats with T2D. Moreover, whole egg consumption attenuated weight gain and reduced percentage of body fat in ZDF rats. These data may support new dietary recommendations targeting the prevention of vitamin D insufficiency in T2D. © 2017 American Society for Nutrition.

Unique Sensor Plane Maps Invisible Toxins for First Responders

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

Kroutil, Robert; Thomas, Mark; Aten, Keith

A unique airborne emergency response tool, ASPECT is a Los Alamos/U.S. Environmental Protection Agency project that can put chemical and radiological mapping tools in the air over an accident scene. The name ASPECT is an acronym for Airborne Spectral Photometric Environmental Collection Technology. Update, Sept. 19, 2008: Flying over storm-damaged refineries and chemical factories, a twin-engine plane carrying the ASPECT (Airborne Spectral Photometric Environmental Collection Technology) system has been on duty throughout the recent hurricanes that have swept the Florida and Gulf Coast areas. ASPECT is a project of the U.S. U.S. Environmental Protection Agencys National Decontamination Team. Los Alamosmore » National Laboratory leads a science and technology program supporting the EPA and the ASPECT aircraft. Casting about with a combination of airborne photography and infrared spectroscopy, the highly instrumented plane provides emergency responders on the ground with a clear concept of where danger lies, and the nature of the sometimes-invisible plumes that could otherwise kill them. ASPECT is the nations only 24/7 emergency response aircraft with chemical plume mapping capability. Bob Kroutil of Bioscience Division is the project leader, and while he said the team has put in long hours, both on the ground and in the air, its a worthwhile effort. The plane flew over 320 targeted sites in four days, he noted. Prior to the deployment to the Gulf Coast, the plane had been monitoring the Democratic National Convention in Denver, Colorado. Los Alamos National Laboratory Divisions that are supporting ASPECT include, in addition to B-Division, CTN-5: Networking Engineering and IRM-CAS: Communication, Arts, and Services. Leslie Mansell, CTN-5, and Marilyn Pruitt, IRM-CAS, were recognized the the U.S. EPA for their outstanding support to the hurricane response of Gustav in Louisiana and Ike in Texas. The information from the data collected in the most recent event, Hurricane Ike, was sent to the EPA Region 6 Rapid Needs Assessment and the State of Texas Joint Field Office in Austin, Texas. It appears that though there is considerable damage in Galveston and Texas City, there are fewer chemical leaks than during either hurricanes Katrina or Rita. Specific information gathered from the data was reported out to the U.S. Environmental Protection Agency Headquarters, the Federal Emergency Management Agency, the Department of Homeland Security, and the State of Texas Emergency Management Agency.« less

Factors related to the development of health-promoting community activities in Spanish primary healthcare: two case–control studies

PubMed Central

March, Sebastià; Ripoll, Joana; Jordan Martin, Matilde; Zabaleta-del-Olmo, Edurne; Benedé Azagra, Carmen Belén; Elizalde Soto, Lázaro; Vidal, Mª Clara; Bauzà Amengual, María de Lluc; Planas Juan, Trinidad; Pérez Mariano, Damiana Maria; Llull Sarralde, Micaela; Ruiz-Giménez, Juan Luís; Bajo Viñas, Rosa; Solano Villarubia, Carmen; Rodriguez Bajo, Maria; Cordoba Victoria, Manuela; Badia Capdevila, Marta; Serrano Ferrandez, Elena; Bosom Diumenjo, Maria; Montaner-Gomis, Isabel; Bolibar-Ribas, Buenaventura; Antoñanzas Lombarte, Angel; Bregel Cotaina, Samantha; Calvo Tocado, Ana; Olivan Blázquez, Barbara; Magallon Botaya, Rosa; Marín Palacios, Pilar; Echauri Ozcoidi, Margarita; Perez - arauta, María Jose; Llobera, Joan; Ramos, Maria

2017-01-01

Objective Spanish primary healthcare teams have the responsibility of performing health-promoting community activities (CAs), although such activities are not widespread. Our aim was to identify the factors related to participation in those activities. Design Two case–control studies. Setting Performed in primary care of five Spanish regions. Subjects In the first study, cases were teams that performed health-promoting CAs and controls were those that did not. In the second study (on case teams from the first study), cases were professionals who developed these activities and controls were those who did not. Main outcome measures Team, professional and community characteristics collected through questionnaires (team managers/professionals) and from secondary sources. Results The first study examined 203 teams (103 cases, 100 controls). Adjusted factors associated with performing CAs were percentage of nurses (OR 1.07, 95% CI 1.01 to 1.14), community socioeconomic status (higher vs lower OR 2.16, 95% CI 1.18 to 3.95) and performing undergraduate training (OR 0.44, 95% CI 0.21 to 0.93). In the second study, 597 professionals responded (254 cases, 343 controls). Adjusted factors were professional classification (physicians do fewer activities than nurses and social workers do more), training in CAs (OR 1.9, 95% CI 1.2 to 3.1), team support (OR 2.9, 95% CI 1.5 to 5.7), seniority (OR 1.06, 95% CI 1.03 to 1.09), nursing tutor (OR 2.0, 95% CI 1.1 to 3.5), motivation (OR 3.7, 95% CI 1.8 to 7.5), collaboration with non-governmental organisations (OR 1.9, 95% CI 1.2 to 3.1) and participation in neighbourhood activities (OR 3.1, 95% CI 1.9 to 5.1). Conclusions Professional personal characteristics, such as social sensitivity, profession, to feel team support or motivation, have influence in performing health-promoting CAs. In contrast to the opinion expressed by many professionals, workload is not related to performance of health-promoting CAs. PMID:28993380

Determining the Specificity of Cascade Binding, Interference, and Primed Adaptation In Vivo in the Escherichia coli Type I-E CRISPR-Cas System.

PubMed

Cooper, Lauren A; Stringer, Anne M; Wade, Joseph T

2018-04-17

In clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) immunity systems, short CRISPR RNAs (crRNAs) are bound by Cas proteins, and these complexes target invading nucleic acid molecules for degradation in a process known as interference. In type I CRISPR-Cas systems, the Cas protein complex that binds DNA is known as Cascade. Association of Cascade with target DNA can also lead to acquisition of new immunity elements in a process known as primed adaptation. Here, we assess the specificity determinants for Cascade-DNA interaction, interference, and primed adaptation in vivo , for the type I-E system of Escherichia coli Remarkably, as few as 5 bp of crRNA-DNA are sufficient for association of Cascade with a DNA target. Consequently, a single crRNA promotes Cascade association with numerous off-target sites, and the endogenous E. coli crRNAs direct Cascade binding to >100 chromosomal sites. In contrast to the low specificity of Cascade-DNA interactions, >18 bp are required for both interference and primed adaptation. Hence, Cascade binding to suboptimal, off-target sites is inert. Our data support a model in which the initial Cascade association with DNA targets requires only limited sequence complementarity at the crRNA 5' end whereas recruitment and/or activation of the Cas3 nuclease, a prerequisite for interference and primed adaptation, requires extensive base pairing. IMPORTANCE Many bacterial and archaeal species encode CRISPR-Cas immunity systems that protect against invasion by foreign DNA. In the Escherichia coli CRISPR-Cas system, a protein complex, Cascade, binds 61-nucleotide (nt) CRISPR RNAs (crRNAs). The Cascade complex is directed to invading DNA molecules through base pairing between the crRNA and target DNA. This leads to recruitment of the Cas3 nuclease, which destroys the invading DNA molecule and promotes acquisition of new immunity elements. We made the first in vivo measurements of Cascade binding to DNA targets. Thus, we show that Cascade binding to DNA is highly promiscuous; endogenous E. coli crRNAs can direct Cascade binding to >100 chromosomal locations. In contrast, we show that targeted degradation and acquisition of new immunity elements require highly specific association of Cascade with DNA, limiting CRISPR-Cas function to the appropriate targets. Copyright © 2018 Cooper et al.

Dramatic Improvement of CRISPR/Cas9 Editing in Candida albicans by Increased Single Guide RNA Expression.

PubMed

Ng, Henry; Dean, Neta

2017-01-01

The clustered regularly interspaced short palindromic repeat system with CRISPR-associated protein 9 nuclease (CRISPR/Cas9) has emerged as a versatile tool for genome editing in Candida albicans . Mounting evidence from other model systems suggests that the intracellular levels of single guide RNA (sgRNA) limit the efficiency of Cas9-dependent DNA cleavage. Here, we tested this idea and describe a new means of sgRNA delivery that improves previously described methods by ~10-fold. The efficiency of Cas9/sgRNA-dependent cleavage and repair of a single-copy yeast enhanced monomeric red fluorescent protein ( RFP ) gene was measured as a function of various parameters that are hypothesized to affect sgRNA accumulation, including transcriptional and posttranscriptional processing. We analyzed different promoters ( SNR52 , ADH1 , and tRNA ), as well as different posttranscriptional RNA processing schemes that serve to generate or stabilize mature sgRNA with precise 5' and 3' ends. We compared the effects of flanking sgRNA with self-cleaving ribozymes or by tRNA, which is processed by endogenous RNases. These studies demonstrated that sgRNA flanked by a 5' tRNA and transcribed by a strong RNA polymerase II ADH1 promoter increased Cas9-dependent RFP mutations by 10-fold. Examination of double-strand-break (DSB) repair in strains hemizygous for RFP demonstrated that both homology-directed and nonhomologous end-joining pathways were used to repair breaks. Together, these results support the model that gRNA expression can be rate limiting for efficient CRISPR/Cas mutagenesis in C. albicans . IMPORTANCE Candida albicans is an important human fungal pathogen. An understanding of fungal virulence factors has been slow because C. albicans is genetically intractable. The recent development of CRISPR/Cas in C. albicans (V. K. Vyas, M. I. Barrasa, G. R. Fink, Sci Adv 1:e1500248, 2015, https://doi.org/10.1126/sciadv.1500248) has the potential to circumvent this problem. However, as has been found in other organisms, CRISPR/Cas mutagenesis efficiency can be frustratingly variable. Here, we systematically examined parameters hypothesized to alter sgRNA intracellular levels in order to optimize CRISPR/Cas in C. albicans . Our most important conclusion is that increased sgRNA expression and maturation dramatically improve efficiency of CRISPR/Cas mutagenesis in C. albicans by ~10-fold. Thus, we anticipate that the modifications described here will further advance the application of CRISPR/Cas for genome editing in C. albicans .

Description and Evaluation of IAP-AACM: A Global-regional Aerosol Chemistry Model for the Earth System Model CAS-ESM

NASA Astrophysics Data System (ADS)

Wei, Y.; Chen, X.

2017-12-01

We present a first description and evaluation of the IAP Atmospheric Aerosol Chemistry Model (IAP-AACM) which has been integrated into the earth system model CAS-ESM. In this way it is possible to research into interaction of clouds and aerosol by its two-way coupling with the IAP Atmospheric General Circulation Model (IAP-AGCM). The model has a nested global-regional grid based on the Global Environmental Atmospheric Transport Model (GEATM) and the Nested Air Quality Prediction Modeling System (NAQPMS). The AACM provides two optional gas chemistry schemes, the CBM-Z gas chemistry as well as a sulfur oxidize box designed specifically for the CAS-ESM. Now the model driven by AGCM has been applied to a 1-year simulation of tropospheric chemistry both on global and regional scales for 2014, and been evaluated against various observation datasets, including aerosol precursor gas concentration, aerosol mass and number concentrations. Furthermore, global budgets in AACM are compared with other global aerosol models. Generally, the AACM simulations are within the range of other global aerosol model predictions, and the model has a reasonable agreement with observations of gases and particles concentration both on global and regional scales.

The Hypothesis of Apraxia of Speech in Children with Autism Spectrum Disorder

PubMed Central

Shriberg, Lawrence D.; Paul, Rhea; Black, Lois M.; van Santen, Jan P.

2010-01-01

In a sample of 46 children aged 4 to 7 years with Autism Spectrum Disorder (ASD) and intelligible speech, there was no statistical support for the hypothesis of concomitant Childhood Apraxia of Speech (CAS). Perceptual and acoustic measures of participants’ speech, prosody, and voice were compared with data from 40 typically-developing children, 13 preschool children with Speech Delay, and 15 participants aged 5 to 49 years with CAS in neurogenetic disorders. Speech Delay and Speech Errors, respectively, were modestly and substantially more prevalent in participants with ASD than reported population estimates. Double dissociations in speech, prosody, and voice impairments in ASD were interpreted as consistent with a speech attunement framework, rather than with the motor speech impairments that define CAS. Key Words: apraxia, dyspraxia, motor speech disorder, speech sound disorder PMID:20972615

Corrective Action Investigation Plan for Corrective Action Unit 127: Areas 25 and 26 Storage Tanks, Nevada Test Site, Nevada (Rev. No.: 0, August 2002)

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

NNSA /NV

This Corrective Action Investigation Plan (CAIP) contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Offices's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 127 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 127 is located on the Nevada Test Site approximately 65 miles northwest of Las Vegas, Nevada. This CAU is comprised of 12 Corrective Action Sites (CASs) located at Test Cell C; the Engine Maintenance, Assembly, and Disassembly (E-MAD) Facility; the X-Tunnel in Area 25; the Pluto Disassembly Facility; themore » Pluto Check Station; and the Port Gaston Training Facility in Area 26. These CASs include: CAS 25-01-05, Aboveground Storage Tank (AST); CAS 25-02-02, Underground Storage Tank (UST); CAS 25-23-11, Contaminated Materials; CAS 25-12-01, Boiler; CAS 25-01-06, AST; CAS 25-01-07, AST; CAS 25-02-13, UST; CAS 26- 01-01, Filter Tank (Rad) and Piping; CAS 26-01-02, Filter Tank (Rad); CAS 26-99-01, Radioactively Contaminated Filters; CAS 26-02-01, UST; CAS 26-23-01, Contaminated Liquids Spreader. Based on site history, process knowledge, and previous field efforts, contaminants of potential concern for CAU 127 include radionuclides, metals, total petroleum hydrocarbons, volatile organic compounds, asbestos, and polychlorinated biphenyls. Additionally, beryllium may be present at some locations. The sources of potential releases are varied, but releases of contaminated liquids may have occurred and may have migrated into and impacted soil below and surrounding storage vessels at some of the CASs. Also, at several CASs, asbestos-containing materials may be present on the aboveground structures and may be friable. Exposure pathways are limited to ingestion, inhalation, and dermal contact (adsorption) of soils/sediments or liquids, or inhalation of contaminants by site workers due to disturbance of contaminated materials. Future land-use scenarios limit subsequent uses of the CASs to various nonresidential (i.e., industrial) activities. Field activities will consist of radiological walkover and screening surveys, and field-screening and collecting of both tank content and soil samples, and further sample testing as appropriate. A two-step data quality objective strategy will be followed: (1) Phase I will be to collect environmental samples for laboratory analysis to confirm the presence or absence of contaminants at concentrations exceeding preliminary action levels; and (2) Phase II will be to collect additional environmental samples for laboratory analysis to determine the extent of contamination identified in Phase I. The results of this field investigation will support a defensible evaluation of corrective action alternatives in the corrective action decision document.« less

A conflict analysis of 4D descent strategies in a metered, multiple-arrival route environment

NASA Technical Reports Server (NTRS)

Izumi, K. H.; Harris, C. S.

1990-01-01

A conflict analysis was performed on multiple arrival traffic at a typical metered airport. The Flow Management Evaluation Model (FMEM) was used to simulate arrival operations using Denver Stapleton's arrival route structure. Sensitivities of conflict performance to three different 4-D descent strategies (clear-idle Mach/Constant AirSpeed (CAS), constant descent angle Mach/CAS and energy optimal) were examined for three traffic mixes represented by those found at Denver Stapleton, John F. Kennedy and typical en route metering (ERM) airports. The Monte Carlo technique was used to generate simulation entry point times. Analysis results indicate that the clean-idle descent strategy offers the best compromise in overall performance. Performance measures primarily include susceptibility to conflict and conflict severity. Fuel usage performance is extrapolated from previous descent strategy studies.

Carbonic Anhydrases in Cnidarians: Novel Perspectives from the Octocorallian Corallium rubrum

PubMed Central

Le Goff, Carine; Ganot, Philippe; Zoccola, Didier; Caminiti-Segonds, Natacha; Allemand, Denis; Tambutté, Sylvie

2016-01-01

Although the ability to elaborate calcium carbonate biominerals was apparently gained independently during animal evolution, members of the alpha carbonic anhydrases (α-CAs) family, which catalyze the interconversion of CO2 into HCO3-, are involved in the biomineralization process across metazoans. In the Mediterranean red coral Corallium rubrum, inhibition studies suggest an essential role of CAs in the synthesis of two biominerals produced in this octocoral, the axial skeleton and the sclerites. Hitherto no molecular characterization of these enzymes was available. In the present study we determined the complete set of α-CAs in C. rubrum by data mining the genome and transcriptome, and measured their differential gene expression between calcifying and non-calcifying tissues. We identified six isozymes (CruCA1-6), one cytosolic and five secreted/membrane-bound among which one lacked two of the three zinc-binding histidines and was so referred to as a carbonic anhydrase related protein (CARP). One secreted isozyme (CruCA4) showed specific expression both by qPCR and western-blot in the calcifying tissues, suggesting its involvement in biomineralization. Moreover, phylogenetic analyses of α-CAs, identified in six representative cnidarians with complete genome, support an independent recruitment of α-CAs for biomineralization within anthozoans. Finally, characterization of cnidarian CARPs highlighted two families: the monophyletic cytosolic CARPs, and the polyphyletic secreted CARPs harboring a cnidarian specific cysteine disulfide bridge. Alignment of the cytosolic CARPs revealed an evolutionary conserved R-H-Q motif in place of the characteristic zinc-binding H-H-H necessary for the catalytic function of α-CAs. PMID:27513959

IRIS Toxicological Review of 1,4-Dioxane (Final Report)

EPA Science Inventory

EPA announced the release of the final report, Toxicological Review of 1,4-Dioxane (CAS No. 123-91-1): In Support of Summary Information on the Integrated Risk Information System (IRIS). The final Toxicological Review of 1,4-dioxane provides scientific support and rationa...

CALM: Complex Adaptive System (CAS)-Based Decision Support for Enabling Organizational Change

NASA Astrophysics Data System (ADS)

Adler, Richard M.; Koehn, David J.

Guiding organizations through transformational changes such as restructuring or adopting new technologies is a daunting task. Such changes generate workforce uncertainty, fear, and resistance, reducing morale, focus and performance. Conventional project management techniques fail to mitigate these disruptive effects, because social and individual changes are non-mechanistic, organic phenomena. CALM (for Change, Adaptation, Learning Model) is an innovative decision support system for enabling change based on CAS principles. CALM provides a low risk method for validating and refining change strategies that combines scenario planning techniques with "what-if" behavioral simulation. In essence, CALM "test drives" change strategies before rolling them out, allowing organizations to practice and learn from virtual rather than actual mistakes. This paper describes the CALM modeling methodology, including our metrics for measuring organizational readiness to respond to change and other major CALM scenario elements: prospective change strategies; alternate futures; and key situational dynamics. We then describe CALM's simulation engine for projecting scenario outcomes and its associated analytics. CALM's simulator unifies diverse behavioral simulation paradigms including: adaptive agents; system dynamics; Monte Carlo; event- and process-based techniques. CALM's embodiment of CAS dynamics helps organizations reduce risk and improve confidence and consistency in critical strategies for enabling transformations.

Pan genome and CRISPR analyses of the bacterial fish pathogen Moritella viscosa.

PubMed

Karlsen, Christian; Hjerde, Erik; Klemetsen, Terje; Willassen, Nils Peder

2017-04-20

Winter-ulcer Moritella viscosa infections continue to be a significant burden in Atlantic salmon (Salmo salar L.) farming. M. viscosa comprises two main clusters that differ in genetic variation and phenotypes including virulence. Horizontal gene transfer through acquisition and loss of mobile genetic elements (MGEs) is a major driving force of bacterial diversification. To gain insight into genomic traits that could affect sublineage evolution within this bacterium we examined the genome sequences of twelve M. viscosa strains. Matches between M. viscosa clustered, regularly interspaced, short palindromic, repeats and associated cas genes (CRISPR-Cas) were analysed to correlate CRISPR-Cas with adaptive immunity against MGEs. The comparative genomic analysis of M. viscosa isolates from across the North Atlantic region and from different fish species support delineation of M. viscosa into four phylogenetic lineages. The results showed that M. viscosa carries two distinct variants of the CRISPR-Cas subtype I-F systems and that CRISPR features follow the phylogenetic lineages. A subset of the spacer content match prophage and plasmid genes dispersed among the M. viscosa strains. Further analysis revealed that prophage and plasmid-like element distribution were reflected in the content of the CRISPR-spacer profiles. Our data suggests that CRISPR-Cas mediated interactions with MGEs impact genome properties among M. viscosa, and that patterns in spacer and MGE distributions are linked to strain relationships.

Correction of a genetic disease by CRISPR-Cas9-mediated gene editing in mouse spermatogonial stem cells.

PubMed

Wu, Yuxuan; Zhou, Hai; Fan, Xiaoying; Zhang, Ying; Zhang, Man; Wang, Yinghua; Xie, Zhenfei; Bai, Meizhu; Yin, Qi; Liang, Dan; Tang, Wei; Liao, Jiaoyang; Zhou, Chikai; Liu, Wujuan; Zhu, Ping; Guo, Hongshan; Pan, Hong; Wu, Chunlian; Shi, Huijuan; Wu, Ligang; Tang, Fuchou; Li, Jinsong

2015-01-01

Spermatogonial stem cells (SSCs) can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc (Crygc(-/-)) that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Spacer capture and integration by a type I-F Cas1-Cas2-3 CRISPR adaptation complex.

PubMed

Fagerlund, Robert D; Wilkinson, Max E; Klykov, Oleg; Barendregt, Arjan; Pearce, F Grant; Kieper, Sebastian N; Maxwell, Howard W R; Capolupo, Angela; Heck, Albert J R; Krause, Kurt L; Bostina, Mihnea; Scheltema, Richard A; Staals, Raymond H J; Fineran, Peter C

2017-06-27

CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas1 4 -Cas2-3 2 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.

Weak connections form an infinite number of patterns in the brain

NASA Astrophysics Data System (ADS)

Ren, Hai-Peng; Bai, Chao; Baptista, Murilo S.; Grebogi, Celso

2017-04-01

Recently, much attention has been paid to interpreting the mechanisms for memory formation in terms of brain connectivity and dynamics. Within the plethora of collective states a complex network can exhibit, we show that the phenomenon of Collective Almost Synchronisation (CAS), which describes a state with an infinite number of patterns emerging in complex networks for weak coupling strengths, deserves special attention. We show that a simulated neuron network with neurons weakly connected does produce CAS patterns, and additionally produces an output that optimally model experimental electroencephalograph (EEG) signals. This work provides strong evidence that the brain operates locally in a CAS regime, allowing it to have an unlimited number of dynamical patterns, a state that could explain the enormous memory capacity of the brain, and that would give support to the idea that local clusters of neurons are sufficiently decorrelated to independently process information locally.

RIFM fragrance ingredient safety assessment, 3,7-dimethyl-1,6-nonadien-3-ol, CAS Registry Number 10339-55-6.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Miyachi, Y; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data from the suitable read across analog linalool (CAS # 78-70-6) show that this material is not genotoxic nor does it have skin sensitization potential and also provided a MOE > 100 for the local respiratory endpoint. The repeated dose, developmental and reproductive toxicity endpoints were completed using nerolidol (isomer unspecified, CAS # 7212-44-4) as a suitable read across analog, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

AFTI/F-16 in flight

NASA Technical Reports Server (NTRS)

1989-01-01

Overhead photograph of the AFTI F-16 painted in a non-standard gray finish, taken during a research flight in 1989. The two sensor pods are visible on the fuselage just forward of the wings and one of the two chin canards can be seen as a light-colored triangle ahead of one of the pods. A Sidewinder air-to-air missile is mounted on each wing tip. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

Nutritional status, cachexia, and anorexia in women with peritoneal metastasis and intraperitoneal chemotherapy: a longitudinal analysis.

PubMed

Hilal, Ziad; Rezniczek, Günther A; Klenke, Robert; Dogan, Askin; Tempfer, Clemens B

2017-11-01

To describe the nutritional status of women with peritoneal metastasis (PM) from recurrent ovarian, fallopian, or peritoneal cancer and to assess longitudinal variations of the cachexia-anorexia syndrome (CAS) during palliative pressurized intraperitoneal aerosol chemotherapy (PIPAC). Nutritional assessment included body mass index (BMI), bioelectrical impedance analysis (BIA), and blood chemistry. CAS presence/absence was recorded before and during repeated cycles (1-11) of PIPAC. Eighty-four patients with peritoneal cancer (n=5) or PM from recurrent ovarian (n=77) or fallopian tube (n=2) cancer were included. At baseline, resting metabolism (RM) (1,432±172 kcal/day), visceral fat level (7.5±3.2), skeletal muscle mass (27.2%±4.6%), upper arm circumference (27.9±4.6 cm), lower leg circumference (35.1±3.9 cm), serum parameters (albumin [3.5±0.7 g/dL], total protein [6.3±0.9 g/dL], and transferrin [202±60 mg/dL]) were below normal limits. C-reactive protein (CRP) (4.3±6.8 mg/dL), caliper body fat (35.7%±6.3%), and total body fat mass (35.6%±8.5%) were above normal limits. Nineteen/84 (23%) patients had CAS at baseline. Deterioration or stabilization/improvement of CAS was observed in 9/55 (16.4%) and 46/55 (83.6%) patients with follow-up data, respectively. Baseline body fat mass, visceral fat level, skeletal muscle mass, caliper body fat, BMI, ascites, Karnofsky index, RM, and CRP, as well as tumor response were not predictive of CAS deterioration. Nutritional decline and onset or deterioration of CAS are difficult to predict. Careful measuring and monitoring of nutritional parameters and CAS in all patients seems to be necessary in order to identify those patients in need of enteral/parenteral nutrition support. Copyright © 2017. Asian Society of Gynecologic Oncology, Korean Society of Gynecologic Oncology

A computational framework for supporting environmental ...

EPA Pesticide Factsheets

GLIMPSE is a effort in which the U.S. EPA Office of Research and Development is developing tools to support long-term, coordinated environmental, climate, and energy planning. The purpose of this presentation is to discuss the underlying science questions; provide an overview of current and future GLIMPSE capabilities; introduce GCAM, the computational engine behind GLIMPSE; and, highlight relevant activities in China, including the ABaCAS framework and GCAM-China. A group of Chinese visitors will be on the EPA RTP campus July 28, 9-noon. The visitors are from the PowerChina Huadong Engineering Corporation (weblink is here: http://www.ecidi.com/en/introduction.aspx) and are in US for a training program at Duke. The group is interested in broad management topics such as international business development and managing environmental projects as well as interacting with practitioners to understand “real world” case studies and issues. Their background is primarily related to hydro power but their corporate mission is “Providing engineering services and promoting harmonious development between Man and Nature,” implying a broad interest in the environment. Several researchers with projects with connections to China have been asked to provide an overview of their research to the visitors. I will be talking about the GLIMPSE air-climate-energy decision support project.

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

Cho, H; Ding, H; Molloi, S

Purpose: To investigate the feasibility of micro-calcification (μCa) detectability by using an energy-resolved photon-counting Si strip detector for spectral breast computed tomography (CT). Methods: A bench-top CT system was constructed using a tungsten anode x-ray source with a focal spot size of 0.8 mm and a single line 256-pixel Si strip photon counting detector with a pixel pitch of 100 μm. The slice thickness was 0.5 mm. Five different size groups of calcium carbonate grains, from 105 to 215 μm in diameter, were embedded in a cylindrical resin phantom with a diameter of 16 mm to simulate μCas. The phantomsmore » were imaged at 65 kVp with an Entrance Skin Air Kerma (ESAK) of 1.2, 3, 6, and 8 mGy. The images were reconstructed using a standard filtered back projection (FBP) with a ramp filter. A total of 200 μCa images (5 different sizes of μCas × 4 different doses × 10 images for each setting) were combined with another 200 control images without μCas, to ultimately form 400 images for the reader study. The images were displayed in random order to three blinded observers, who were asked to give a binary score on each image regarding the presence of μCas. The μCa detectability for each image was evaluated in terms of binary decision theory metrics. The sensitivity, specificity, and accuracy were calculated to study the size and dose-dependence for μCa detectability. Additionally, the influence of the partial volume effect on the μCa detectability was investigated by simulation. Results: For a μCa larger than 140 μm in diameter, detection accuracy of above 90 % was achieved with the investigated prototype spectral CT system at ESAK of 1.2 mGy. Conclusion: The proposed Si strip detector is expected to offer superior image quality with the capability to detect μCas for low dose breast imaging.« less

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair

PubMed Central

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-01-01

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. PMID:26850641

Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair.

PubMed

He, Xiangjun; Tan, Chunlai; Wang, Feng; Wang, Yaofeng; Zhou, Rui; Cui, Dexuan; You, Wenxing; Zhao, Hui; Ren, Jianwei; Feng, Bo

2016-05-19

CRISPR/Cas9-induced site-specific DNA double-strand breaks (DSBs) can be repaired by homology-directed repair (HDR) or non-homologous end joining (NHEJ) pathways. Extensive efforts have been made to knock-in exogenous DNA to a selected genomic locus in human cells; which, however, has focused on HDR-based strategies and was proven inefficient. Here, we report that NHEJ pathway mediates efficient rejoining of genome and plasmids following CRISPR/Cas9-induced DNA DSBs, and promotes high-efficiency DNA integration in various human cell types. With this homology-independent knock-in strategy, integration of a 4.6 kb promoterless ires-eGFP fragment into the GAPDH locus yielded up to 20% GFP+ cells in somatic LO2 cells, and 1.70% GFP+ cells in human embryonic stem cells (ESCs). Quantitative comparison further demonstrated that the NHEJ-based knock-in is more efficient than HDR-mediated gene targeting in all human cell types examined. These data support that CRISPR/Cas9-induced NHEJ provides a valuable new path for efficient genome editing in human ESCs and somatic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

RIFM fragrance ingredient safety assessment, ethylene brassylate, CAS Registry Number 105-95-3.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

: The use of this material under current conditions is supported by existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data show that this material is not genotoxic nor does it have skin sensitization potential. The local respiratory toxicity endpoint was completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class I material (1.4 mg/day). The repeated dose toxicity endpoint was completed using ethylene dodecanedioate (CAS # 54982-83-1) as a suitable read across analog, which provided a MOE > 100. The developmental and reproductive toxicity endpoint was completed using oxacyclohexadec-12-en-2-one, (12E)- (CAS # 111879-80-2) as a suitable read across analog, which provided a MOE > 100. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra along with data on the target material. The environmental endpoint was completed as described in the RIFM Framework along with data on the suitable read across analog oxacyclohexadec-12-en-2-one, (12E)- (CAS # 111879-80-2). Copyright © 2016 Elsevier Ltd. All rights reserved.

AFTI/F-16 50th flight team photo

NASA Technical Reports Server (NTRS)

1983-01-01

An early (1983) photograph of the AFTI F-16 team, commemorating the aircraft's 50th flight. It shows the initial configuration and paint finish of the AFTI F-16, as well as the forward mounted canards and the spin chute. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

A New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System of Pseudomonas aeruginosa

PubMed Central

Pawluk, April; Bondy-Denomy, Joseph; Cheung, Vivian H. W.; Maxwell, Karen L.; Davidson, Alan R.

2014-01-01

ABSTRACT CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. PMID:24736222

Chlordane (Technical)

Integrated Risk Information System (IRIS)

TOXICOLOGICAL REVIEW of CHLORDANE ( TECHNICAL ) ( CAS No . 12789 - 03 - 6 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) December 1997 U.S . Environmental Protection Agency Washington , DC TABLE OF CONTENTS Authors and Reviewers . . . . . . . . . . . . .

Comparative transcriptome analysis of the CO2 sensing pathway via differential expression of carbonic anhydrase in Cryptococcus neoformans.

PubMed

Kim, Min Su; Ko, Young-Joon; Maeng, Shinae; Floyd, Anna; Heitman, Joseph; Bahn, Yong-Sun

2010-08-01

Carbon dioxide (CO(2)) sensing and metabolism via carbonic anhydrases (CAs) play pivotal roles in survival and proliferation of pathogenic fungi infecting human hosts from natural environments due to the drastic difference in CO(2) levels. In Cryptococcus neoformans, which causes fatal fungal meningoencephalitis, the Can2 CA plays essential roles during both cellular growth in air and sexual differentiation of the pathogen. However the signaling networks downstream of Can2 are largely unknown. To address this question, the present study employed comparative transcriptome DNA microarray analysis of a C. neoformans strain in which CAN2 expression is artificially controlled by the CTR4 (copper transporter) promoter. The P(CTR4)CAN2 strain showed growth defects in a CO(2)-dependent manner when CAN2 was repressed but resumed normal growth when CAN2 was overexpressed. The Can2-dependent genes identified by the transcriptome analysis include FAS1 (fatty acid synthase 1) and GPB1 (G-protein beta subunit), supporting the roles of Can2 in fatty acid biosynthesis and sexual differentiation. Cas3, a capsular structure designer protein, was also discovered to be Can2-dependent and yet was not involved in CO(2)-mediated capsule induction. Most notably, a majority of Can2-dependent genes were environmental stress-regulated (ESR) genes. Supporting this, the CAN2 overexpression strain was hypersensitive to oxidative and genotoxic stress as well as antifungal drugs, such as polyene and azole drugs, potentially due to defective membrane integrity. Finally, an oxidative stress-responsive Atf1 transcription factor was also found to be Can2-dependent. Atf1 not only plays an important role in diverse stress responses, including thermotolerance and antifungal drug resistance, but also represses melanin and capsule production in C. neoformans. In conclusion, this study provides insights into the comprehensive signaling networks orchestrated by CA/CO(2)-sensing pathways in pathogenic fungi.

Electro-optical characterization system develped for ATLIDCAS AIV: flat field and collimated beam injections

NASA Astrophysics Data System (ADS)

Ramos, G.; Laguna, H.; Torres, J.; Belenguer, T.

2017-11-01

In the framework of the ESA EarthCare Mission, an atmospheric LIDAR (ATLID) was included as a payload. CAS is the co-alignment system of such a LIDAR instrument, the system responsible of guaranteeing the proper alignment of the projected laser beam and the reflected light collected. Within CAS, in which a consortium leaded by ASTRIUM France is working in, as well as CRISA (electronics) and LIDAX (mechanical engineering), INTA is in charge of the development of the instrumentation to be used on ground (on ground support equipments, OGSEs) needed for the proper electro-optical characterization.

Corrective Action Plan for Corrective Action Unit 271: Areas 25, 26, and 27 Septic Systems, Nevada Test Site, Nevada

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

R. B. Jackson

2003-05-01

The Areas 25, 26 and 27 Septic Systems are in the Federal Facility Agreement and Consent Order (FFACO) of 1996 as Corrective Action Unit (CAU) 271. This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for CAU 271. CAU 271 is located on the Nevada Test Site (NTS) approximately 105 kilometers (65 miles) northwest of Las Vegas, Nevada, and consists of the following 15 Corrective Action Sites (CAS): CAS 25-04-1, Septic System; CAS 25-04-03, Septic System; CAS25-04-04, Septic System; CAS 25-04-08, Septic System; CAS 25-04-09, Septic System; CAS 25-04-10, Septic System; CAS 25-04-11, Septicmore » System; CAS 26-03-01, Contaminated Water Reservoir; CAS 26-04-1, Septic System; CAS 26-04-02, Septic System; CAS 26-05-01, Radioactive Leachfield; CAS-26-05-03, Septic System; CAS 26-05-04, Septic System; CAS 26-05-05, Septic System; and CAS 27-05-02, Leachfield.« less

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays.

PubMed

Lee, Hayun; Zhou, Yi; Taylor, David W; Sashital, Dipali G

2018-04-05

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specific endonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration. Copyright © 2018 Elsevier Inc. All rights reserved.

Crystal Structure of Streptococcus pyogenes Cas1 and Its Interaction with Csn2 in the Type II CRISPR-Cas System.

PubMed

Ka, Donghyun; Lee, Hasup; Jung, Yi-Deun; Kim, Kyunggon; Seok, Chaok; Suh, Nayoung; Bae, Euiyoung

2016-01-05

CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Pharmacy component of a hospital end-product cost-accounting system.

PubMed

Smith, J E; Sheaffer, S L; Meyer, G E; Giorgilli, F

1988-04-01

Determination of pharmacy department standard costs for providing drug products to patients at Thomas Jefferson University Hospital in Philadelphia is described. The hospital is implementing a cost-accounting system (CAS) that uses software developed at the New England Medical Center, Boston. The pharmacy identified nine categories of intermediate products on the basis of labor consumption. Standard labor times for each product category are based on measurement or estimation of time for each task in the preparation and distribution of a dose. Variable-labor standard time was determined by adjusting the cumulative time for the tasks to account for nonproductive time and nonroutine activities, and a variable-labor standard cost for each category was calculated. The standard cost per dose included the costs of labor and supplies (variable and fixed) and equipment; this standard cost plus the acquisition cost of a drug line item is the total intermediate product cost. Because the CAS is based on the hospital's patient charges, clinical pharmacy services are excluded. Intermediate products that substantially affect end-product costs (costs per patient case) will be identified for inclusion in CAS reports. The CAS will give a more accurate picture of resource consumption, enabling managers to focus their efforts to improve efficiency and productivity and reduce supply use; it could also improve the accuracy of the budgeting process. The CAS will support hospital administration decisions about marketing end products and department managers' decisions about controlling intermediate-product costs.

Simulation of acute haemodynamic outcomes of the surgical strategies for the right ventricular failure treatment in pediatric LVAD.

PubMed

Di Molfetta, Arianna; Ferrari, Gianfranco; Iacobelli, Roberta; Filippelli, Sergio; Fresiello, Libera; Gagliardi, Maria G; Toscano, Alessandro; Trivella, Maria G; Amodeo, Antonio

2015-12-01

Right ventricular failure (RVF) is one of the major complications during LVAD. Apart from drug therapy, the most reliable option is the implantation of RVAD. However, BIVAD have a poor prognosis and increased complications. Experiments have been conducted on alternative approaches, such as the creation of an atrial septal defect (ASD), a cavo-aortic shunt (CAS) including the LVAD and a cavo-pulmonary connection (CPC). This work aims at realizing a lumped parameter model (LPM) to compare the acute hemodynamic effects of ASD, CPC, CAS, RVAD in LVAD pediatric patients with RVF. Data of 5 pediatric patients undergoing LVAD were retrospectively collected to reproduce patients baseline hemodynamics with the LPM. The effects of continuous flow LVAD implantation complicated by RVF was simulated and then the effects of ASD, CPC, CAS and RVAD treatments were simulated for each patient. The model successfully reproduced patients' baseline and the hemodynamic effects of the surgical strategies. Simulating the different surgical strategies, an unloading of the right ventricle and an increment of left ventricular preload were observed with an improvement of the hemodynamics (total cardiac output: ASD +15%, CPC +10%, CAS +70% RVAD +20%; right ventricular external work: ASD -19%, CPC -46%, CAS -76%, RVAD -32%; left ventricular external work: ASD +12%, CPC +28%, RVAD +64%). The use of numerical model could offer an additional support for clinical decision-making, also potentially reducing animal experiments, to compare the outcome of different surgical strategies to treat RVF in LVAD.

β-Carbonic Anhydrases Play a Role in Fruiting Body Development and Ascospore Germination in the Filamentous Fungus Sordaria macrospora

PubMed Central

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO2) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO3 −) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into α-, β-, γ-, δ- and ζ-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of β-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding β-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Δcas1, Δcas2, and Δcas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Δcas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Δcas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO2 levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions. PMID:19365544

Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

PubMed Central

Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-01-01

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

The CRISPR-Associated Gene cas2 of Legionella pneumophila Is Required for Intracellular Infection of Amoebae

PubMed Central

Gunderson, Felizza F.; Cianciotto, Nicholas P.

2013-01-01

ABSTRACT Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. PMID:23481601

[Detection of CRSPR-Cas system in Streptococcus thermophiles].

PubMed

Li, Wan; Liang, Hongzhang; Zhang, Danqing; Wang, Nana; Tang, Yaru; Li, Bailiang; Huo, Guicheng

2016-04-14

We aimed to detect the CRSPR-Cas system of six Streptococcus thermophilus. Bioinformatics method was used to predict CRSPR-Cas system of nine S. thermophilus that published in National Center for Biotechnology Information. Four primers were designed according to the flanking sequences of standard strains and the CRISPR-Cas system of six S. thermophilus have been detected by PCR method. S. thermophilus S4 had a Cas9 gene, others all had Cas9 gene, Cas10 gene and Cas9* gene. In addition, 79 and KLDS3.0207 still had Cas3 gene. Signature genes amplification of CRSPR-Cas system could predict the type of CRSPR-Cas system in unsequenced strains, these findings will help establish the foundation for the study of CRSPR-Cas system in lactic acid bacteria.

Hexachlorocyclopentadiene (HCCPD)

Integrated Risk Information System (IRIS)

EPA / 600 / R - 01 / 013 TOXICOLOGICAL REVIEW OF HEXACHLOROCYCLOPENTADIENE ( CAS No . 77 - 47 - 4 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) June 2001 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed

Tetrachloroethylene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 08 / 011F February 2012 TOXICOLOGICAL REVIEW OF Tetrachloroethylene ( Perchloroethylene ) ( CAS No . 127 - 18 - 4 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) February 2012 U.S . Environmental Protection Agency Washington , DC ii DISCLAIMER T

Hypersexual, Sexually Compulsive, or Just Highly Sexually Active? Investigating Three Distinct Groups of Gay and Bisexual Men and Their Profiles of HIV-Related Sexual Risk

PubMed Central

Rendina, H. Jonathon; Ventuneac, Ana; Moody, Raymond L.; Grov, Christian

2015-01-01

Emerging research supports the notion that sexual compulsivity (SC) and hypersexual disorder (HD) among gay and bisexual men (GBM) might be conceptualized as comprising three groups—Neither SC nor HD; SC only, and Both SC and HD—that capture distinct levels of severity across the SC/HD continuum. We examined data from 370 highly sexually active GBM to assess how the three groups compare across a range of risk factors for HIV infection. Comparisons focused on psychosexual measures—temptation for condomless anal sex (CAS), self-efficacy for avoiding CAS, sexual excitation and inhibition—as well as reports of actual sexual behavior. Nearly half (48.9 %) of this highly sexually active sample was classified as Neither SC nor HD, 30 % as SC Only, and 21.1 % as Both SC and HD. While we found no significant differences between the three groups on reported number of male partners, anal sex acts, or anal sex acts with serodiscordant partners, the Both SC and HD group reported higher numbers of CAS acts and CAS acts with serodiscordant partners and also had a higher proportion of their anal sex acts without condoms compared to the SC Only group. Our findings support the validity of a three-group classification system of SC/HD severity in differentiating psychosexual and HIV-related sexual risk behavior outcomes in a sample of GBM who report similarly high levels of sexual activity. Notwithstanding the need for sex positive HIV prevention programs, interventions that attempt to help Both SC and HD men deal with distress and address their psychosexual needs specifically may derive HIV prevention benefits. PMID:25750052

MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

PubMed Central

Abby, Sophie S.; Néron, Bertrand; Ménager, Hervé; Touchon, Marie; Rocha, Eduardo P. C.

2014-01-01

Motivation Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose. Results Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles. Availability and Implementation MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information. PMID:25330359

e-Learning in Advanced Life Support-What factors influence assessment outcome?

PubMed

Thorne, C J; Lockey, A S; Kimani, P K; Bullock, I; Hampshire, S; Begum-Ali, S; Perkins, G D

2017-05-01

To establish variables which are associated with favourable Advanced Life Support (ALS) course assessment outcomes, maximising learning effect. Between 1 January 2013 and 30 June 2014, 8218 individuals participated in a Resuscitation Council (UK) e-learning Advanced Life Support (e-ALS) course. Participants completed 5-8h of online e-learning prior to attending a one day face-to-face course. e-Learning access data were collected through the Learning Management System (LMS). All participants were assessed by a multiple choice questionnaire (MCQ) before and after the face-to-face aspect alongside a practical cardiac arrest simulation (CAS-Test). Participant demographics and assessment outcomes were analysed. The mean post e-learning MCQ score was 83.7 (SD 7.3) and the mean post-course MCQ score was 87.7 (SD 7.9). The first attempt CAS-Test pass rate was 84.6% and overall pass rate 96.6%. Participants with previous ALS experience, ILS experience, or who were a core member of the resuscitation team performed better in the post-course MCQ, CAS-Test and overall assessment. Median time spent on the e-learning was 5.2h (IQR 3.7-7.1). There was a large range in the degree of access to e-learning content. Increased time spent accessing e-learning had no effect on the overall result (OR 0.98, P=0.367) on simulated learning outcome. Clinical experience through membership of cardiac arrest teams and previous ILS or ALS training were independent predictors of performance on the ALS course whilst time spent accessing e-learning materials did not affect course outcomes. This supports the blended approach to e-ALS which allows participants to tailor their e-learning experience to their specific needs. Copyright © 2017 Elsevier B.V. All rights reserved.

Apport de l'imagerie dans le diagnostic des sacroiliites infectieuses : à propos de 19 cas

PubMed Central

Abid, Hanen; Chaabouni, Salim; Frikha, Faten; Toumi, Nozha; Souissi, Basma; Lahiani, Dorra; Bahloul, Zouhir; Ben Mahfoudh, Khaireddine

2014-01-01

Les sacro-iliites infectieuses méritent d’être mieux connues. Leur diagnostic est souvent retardé en raison d'une symptomatologie trompeuse et des diffcultés d'exploration de l'articulation sacro-iliaque. Notre travail est basé sur une étude rétrospective portant sur les cas de SII, recueillis sur une période comprise entre 1997 et 2008 dans notre centre universitaire Sfax-Tunisie. Le diagnostic de sacro-iliite était retenu en présence d'arguments cliniques et radiologiques d'atteinte sacroiliaque. Nous rapportons dix neuf cas de sacroiliites infectieuses (10 hommes et 9 femmes), avec un âge moyen de 32 ans. L'atteinte était unilatérale dans tous les cas. Les radiographies standard faites dans tous les cas ont été suggestives dans 14 cas et normales dans les autres cas. La TDM faite dans 13 cas a montré, un abcès des parties molles dans 8 cas et un séquestre osseux dans 2 cas. L'IRM réalisée dans 8 cas, a objectivé une infiltration des parties molles dans tous les cas et un abcès dans 3 cas. Le germe a été identifié dans 9 cas (3 cas de tuberculose, 3 cas de brucellose, 2 sacro-iliites à pyogène et un cas de candidose). Cette identification était faite par biopsie dans 3 cas, hémocultures dans 2 cas, prélèvement au niveau de la porte d'entrée dans 1 cas et sérodiagnostic dans 3 cas. Pour les autres cas, l'origine pyogène a été retenue sur des arguments cliniques et biologiques. L'imagerie joue un rôle primordial dans le diagnostic précoce et l'orientation étiologique d'une sacroiliite infectieuse. PMID:25120884

Study on analysis of waste edible oil with deterioration and removal of acid value, carbonyl value, and free fatty acid by a food additive (calcium silicate).

PubMed

Ogata, Fumihiko; Tanaka, Yuko; Tominaga, Hisato; Kangawa, Moe; Inoue, Kenji; Ueda, Ayaka; Iwata, Yuka; Kawasaki, Naohito

2013-01-01

This study investigated the regeneration of waste edible oil using a food additive (calcium silicate, CAS). Waste edible oil was prepared by combined heat and aeration treatment. Moreover, the deterioration of edible oil by combined heat and aeration treatment was greater than that by heat treatment alone. The acid value (AV) and carbonyl value (CV) increased with increasing deterioration; conversely, the tocopherol concentration decreased with increasing deterioration. The specific surface area, pore volume, and mean pore diameter of the 3 CAS formulations used (CAS30, CAS60, and CAS90) were evaluated, and scanning electron microscopic images were taken. The specific surface area increased in the order of CAS30 (115.54 m(2)/g) < CAS60 (163.93 m(2)/g) < CAS90 (187.47 m(2)/g). The mean pore diameter increased in the order of CAS90 (170.59 Å) < CAS60 (211.60 Å) < CAS30 (249.70 Å). The regeneration of waste edible oil was possible with CAS treatment. The AV reduced by 15.2%, 10.8%, and 23.1% by CAS30, CAS60, and CAS90 treatment, respectively, and the CV was reduced by 35.6%, 29.8%, and 31.3% by these 3 treatments, respectively. Moreover, the concentrations of tocopherol and free fatty acids did not change with CAS treatment. The characteristics of CAS were not related to the degree of change of AV and CV. However, the adsorption mechanism of polar and non-polar compounds generated in waste edible oil by CAS was related with the presence of silica gel molecules in CAS. The findings indicated that CAS was useful for the regeneration of waste edible oil.

Closure Report for Corrective Action Unit 574: Neptune, Nevada National Security Site, Nevada

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

NSTec Environmental Restoration

Corrective Action Unit (CAU) 574 is identified in the Federal Facility Agreement and Consent Order (FFACO) as 'Neptune' and consists of the following two Corrective Action Sites (CASs), located in Area 12 of the Nevada National Security Site: (1) CAS 12-23-10, U12c.03 Crater (Neptune); and (2) CAS 12-45-01, U12e.05 Crater (Blanca). This Closure Report presents information supporting closure of CAU 574 according to the FFACO (FFACO, 1996 [as amended March 2010]) and the Streamlined Approach for Environmental Restoration Plan for CAU 574 (U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office [NNSA/NSO], 2011). The following activities were performedmore » to support closure of CAU 574: (1) In situ external dose rate measurements were collected using thermoluminescent dosimeters at CAS 12-45-01, U12e.05 Crater (Blanca). (2) Total effective dose rates were determined at both sites by summing the internal and external dose rate components. (3) A use restriction (UR) was implemented at CAS 12-23-10, U12c.03 Crater (Neptune). Areas that exceed the final action level (FAL) of 25 millirems per year (mrem/yr) based on the Occasional Use Area exposure scenario are within the existing use restricted area for CAU 551. The 25-mrem/yr FAL is not exceeded outside the existing CAU 551 UR for any of the exposure scenarios (Industrial Area, Remote Work Area, and Occasional Use Area). Therefore, the existing UR for CAU 551 is sufficient to bound contamination that exceeds the FAL. (4) An administrative UR was implemented at CAS 12-45-01, U12e.05 Crater (Blanca) as a best management practice (BMP). The 25-mrem/yr FAL was not exceeded for the Remote Work Area or Occasional Use Area exposure scenarios; therefore, a UR is not required. However, because the 25-mrem/yr FAL was exceeded for the Industrial Area exposure scenario, an administrative UR was established as a BMP. UR documentation is included as Appendix B. The UR at CAS 12-23-10, U12c.03 Crater (Neptune), is within the existing UR for CAU 551. Additional postings were not installed, and annual post-closure inspections will be performed in conjunction with the inspections performed for CAU 551. At CAS 12-45-01, U12e.05 Crater (Blanca), the administrative UR does not require postings or inspections. NNSA/NSO requests the following: (1) A Notice of Completion from the Nevada Division of Environmental Protection to NNSA/NSO for closure of CAU 574; and (2) The transfer of CAU 574 from Appendix III to Appendix IV, Closed Corrective Action Units, of the FFACO« less

Corrective Action Investigation Plan for Corrective Action Unit 106: Areas 5, 11 Frenchman Flat Atmospheric Sites, Nevada National Security Site, Nevada

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

Patrick Matthews

2011-07-01

Corrective Action Unit 106 comprises the four corrective action sites (CASs) listed below: • 05-20-02, Evaporation Pond • 05-23-05, Atmospheric Test Site - Able • 05-45-04, 306 GZ Rad Contaminated Area • 05-45-05, 307 GZ Rad Contaminated Area These sites are being investigated because existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives (CAAs). Additional information will be obtained by conducting a corrective action investigation before evaluating CAAs and selecting the appropriate corrective action for each CAS. The results of the field investigation will support a defensible evaluation of viablemore » CAAs that will be presented in the Corrective Action Decision Document. The sites will be investigated based on the data quality objectives (DQOs) developed on January 19, 2010, by representatives of the Nevada Division of Environmental Protection and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office. The DQO process was used to identify and define the type, amount, and quality of data needed to develop and evaluate appropriate corrective actions for CAU 106. The presence and nature of contamination at CAU 106 will be evaluated based on information collected from a field investigation. The CAU includes land areas impacted by the release of radionuclides from groundwater pumping during the Radionuclide Migration study program (CAS 05-20-02), a weapons-related airdrop test (CAS 05-23-05), and unknown support activities at two sites (CAS 05-45-04 and CAS 05-45-05). The presence and nature of contamination from surface-deposited radiological contamination from CAS 05-23-05, Atmospheric Test Site - Able, and other types of releases (such as migration and excavation as well as any potential releases discovered during the investigation) from the remaining three CASs will be evaluated using soil samples collected from the locations most likely containing contamination, if present. Appendix A provides a detailed discussion of the DQO methodology and the DQOs specific to each CAS. The scope of the corrective action investigation for CAU 106 includes the following activities: • Conduct radiological surveys. • Collect and submit environmental samples for laboratory analysis to determine internal dose rates and the presence of contaminants of concern. • If contaminants of concern are present, collect additional samples to define the extent of the contamination and determine the area where the total effective dose at the site exceeds final action levels (i.e., corrective action boundary). • Collect samples of investigation-derived waste, as needed, for waste management purposes.« less

Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

PubMed

Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

2015-10-15

Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

PubMed Central

Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

2014-01-01

The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

Corrective Action Plan for Corrective Action Unit 262: Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada

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

K. B. Campbell

This Corrective Action Plan (CAP) provides selected corrective action alternatives and proposes the closure methodology for Corrective Action Unit (CAU) 262, Area 25 Septic Systems and Underground Discharge Point. CAU 262 is identified in the Federal Facility Agreement and Consent Order (FFACO) of 1996. Remediation of CAU 262 is required under the FFACO. CAU 262 is located in Area 25 of the Nevada Test Site (NTS), approximately 100 kilometers (km) (62 miles [mi]) northwest of Las Vegas, Nevada. The nine Corrective Action Sites (CASs) within CAU 262 are located in the Nuclear Rocket Development Station complex. Individual CASs are locatedmore » in the vicinity of the Reactor Maintenance, Assembly, and Disassembly (R-MAD); Engine Maintenance, Assembly, and Disassembly (E-MAD); and Test Cell C compounds. CAU 262 includes the following CASs as provided in the FFACO (1996); CAS 25-02-06, Underground Storage Tank; CAS 25-04-06, Septic Systems A and B; CAS 25-04-07, Septic System; CAS 25-05-03, Leachfield; CAS 25-05-05, Leachfield; CAS 25-05-06, Leachfield; CAS 25-05-08, Radioactive Leachfield; CAS 25-05-12, Leachfield; and CAS 25-51-01, Dry Well. Figures 2, 3, and 4 show the locations of the R-MAD, the E-MAD, and the Test Cell C CASs, respectively. The facilities within CAU 262 supported nuclear rocket reactor engine testing. Activities associated with the program were performed between 1958 and 1973. However, several other projects used the facilities after 1973. A significant quantity of radioactive and sanitary waste was produced during routine operations. Most of the radioactive waste was managed by disposal in the posted leachfields. Sanitary wastes were disposed in sanitary leachfields. Septic tanks, present at sanitary leachfields (i.e., CAS 25-02-06,2504-06 [Septic Systems A and B], 25-04-07, 25-05-05,25-05-12) allowed solids to settle out of suspension prior to entering the leachfield. Posted leachfields do not contain septic tanks. All CASs located in CAU 262 are inactive or abandoned. However, some leachfields may still receive liquids from runoff during storm events. Results from the 2000-2001 site characterization activities conducted by International Technology (IT) Corporation, Las Vegas Office are documented in the Corrective Action Investigation Report for Corrective Action Unit 262: Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada. This document is located in Appendix A of the Corrective Action Decision Document for CAU 262. Area 25 Septic Systems and Underground Discharge Point, Nevada Test Site, Nevada. (DOE/NV, 2001).« less

Characterizing and Supporting Change in Algebra Students' Representational Fluency in a CAS/Paper-and-Pencil Environment

ERIC Educational Resources Information Center

Fonger, Nicole L.

2012-01-01

Representational fluency (RF) includes an ability to interpret, create, move within and among, and connect tool-based representations of mathematical objects. Taken as an indicator of conceptual understanding, there is a need to better support school algebra students' RF in learning environments that utilize both computer algebra systems…

Instructional Supports for Representational Fluency in Solving Linear Equations with Computer Algebra Systems and Paper-and-Pencil

ERIC Educational Resources Information Center

Fonger, Nicole L.; Davis, Jon D.; Rohwer, Mary Lou

2018-01-01

This research addresses the issue of how to support students' representational fluency--the ability to create, move within, translate across, and derive meaning from external representations of mathematical ideas. The context of solving linear equations in a combined computer algebra system (CAS) and paper-and-pencil classroom environment is…

A Broad-Spectrum Inhibitor of CRISPR-Cas9.

PubMed

Harrington, Lucas B; Doxzen, Kevin W; Ma, Enbo; Liu, Jun-Jie; Knott, Gavin J; Edraki, Alireza; Garcia, Bianca; Amrani, Nadia; Chen, Janice S; Cofsky, Joshua C; Kranzusch, Philip J; Sontheimer, Erik J; Davidson, Alan R; Maxwell, Karen L; Doudna, Jennifer A

2017-09-07

CRISPR-Cas9 proteins function within bacterial immune systems to target and destroy invasive DNA and have been harnessed as a robust technology for genome editing. Small bacteriophage-encoded anti-CRISPR proteins (Acrs) can inactivate Cas9, providing an efficient off switch for Cas9-based applications. Here, we show that two Acrs, AcrIIC1 and AcrIIC3, inhibit Cas9 by distinct strategies. AcrIIC1 is a broad-spectrum Cas9 inhibitor that prevents DNA cutting by multiple divergent Cas9 orthologs through direct binding to the conserved HNH catalytic domain of Cas9. A crystal structure of an AcrIIC1-Cas9 HNH domain complex shows how AcrIIC1 traps Cas9 in a DNA-bound but catalytically inactive state. By contrast, AcrIIC3 blocks activity of a single Cas9 ortholog and induces Cas9 dimerization while preventing binding to the target DNA. These two orthogonal mechanisms allow for separate control of Cas9 target binding and cleavage and suggest applications to allow DNA binding while preventing DNA cutting by Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

Sexual Risk-Taking in HIV-Negative Gay and Bisexual Men Increases with Depression: Results from a U.S. National Study

PubMed Central

Millar, Brett M.; Starks, Tyrel J.; Grov, Christian; Parsons, Jeffrey T.

2017-01-01

The link between depression and sexual risk-taking has received mixed findings in the literature. The current study analyzed the links between depression and recent condomless anal sex (CAS) with casual partners among 1033 HIV-negative, non-PrEP-using, gay and bisexual men. When CAS was dichotomized as either none or some, depression was not associated with the odds of CAS (with receptive and insertive combined) or insertive CAS only, but was positively associated with the odds of receptive CAS. When CAS was tallied as a count variable of events, depression was positively associated with total CAS, receptive CAS, and insertive CAS. With the addition of a quadratic term for depression, a positive quadratic effect was only found for total CAS and receptive CAS, but not for insertive CAS. These findings highlight the utility of using count data for CAS events and treating CAS separately with regard to receptive and insertive positioning when considering the role of depression among gay and bisexual men. PMID:27475943

Guidance, Control and Positioning of Future Precision Guided Stand-off Weapons Systems

DTIC Science & Technology

1986-06-01

environment tests. The programme consists of approximately ten fl ights, the firsts having a passive nature . These are followed by progressive...Limited _. .. , 40 Chigwell Lane, Loughton, Essex IGIO 3TZ PREFACE The environment in which tactical air forces must be able to operate is becoming...GUIDANCE SYSTEM CONCEPT FOR HIGH-DYNAMIC ENVIRONMENT * by U.K.Krogmann 17 APPLICATIONS DES CENTRALES A COMPOSANTS LIES AUX MISSILES TACTIQUES: CAS DES

High Frontier, The Journal for Space & Missile Professionals. Volume 5, Number 1

DTIC Science & Technology

2008-11-01

competency of CAS integration and control. There are also air mobility liaison officers ( AMLOs ) corps and divisions levels. These officers are...deliberately assigned to a TACP similar to AMLOs and ISR LNOs. The Army identified this knowledge gap as an issue and developed Functional Area 40...An obvious way to address this need is by adding space professionals, similar to AMLOs and ISR LNOs, to TACPs. Because this follows an established

CAS - A Turboprop Solution for the COIN Fight

DTIC Science & Technology

2009-01-01

Worlds First Civilian Tiltrotor. http://www.bellagusta.com/air ba main.cfm. Accessed 13 Jan 2009. Blake, Brett R , AT-6-The Best Investmentfor the Long War...Terroism and Counterinsurgency, CRS for Congress RL32737, Washington DC: Congressional Research Service, 24 Jan. 2005. Blake, Brett R Major USAF. AT-6...www.aviationweek.com/aw/generic/channel .jsp?channel= awst # (accessed 15 Jan 2009). 7 Bolkcom and Katzman, 24. 8 U.S. Congress, Office ofTechnology

Benzene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 02 / 001F TOXICOLOGICAL REVIEW OF BENZENE ( NONCANCER EFFECTS ) ( CAS No . 71 - 43 - 2 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) October 2002 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been

Chromium(VI)

Integrated Risk Information System (IRIS)

TOXICOLOGICAL REVIEW OF HEXAVALENT CHROMIUM ( CAS No . 18540 - 29 - 9 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) August 1998 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in accordance with U.S . Env

Nitrobenzene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 08 / 004F www.epa.gov / iris TOXICOLOGICAL REVIEW OF NITROBENZENE ( CAS No . 98 - 95 - 3 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) January 2009 U.S . Environmental Protection Agency Washington , DC i DISCLAIMER This documen

Bromate

Integrated Risk Information System (IRIS)

EPA / 635 / R - 01 / 002 TOXICOLOGICAL REVIEW OF BROMATE ( CAS No . 15541 - 45 - 4 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) March 2001 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in accordance

Toluene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 05 / 004 TOXICOLOGICAL REVIEW OF TOLUENE ( CAS No . 108 - 88 - 3 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) September 2005 U.S . Environmental Protection Agency W ashington D.C . DISCLAIMER This docum ent has been review

Corrective Action Investigation Plan for Corrective Action Unit 165: Areas 25 and 26 Dry Well and Washdown Areas, Nevada Test Site, Nevada (including Record of Technical Change Nos. 1, 2, and 3) (January 2002, Rev. 0)

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

U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office

This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Operations Office's approach to collect the data necessary to evaluate corrective action alternatives appropriate for the closure of Corrective Action Unit (CAU) 165 under the Federal Facility Agreement and Consent Order. Corrective Action Unit 165 consists of eight Corrective Action Sites (CASs): CAS 25-20-01, Lab Drain Dry Well; CAS 25-51-02, Dry Well; CAS 25-59-01, Septic System; CAS 26-59-01, Septic System; CAS 25-07-06, Train Decontamination Area; CAS 25-07-07, Vehicle Washdown; CAS 26-07-01, Vehicle Washdown Station; and CAS 25-47-01, Reservoir and French Drain. All eight CASsmore » are located in the Nevada Test Site, Nevada. Six of these CASs are located in Area 25 facilities and two CASs are located in Area 26 facilities. The eight CASs at CAU 165 consist of dry wells, septic systems, decontamination pads, and a reservoir. The six CASs in Area 25 are associated with the Nuclear Rocket Development Station that operated from 1958 to 1973. The two CASs in Area 26 are associated with facilities constructed for Project Pluto, a series of nuclear reactor tests conducted between 1961 to 1964 to develop a nuclear-powered ramjet engine. Based on site history, the scope of this plan will be a two-phased approach to investigate the possible presence of hazardous and/or radioactive constituents at concentrations that could potentially pose a threat to human health and the environment. The Phase I analytical program for most CASs will include volatile organic compounds, semivolatile organic compounds, Resource Conservation and Recovery Act metals, total petroleum hydrocarbons, polychlorinated biphenyls, and radionuclides. If laboratory data obtained from the Phase I investigation indicates the presence of contaminants of concern, the process will continue with a Phase II investigation to define the extent of contamination. Based on the results of Phase I sampling, the analytical program for Phase II investigation may be reduced. The results of this field investigation will support a defensible evaluation of corrective action alternatives in the corrective action decision document.« less

Computer-assisted learning and simulation systems in dentistry--a challenge to society.

PubMed

Welk, A; Splieth, Ch; Wierinck, E; Gilpatrick, R O; Meyer, G

2006-07-01

Computer technology is increasingly used in practical training at universities. However, in spite of their potential, computer-assisted learning (CAL) and computer-assisted simulation (CAS) systems still appear to be underutilized in dental education. Advantages, challenges, problems, and solutions of computer-assisted learning and simulation in dentistry are discussed by means of MEDLINE, open Internet platform searches, and key results of a study among German dental schools. The advantages of computer-assisted learning are seen for example in self-paced and self-directed learning and increased motivation. It is useful for both objective theoretical and practical tests and for training students to handle complex cases. CAL can lead to more structured learning and can support training in evidence-based decision-making. The reasons for the still relatively rare implementation of CAL/CAS systems in dental education include an inability to finance, lack of studies of CAL/CAS, and too much effort required to integrate CAL/CAS systems into the curriculum. To overcome the reasons for the relative low degree of computer technology use, we should strive for multicenter research and development projects monitored by the appropriate national and international scientific societies, so that the potential of computer technology can be fully realized in graduate, postgraduate, and continuing dental education.

MIDAS-FAST: Design and Validation of a Model-Based Tool to Predict Operator Performance with Robotic Arm Automation

NASA Technical Reports Server (NTRS)

Sebok, Angelia (Principal Investigator); Wickens, Christopher; Gacy, Marc; Brehon, Mark; Scott-Nash, Shelly; Sarter, Nadine; Li, Huiyang; Gore, Brian; Hooey, Becky

2017-01-01

The Coalition for Aerospace and Science (CAS) is hosting an exhibition on Capitol Hill on June 14, 2017, to highlight the contributions of CAS members to NASAs portfolio of activities. This exhibition represents an opportunity for an HFES members ground breaking work to be displayed and to build on support within Congress for NASAs human research program including in those areas that are of specific interest to the HFE community. The intent of this poster presentation is to demonstrate the positive outcome that comes from funding HFE related research on a project like the one exemplified by MIDAS-FAST.

Cultural diversity among nursing students: reanalysis of the cultural awareness scale.

PubMed

Rew, Lynn; Becker, Heather; Chontichachalalauk, Jiraporn; Lee, H Y

2014-02-01

Nurses are educated to provide culturally competent care. Cultural competence begins with cultural awareness, a concept previously measured with the Cultural Awareness Scale (CAS). The purpose of this study was to reanalyze the CAS to determine construct validity and differences in cultural awareness among students of varying educational levels and experiences. The sample consisted of 150 nursing students (92% female, 33.6% racial minorities). Confirmatory factor analysis yielded three factors (CFI = 0.868, TLI = 0.854, RMSEA = 0.065, and SRMR = 0.086). Cronbach's alpha ranged from 0.70 to 0.89. There were significant differences among educational levels, with lower division BSN students generally scoring higher than upper division and master's of science in nursing students. Students who had taken courses on cultural diversity or global health generally outscored those who had not taken such courses. Findings support the validity of the CAS and its applicability to research studies of cultural awareness in nursing. Copyright 2014, SLACK Incorporated.

A Cas9 transgenic Plasmodium yoelii parasite for efficient gene editing.

PubMed

Qian, Pengge; Wang, Xu; Yang, Zhenke; Li, Zhenkui; Gao, Han; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

2018-06-01

The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage. By introducing the plasmid (pYCs) containing only sgRNA and homologous template elements, we successfully achieved both deletion and tagging modifications for different endogenous genes in the genome of PyCas9ki parasite. This cas9-knockin PyCas9ki parasite provides a new platform facilitating gene functions study in the rodent malaria parasite P. yoelii. Copyright © 2018 Elsevier B.V. All rights reserved.

A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii*

PubMed Central

Brendel, Jutta; Stoll, Britta; Lange, Sita J.; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A.; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-01-01

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1–8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA. PMID:24459147

A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of clustered regularly interspaced short palindromic repeats (crispr)-derived rnas (crrnas) in Haloferax volcanii.

PubMed

Brendel, Jutta; Stoll, Britta; Lange, Sita J; Sharma, Kundan; Lenz, Christof; Stachler, Aris-Edda; Maier, Lisa-Katharina; Richter, Hagen; Nickel, Lisa; Schmitz, Ruth A; Randau, Lennart; Allers, Thorsten; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita

2014-03-07

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

Beta-carbonic anhydrases play a role in fruiting body development and ascospore germination in the filamentous fungus Sordaria macrospora.

PubMed

Elleuche, Skander; Pöggeler, Stefanie

2009-01-01

Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Corrective Action Investigation Plan for Corrective Action Unit 555: Septic Systems Nevada Test Site, Nevada, Rev. No.: 0 with Errata

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

Pastor, Laura

2005-12-01

This Corrective Action Investigation Plan (CAIP) contains project-specific information including facility descriptions, environmental sample collection objectives, and criteria for conducting site investigation activities at Corrective Action Unit (CAU) 555: Septic Systems, Nevada Test Site (NTS), Nevada. This CAIP has been developed in accordance with the ''Federal Facility Agreement and Consent Order'' (FFACO) (1996) that was agreed to by the State of Nevada, the U.S. Department of Energy (DOE), and the U.S. Department of Defense. Corrective Action Unit 555 is located in Areas 1, 3 and 6 of the NTS, which is approximately 65 miles (mi) northwest of Las Vegas, Nevada,more » and is comprised of the five corrective action sites (CASs) shown on Figure 1-1 and listed below: (1) CAS 01-59-01, Area 1 Camp Septic System; (2) CAS 03-59-03, Core Handling Building Septic System; (3) CAS 06-20-05, Birdwell Dry Well; (4) CAS 06-59-01, Birdwell Septic System; and (5) CAS 06-59-02, National Cementers Septic System. An FFACO modification was approved on December 14, 2005, to include CAS 06-20-05, Birdwell Dry Well, as part of the scope of CAU 555. The work scope was expanded in this document to include the investigation of CAS 06-20-05. The Corrective Action Investigation (CAI) will include field inspections, radiological surveys, geophysical surveys, sampling of environmental media, analysis of samples, and assessment of investigation results, where appropriate. Data will be obtained to support corrective action alternative evaluations and waste management decisions. The CASs in CAU 555 are being investigated because hazardous and/or radioactive constituents may be present in concentrations that could potentially pose a threat to human health and the environment. Existing information on the nature and extent of potential contamination is insufficient to evaluate and recommend corrective action alternatives for the CASs. Additional information will be generated by conducting a CAI before the evaluation and selection of corrective action alternatives.« less

The Effects of the Contact Activation System on Hemorrhage

PubMed Central

Simão, Fabrício; Feener, Edward P.

2017-01-01

The contact activation system (CAS) exerts effects on coagulation via multiple mechanisms, which modulate both the intrinsic and extrinsic coagulation cascades as well as fibrinolysis and platelet activation. While the effects of the CAS on blood coagulation measured as activated partial thromboplastin time shortening are well documented, genetic mutations that result in deficiencies in the expression of either plasma prekallikrein (PPK) or factor XII (FXII) are not associated with spontaneous bleeding or increased bleeding risk during surgery. Deficiencies in these proteins are often undiagnosed for decades and detected later in life during routine coagulation assays without an apparent clinical phenotype. Increased interest in the CAS as a potentially safe target for antithrombotic therapies has emerged, in large part, from studies on animal models with provoked thrombosis, which have shown that deficiencies in PPK or FXII can reduce thrombus formation without increasing bleeding. Gene targeting and pharmacological studies in healthy animals have confirmed that PPK and FXII blockade does not cause coagulopathies. These findings support the conclusion that CAS is not required for hemostasis. However, while deficiencies in FXII and PPK do not significantly affect bleeding associated with peripheral wounds, recent reports have demonstrated that these proteins can promote hemorrhage in the retina and brain. Intravitreal injection of plasma kallikrein (PKal) induces retinal hemorrhage and intracerebral injection of PKal increases intracranial bleeding. PPK deficiency and PKal inhibition ameliorates hematoma formation following cerebrovascular injury in diabetic animals. Moreover, both PPK and FXII deficiency are protective against intracerebral hemorrhage caused by tissue plasminogen activator-mediated thrombolytic therapy in mice with thrombotic middle cerebral artery occlusion. Thus, while the CAS is not required for hemostasis, its inhibition may provide an opportunity to reduce hemorrhage in the retina and brain. Characterization of the mechanisms and potential clinical implications associated with the effects of the CAS on hemorrhage requires further consideration of the effects of PPK and FXII on hemorrhage beyond their putative effects on coagulation cascades. Here, we review the experimental and clinical evidence on the effects of the CAS on bleeding and hemostatic mechanisms. PMID:28824910

The role of Cas8 in type I CRISPR interference.

PubMed

Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

2015-05-05

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA. © 2015 Authors.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation.

PubMed

Kieper, Sebastian N; Almendros, Cristóbal; Behler, Juliane; McKenzie, Rebecca E; Nobrega, Franklin L; Haagsma, Anna C; Vink, Jochem N A; Hess, Wolfgang R; Brouns, Stan J J

2018-03-27

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Somatic Marker Hypothesis and Sexual Decision Making: Understanding the Role of Iowa Gambling Task Performance and Daily Sexual Arousal on the Sexual Behavior of Gay and Bisexual Men.

PubMed

Rendina, H Jonathon; Millar, Brett M; Dash, Genevieve; Feldstein Ewing, Sarah W; Parsons, Jeffrey T

2018-04-19

The sexual decision-making literature suggests that sexual arousal and behavior are associated. The somatic marker hypothesis suggests that individual neuropsychological differences in decision making, as measured by the Iowa Gambling Task (IGT), may moderate these associations; however, this hypothesis has yet to be tested with event-level sexual behavior data. We hypothesized that (a) daily sexual arousal would be positively associated with likelihood of engaging in sex and condomless anal sex (CAS) and (b) IGT scores would moderate these associations such that the associations would be stronger among those with higher IGT scores. We used daily diary data from 334 highly sexually active gay and bisexual men to examine the main and interaction effects of sexual arousal and IGT scores on sexual engagement and CAS. As hypothesized, daily sexual arousal was positively associated with greater odds of both sexual engagement and CAS with casual male partners. Individual-level IGT performance significantly moderated the day-level association between arousal and sexual engagement, which was stronger for men with higher IGT scores. There was no main effect of IGT scores on either sexual behavior outcome, nor did it moderate the association between arousal and CAS. These findings highlight the influence of sexual arousal on sexual engagement, which differed by IGT scores; the effect of arousal on CAS was much less variable and may not be moderated by neurocognitive factors. This study supports the importance of exploring integrated behavioral/biomedical interventions to improve individual decision making to prevent HIV infection.

Naturally Occurring Off-Switches for CRISPR-Cas9.

PubMed

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J; Maxwell, Karen L; Davidson, Alan R

2016-12-15

CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT. Copyright © 2016 Elsevier Inc. All rights reserved.

Naturally occurring off-switches for CRISPR-Cas9

PubMed Central

Pawluk, April; Amrani, Nadia; Zhang, Yan; Garcia, Bianca; Hidalgo-Reyes, Yurima; Lee, Jooyoung; Edraki, Alireza; Shah, Megha; Sontheimer, Erik J.; Maxwell, Karen L.; Davidson, Alan R.

2017-01-01

Summary CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These “anti-CRISPRs” were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9), and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable “off-switches” for CRISPR-Cas9 activity, and provide a genetically-encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. PMID:27984730

The Revolution Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit.

PubMed

Murugan, Karthik; Babu, Kesavan; Sundaresan, Ramya; Rajan, Rakhi; Sashital, Dipali G

2017-10-05

CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.

PubMed

Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

2017-10-05

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer. The prespacer is bound by Cas1-Cas2 as a dual-forked DNA, and the terminal 3'-OH of each 3' overhang serves as an attacking nucleophile during integration. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat. Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor. In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers. Here we present four structural snapshots from the type II-A system of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3' overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.

How Type II CRISPR-Cas establish immunity through Cas1-Cas2 mediated spacer integration

PubMed Central

Xiao, Yibei; Ng, Sherwin; Nam, Ki Hyun; Ke, Ailong

2017-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1–5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6–9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical7–9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle6,10. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3′-OH of each 3′-overhang serves as an attacking nucleophile during integration11–14. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat7,15–20. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF)21,22. In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro18; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis17,23. Focusing on the Enterococcus faecalis Type II-A system24, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3′-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference. PMID:28869593

Serious Gaming for Test & Evaluation of Clean-Slate (Ab Initio) National Airspace System (NAS) Designs

NASA Technical Reports Server (NTRS)

Allen, B. Danette; Alexandrov, Natalia

2016-01-01

Incremental approaches to air transportation system development inherit current architectural constraints, which, in turn, place hard bounds on system capacity, efficiency of performance, and complexity. To enable airspace operations of the future, a clean-slate (ab initio) airspace design(s) must be considered. This ab initio National Airspace System (NAS) must be capable of accommodating increased traffic density, a broader diversity of aircraft, and on-demand mobility. System and subsystem designs should scale to accommodate the inevitable demand for airspace services that include large numbers of autonomous Unmanned Aerial Vehicles and a paradigm shift in general aviation (e.g., personal air vehicles) in addition to more traditional aerial vehicles such as commercial jetliners and weather balloons. The complex and adaptive nature of ab initio designs for the future NAS requires new approaches to validation, adding a significant physical experimentation component to analytical and simulation tools. In addition to software modeling and simulation, the ability to exercise system solutions in a flight environment will be an essential aspect of validation. The NASA Langley Research Center (LaRC) Autonomy Incubator seeks to develop a flight simulation infrastructure for ab initio modeling and simulation that assumes no specific NAS architecture and models vehicle-to-vehicle behavior to examine interactions and emergent behaviors among hundreds of intelligent aerial agents exhibiting collaborative, cooperative, coordinative, selfish, and malicious behaviors. The air transportation system of the future will be a complex adaptive system (CAS) characterized by complex and sometimes unpredictable (or unpredicted) behaviors that result from temporal and spatial interactions among large numbers of participants. A CAS not only evolves with a changing environment and adapts to it, it is closely coupled to all systems that constitute the environment. Thus, the ecosystem that contains the system and other systems evolves with the CAS as well. The effects of the emerging adaptation and co-evolution are difficult to capture with only combined mathematical and computational experimentation. Therefore, an ab initio flight simulation environment must accommodate individual vehicles, groups of self-organizing vehicles, and large-scale infrastructure behavior. Inspired by Massively Multiplayer Online Role Playing Games (MMORPG) and Serious Gaming, the proposed ab initio simulation environment is similar to online gaming environments in which player participants interact with each other, affect their environment, and expect the simulation to persist and change regardless of any individual player's active participation.

Procurement Opportunitites in: Afghanistan and the Central Asian States (CAS)

DTIC Science & Technology

2012-08-01

barriers) Furniture (office and household) Prefabricated Structures (CONEXs, containerized housing units, re-locatable buildings) Office Supplies... Concrete Foundation and Structure Road Support Activities Minor Building Renovation Water and Sewer Line and Related Construction Heavy and Civil

Methanol

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A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

PubMed

Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

2016-01-28

The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

Cas9 versus Cas12a/Cpf1: Structure-function comparisons and implications for genome editing.

PubMed

Swarts, Daan C; Jinek, Martin

2018-05-22

Cas9 and Cas12a are multidomain CRISPR-associated nucleases that can be programmed with a guide RNA to bind and cleave complementary DNA targets. The guide RNA sequence can be varied, making these effector enzymes versatile tools for genome editing and gene regulation applications. While Cas9 is currently the best-characterized and most widely used nuclease for such purposes, Cas12a (previously named Cpf1) has recently emerged as an alternative for Cas9. Cas9 and Cas12a have distinct evolutionary origins and exhibit different structural architectures, resulting in distinct molecular mechanisms. Here we compare the structural and mechanistic features that distinguish Cas9 and Cas12a, and describe how these features modulate their activity. We discuss implications for genome editing, and how they may influence the choice of Cas9 or Cas12a for specific applications. Finally, we review recent studies in which Cas12a has been utilized as a genome editing tool. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes. © 2018 Wiley Periodicals, Inc.

[Clustered regularly interspaced short palindromic repeat associated protein genes cas1 and cas2 in Shigella].

PubMed

Xue, Zerun; Wang, Yingfang; Duan, Guangcai; Wang, Pengfei; Wang, Linlin; Guo, Xiangjiao; Xi, Yuanlin

2014-05-01

To detect the distribution of clustered regularly interspaced short palindromic repeat (CRISPR) associated protein genes cas1 and cas2 in Shigella and to understand the characteristics of CRISPR with relationship between CRISPR and related characteristics on drug resistance. CRISPR associated protein genes cas1 and cas2 in Shigella were detected by PCR, with its products sequenced and compared. The CRISPR-associated protein genes cas1 and cas2 were found in all the 196 Shigella isolates which were isolated at different times and locations in China. Consistencies showed through related sequencing appeared as follows: cas2, cas1 (a) and cas1 (b) were 96.44%, 97.61% and 96.97%, respectively. There were two mutations including 3177129 site(C→G)and 3177126 site (G→C) of cas1 (b) gene in 2003135 strain which were not found in the corresponding sites of Z23 and 2008113. showed that in terms of both susceptibility and antibiotic-resistance, strain 2003135 was stronger than Z23 and 2008113. CRISPR system widely existed in Shigella, with the level of drug resistance in cas1 (b) gene mutant strains higher than in wild strains. Cas1 (b) gene mutation might be one of the reasons causing the different levels of resistance.

Lighter-Than-Air (LTA) "AirStation": Unmanned Aircraft System (UAS) Carrier Concept

NASA Technical Reports Server (NTRS)

Hochstetler, Ron; Chachad, Girish; Blanken, Matthew; Bosma, John

2016-01-01

Proposals for adapting modern airship technologies for military missions have mostly focused on exploiting the airships high flight endurance and low fuel requirement to conduct direct surveillance missions requiring high degrees of persistence over the areas to be observed. While this mission has value, it constrains the airship in two regards. (1) It places all the surveillance sensors, communication systems, and other mission equipment in the airship itself. (2) It requires the airship to be physically in the vicinity of the areas to be directly observed. A more advanced utilization of airship technology would be to add the capability to deploy a separate set of surveillance equipment, thereby enabling indirect and distributed observation operations. This can be undertaken by installing surveillance equipment in a squadron of unmanned aircraft systems (UAS) that can be carried and operated remotely from the airship, and then return to the airship as a base of support. This could be accomplished by deploying 20-30 UASs on an optionally manned (5 person crew) airship. The mission focus of the airship UAS carrier would be for support of distributed intelligence, surveillance and reconnaissance (ISR), close air support (CAS), maritime patrol and interdiction, electronic warfare (EW), persistent area dominance and missile defense. The logic for utilizing an airship carrier over a ground base to deploy UAS will be examined. Whether to be used as a stand-alone platform or in concert with conventional intelligence gathering techniques, the airship UAS carrier can provide the following benefits: a mobile base that will remain accessible despite political fallout which may render a ground base unavailable for use, the psychological impact of a power projection tool that has no geographical limits (imagined in the same way a naval carrier group projects power), cost-saving intelligence gathering over manned alternatives (assumption), and a wider area of influence when compared to an immobile ground base that must facilitate the transfer of UAS to other bases in order to overfly particular areas (all operations, launch, recover, etc. take place from the airship).

Expression of MdCAS1 and MdCAS2, encoding apple beta-cyanoalanine synthase homologs, is concomitantly induced during ripening and implicates MdCASs in the possible role of the cyanide detoxification in Fuji apple (Malus domestica Borkh.) fruits.

PubMed

Han, Sang Eun; Seo, Young Sam; Kim, Daeil; Sung, Soon-Kee; Kim, Woo Taek

2007-08-01

Fruit ripening involves complex biochemical and physiological changes. Ethylene is an essential hormone for the ripening of climacteric fruits. In the process of ethylene biosynthesis, cyanide (HCN), an extremely toxic compound, is produced as a co-product. Thus, most cyanide produced during fruit ripening should be detoxified rapidly by fruit cells. In higher plants, the key enzyme involved in the detoxification of HCN is beta-cyanoalanine synthase (beta-CAS). As little is known about the molecular function of beta-CAS genes in climacteric fruits, we identified two homologous genes, MdCAS1 and MdCAS2, encoding Fuji apple beta-CAS homologs. The structural features of the predicted polypeptides as well as an in vitro enzyme activity assay with bacterially expressed recombinant proteins indicated that MdCAS1 and MdCAS2 may indeed function as beta-CAS isozymes in apple fruits. RNA gel-blot studies revealed that both MdCAS1 and MdCAS2 mRNAs were coordinately induced during the ripening process of apple fruits in an expression pattern comparable with that of ACC oxidase and ethylene production. The MdCAS genes were also activated effectively by exogenous ethylene treatment and mechanical wounding. Thus, it seems like that, in ripening apple fruits, expression of MdCAS1 and MdCAS2 genes is intimately correlated with a climacteric ethylene production and ACC oxidase activity. In addition, beta-CAS enzyme activity was also enhanced as the fruit ripened, although this increase was not as dramatic as the mRNA induction pattern. Overall, these results suggest that MdCAS may play a role in cyanide detoxification in ripening apple fruits.

Behavioral effects induced by antitumor cleronade diterpenes from Casearia sylvestris and in silico interactions with neuron receptors.

PubMed

de Araújo, Éverton José Ferreira; de Almeida, Antônia Amanda Cardoso; Silva, Oskar Almeida; da Costa, Iwyson Henrique Fernandes; Rezende-Júnior, Luis Mário; Lima, Francisco das Chagas Alves; Cavalheiro, Alberto José; Pessoa, Cláudia; de Moraes, Manoel Odorico; Ferreira, Paulo Michel Pinheiro

2017-02-23

Casearia sylvestris is a medicinal plant traditionally used to treat snakebites, wounds, inflammation and gastric ulcers and scientific supports for have demonstrated its antitumor, antihyperlipidemic and antiparasitic properties. To assess the effects of a fraction with casearins (FC) on adult mice using classical experimental models of animal behavior and theoretical calculations to verify the interaction of Casearin X (Cas X) with neuron receptors. Animals divided in 6 groups (n=9/group) were intraperitoneally treated with vehicle (DMSO 4%), FC (2.5, 5, 10 and 25mg/kg/day) and diazepam (2mg/kg) for 7 days. Thirty minutes after the last dose of treatment, acute toxicity and behavioral experiments were performed. The highest dose of FC (25mg/kg/day) caused diarrhea, weight loss and death of one animal. Elevated plus maze test showed that lower doses [2.5mg/kg/day (36.4±5.1s) and 5mg/kg/day (43.9±6.2s)] increased the time spent in open arms (TSOA). Open field test revealed reduction in the number of crossings (54.9%, 51.1%, 48% and 67.7% for 2.5, 5, 10 and 25mg/kg/day, respectively) in all doses of FC studied and decrease of rearings at 25mg/kg/day (p<0.05). Computational calculations showed that the inhibition constant (Ki) for the Cas X-D 1 complex is up to 1000-fold more favourable than the Cas X-GABA A complex. All ∆G° values obtained for Cas X-D 1 complexes were more negative than those seen with Cas X-GABA A complexes. Findings indicate a probable anxiolytic action of the FC since it reduces the number of crossings and rearings and prolonged the time spent in open arms, without sedative and myorelaxant effects, probably due to the interaction of Cas X with dopaminergic system. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Evaluation of the inhibitory effect of dihydropyridines on N-type calcium channel by virtual three-dimensional pharmacophore modeling.

PubMed

Ogihara, Takuo; Kano, Takashi; Kakinuma, Chihaya

2009-01-01

Currently, a new type of calcium channel blockers, which can inhibit not only L-type calcium channels abundantly expressed in vascular smooth muscles, but also N-type calcium channels that abound in the sympathetic nerve endings, have been developed. In this study, analysis on a like-for-like basis of the L- and N-type calcium channel-inhibitory activity of typical dihydropyridine-type calcium-channel blockers (DHPs) was performed. Moreover, to understand the differences of N-type calcium channel inhibition among DHPs, the binding of DHPs to the channel was investigated by means of hypothetical three-dimensional pharmacophore modeling using multiple calculated low-energy conformers of the DHPs. All of the tested compounds, i.e. cilnidipine (CAS 132203-70-4), efonidipine (CAS 111011-76-8), amlodipine (CAS 111470-99-6), benidipine (CAS 85387-35-5), azelnidipine (CAS 123524-52-7) and nifedipine (CAS 21829-25-4), potently inhibited the L-type calcium channel, whereas only cilnidipine inhibited the N-type calcium channel (IC50 value: 51.2 nM). A virtual three-dimensional structure of the N-type calcium channel was generated by using the structure of the peptide omega-conotoxin GVIA, a standard inhibitor of the channel, and cilnidipine was found to fit well into this pharmacophore model. Lipophilic potential maps of omega-conotoxin GVIA and cilnidipine supported this finding. Conformational overlay of cilnidipine and the other DHPs indicated that amlodipine and nifedipine were not compatible with the pharmacophore model because they did not contain an aromatic ring that was functionally equivalent to Tyr13 of omega-conotoxin GVIA. Azelnidipine, benidipine, and efonidipine, which have this type of aromatic ring, were not positively identified due to intrusions into the excluded volume. Estimation of virtual three-dimensional structures of proteins, such as ion channels, by using standard substrates and/or inhibitors may be a useful method to explore the mechanisms of pharmacological and toxicological effects of substrates and/or inhibitors, and to discover new drugs.

Reframing the challenges to integrated care: a complex-adaptive systems perspective.

PubMed

Tsasis, Peter; Evans, Jenna M; Owen, Susan

2012-01-01

Despite over two decades of international experience and research on health systems integration, integrated care has not developed widely. We hypothesized that part of the problem may lie in how we conceptualize the integration process and the complex systems within which integrated care is enacted. This study aims to contribute to discourse regarding the relevance and utility of a complex-adaptive systems (CAS) perspective on integrated care. In the Canadian province of Ontario, government mandated the development of fourteen Local Health Integration Networks in 2006. Against the backdrop of these efforts to integrate care, we collected focus group data from a diverse sample of healthcare professionals in the Greater Toronto Area using convenience and snowball sampling. A semi-structured interview guide was used to elicit participant views and experiences of health systems integration. We use a CAS framework to describe and analyze the data, and to assess the theoretical fit of a CAS perspective with the dominant themes in participant responses. Our findings indicate that integration is challenged by system complexity, weak ties and poor alignment among professionals and organizations, a lack of funding incentives to support collaborative work, and a bureaucratic environment based on a command and control approach to management. Using a CAS framework, we identified several characteristics of CAS in our data, including diverse, interdependent and semi-autonomous actors; embedded co-evolutionary systems; emergent behaviours and non-linearity; and self-organizing capacity. One possible explanation for the lack of systems change towards integration is that we have failed to treat the healthcare system as complex-adaptive. The data suggest that future integration initiatives must be anchored in a CAS perspective, and focus on building the system's capacity to self-organize. We conclude that integrating care requires policies and management practices that promote system awareness, relationship-building and information-sharing, and that recognize change as an evolving learning process rather than a series of programmatic steps.

Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.

PubMed

Yamada, Mari; Watanabe, Yuto; Gootenberg, Jonathan S; Hirano, Hisato; Ran, F Ann; Nakane, Takanori; Ishitani, Ryuichiro; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2017-03-16

The RNA-guided endonuclease Cas9 generates a double-strand break at DNA target sites complementary to the guide RNA and has been harnessed for the development of a variety of new technologies, such as genome editing. Here, we report the crystal structures of Campylobacter jejuni Cas9 (CjCas9), one of the smallest Cas9 orthologs, in complex with an sgRNA and its target DNA. The structures provided insights into a minimal Cas9 scaffold and revealed the remarkable mechanistic diversity of the CRISPR-Cas9 systems. The CjCas9 guide RNA contains a triple-helix structure, which is distinct from known RNA triple helices, thereby expanding the natural repertoire of RNA triple helices. Furthermore, unlike the other Cas9 orthologs, CjCas9 contacts the nucleotide sequences in both the target and non-target DNA strands and recognizes the 5'-NNNVRYM-3' as the protospacer-adjacent motif. Collectively, these findings improve our mechanistic understanding of the CRISPR-Cas9 systems and may facilitate Cas9 engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of peer-mediated instruction to teach use of speech-generating devices to students with autism in social game routines.

PubMed

Trottier, Nadine; Kamp, Lorraine; Mirenda, Pat

2011-03-01

Supporting social interactions between students with autism spectrum disorders (ASDs) and their typically developing peers presents many challenges. The purpose of this study was to investigate the effects of a peer-mediated intervention designed to teach two students with ASD to use speech-generating devices (SGDs) to engage in interactions with peers in a social context at school. Six peer confederates (three from each student with ASD's general education classroom) were taught to support SGD use during game activities. A multiple baseline design was used to examine the relationship between peer-mediated instruction and an increase in total communicative acts (CAs) by the two students with ASD. Results provide evidence that the confederates acquired the skills needed to support SGD use by students with ASD. The results also suggest that the intervention was effective at increasing total appropriate CAs by students with ASD. In addition, social validity ratings by all of the confederates were positive. Results are discussed regarding educational implications, limitations, and future research.

Structure and Engineering of Francisella novicida Cas9

PubMed Central

Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2016-01-01

Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9.

PubMed

Tsui, Tsz Kin Martin; Hand, Travis H; Duboy, Emily C; Li, Hong

2017-06-16

Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be complementary to the guide region, typically 20-nucleotides in length, of the Cas9-bound guide RNA, and adjacent to a short Cas9-specific element called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends on a 5'-NNNCC-3' PAM and is more efficient in cleaving negative supercoils than relaxed DNA. Kinetic as well as in vivo activity assays reveal that AceCas9 achieves optimal activity when combined with a guide RNA containing a 24-nucleotide complementarity region. The cytosine-specific, DNA topology-sensitive, and extended guide-dependent properties of AceCas9 may be explored for specific genome editing applications.

Structure and Engineering of Francisella novicida Cas9.

PubMed

Hirano, Hisato; Gootenberg, Jonathan S; Horii, Takuro; Abudayyeh, Omar O; Kimura, Mika; Hsu, Patrick D; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

2016-02-25

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox. Copyright © 2016 Elsevier Inc. All rights reserved.

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

PubMed

Nakajima, Osamu; Nishimaki-Mogami, Tomoko; Kondo, Kazunari

2016-01-01

Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

Childhood adversities and first onset of psychiatric disorders in a national sample of adolescents

PubMed Central

McLaughlin, Katie A.; Green, Jennifer Greif; Gruber, Michael J.; Sampson, Nancy A.; Zaslavsky, Alan M.; Kessler, Ronald C.

2012-01-01

Context Although childhood adversities (CAs) are known to be highly co-occurring, most research examines their associations with mental disorders one at a time. Recent evidence from adult studies suggests, though, that the associations of multiple CAs with mental disorders are non-additive, arguing for the importance of multivariate analysis of multiple CAs. No attempt has yet been made to carry out a similar kind of analysis among children or adolescents. Objective To examine the multivariate associations of 12 CAs with first onset of mental disorders in a national sample of US adolescents. Design US national survey of adolescents (ages 13–17) assessing DSM-IV anxiety, mood, behavior, and substance disorders and CAs. The CAs include parental loss (death, divorce, other separations), maltreatment (physical, sexual, and emotional abuse, neglect), parental maladjustment (psychopathology, substance abuse, criminality, violence) and economic adversity. Setting Dual-frame household-school samples. Participants 6,483 adolescents-parent pairs. Main outcome measure Lifetime DSM-IV disorders assessed with the WHO Composite International Diagnostic Interview. Results 58.3% of adolescents reported at least one CA, among whom 59.7% reported multiple CAs. CAs reflecting maladaptive family functioning (MFF) were more strongly associated than other CAs with disorder onsets. The best-fitting model included terms for type and number of CAs and distinguished between MFF and Other CAs. CAs predicted behavior disorders most strongly and fear disorders least strongly. The joint associations of multiple CAs were sub-additive. The population-attributable risk proportions for disorder classes ranged from 15.7% for fear disorders to 40.7% for behavior disorders. CAs were associated with 28.2% of all onsets. Conclusions CAs are common, highly co-occurring, and strongly associated with onset of mental disorders among US adolescents. The sub-additive multivariate associations of CAs with disorder onsets have implications for targeting interventions to reduce exposure to CAs and to mitigate the harmful effects of CAs to improve population mental health. PMID:23117636

Carbon tetrachloride

Integrated Risk Information System (IRIS)

EPA / 635 / R - 08 / 005F www.epa.gov / iris TOXICOLOGICAL REVIEW OF CARBON TETRACHLORIDE ( CAS No . 56 - 23 - 5 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) March 2010 U.S . Environmental Protection Agency Washington , DC ii DISCLAIMER This document has bee

1,3-Dichloropropene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 00 / 001 TOXICOLOGICAL REVIEW OF 1,3 - DICHLOROPROPENE ( CAS No . 542 - 75 - 6 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) May 2000 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in a

Trichloroacetic acid

Integrated Risk Information System (IRIS)

EPA / 635 / R - 09 / 003F www.epa.gov / iris TOXICOLOGICAL REVIEW OF TRICHLOROACETIC ACID ( CAS No . 76 - 03 - 9 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) September 2011 U.S . Environmental Protection Agency Washington , DC ii DISCLAIMER This document has

1,4-Dioxane

Integrated Risk Information System (IRIS)

EPA / 635 / R - 11 / 003F www.epa.gov / iris TOXICOLOGICAL REVIEW OF 1,4 - DIOXANE ( WITH INHALATION UPDATE ) ( CAS No . 123 - 91 - 1 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) September 2013 U.S . Environmental Protection Agency Washington , DC DISCLAIMER

Beryllium and compounds

Integrated Risk Information System (IRIS)

EPA / 635 / R - 98 / 008 TOXICOLOGICAL REVIEW OF BERYLLIUM AND COMPOUNDS ( CAS No . 7440 - 41 - 7 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) April 1998 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has been reviewed in ac

All-in-One CRISPR-Cas9/FokI-dCas9 Vector-Mediated Multiplex Genome Engineering in Cultured Cells.

PubMed

Sakuma, Tetsushi; Sakamoto, Takuya; Yamamoto, Takashi

2017-01-01

CRISPR-Cas9 enables highly convenient multiplex genome engineering in cultured cells, because it utilizes generic Cas9 nuclease and an easily customizable single-guide RNA (sgRNA) for site-specific DNA double-strand break induction. We previously established a multiplex CRISPR-Cas9 assembly system for constructing an all-in-one vector simultaneously expressing multiple sgRNAs and Cas9 nuclease or other Cas9 variants including FokI-dCas9, which supersedes the wild-type Cas9 with regard to high specificity. In this chapter, we describe a streamlined protocol to design and construct multiplex CRISPR-Cas9 or FokI-dCas9 vectors, to introduce them into cultured cells by lipofection or electroporation, to enrich the genomically edited cells with a transient puromycin selection, to validate the mutation efficiency by Surveyor nuclease assay, and to perform off-target analyses. We show that our protocol enables highly efficient multiplex genome engineering even in hard-to-transfect HepG2 cells.

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field.

PubMed

He, Zhi-Yao; Men, Ke; Qin, Zhou; Yang, Yang; Xu, Ting; Wei, Yu-Quan

2017-05-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs (sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies. Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.

Interactions between iron(III)-hydroxide polymaltose complex and commonly used medications / laboratory studies in rats.

PubMed

Funk, Felix; Canclini, Camillo; Geisser, Peter

2007-01-01

Simple iron salts, such as iron sulphate, often interact with food and other medications reducing bioavailability and tolerability. Iron(III)-hydroxide polymaltose complex (IPC, Maltofer) provides a soluble form of non-ionic iron, making it an ideal form of oral iron supplementation. The physicochemical properties of IPC predict a low potential for interactions. The effects of co-administration with aluminium hydroxide (CAS 21645-51-2), acetylsalicylic acid (CAS 50-78-2), bromazepam (CAS 1812-30-2), calcium acetate (CAS 62-54-4), calcium carbonate (CAS 471-34-1), auranofin (CAS 34031-32-8), magnesium-L-aspartate hydrochloride (CAS 28184-71-6), methyldopa sesquihydrate (CAS 41372-08-1), paracetamol (CAS 103-90-2), penicillamine (CAS 52-67-5), sulfasalazine (CAS 599-79-1), tetracycline hydrochloride (CAS 64-75-5), calcium phosphate (CAS 7757-93-9) in combination with vitamin D3 (CAS 67-97-0), and a multi-vitamin preparation were tested in rats fed an iron-deficient diet. Uptake of iron from radiolabelled IPC with and without concomitant medications was compared. None of the medicines tested had a significant effect on iron uptake. Iron-59 retrieval from blood and major storage organs was 64-76% for IPC alone compared with 59-85% following co-administration with other medications. It is concluded that, under normal clinical conditions, IPC does not interact with these medications.

Genome Editing with CRISPR-Cas9: Can It Get Any Better?

PubMed Central

Haeussler, Maximilian; Concordet, Jean-Paul

2017-01-01

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage. PMID:27210042

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish

PubMed Central

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites (tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. PMID:29295818

CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference.

PubMed

Hochstrasser, Megan L; Taylor, David W; Bhat, Prashant; Guegler, Chantal K; Sternberg, Samuel H; Nogales, Eva; Doudna, Jennifer A

2014-05-06

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Rational Design of Mini-Cas9 for Transcriptional Activation.

PubMed

Ma, Dacheng; Peng, Shuguang; Huang, Weiren; Cai, Zhiming; Xie, Zhen

2018-04-20

Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.

Generic Methodology for Verification and Validation (GM-VV) to Support Acceptance of Models, Simulations and Data (Methodologie generale de verification et de validation (GM-VV) visant a soutenir l acceptation des modeles, simulations et donnees)

DTIC Science & Technology

2015-01-01

RTO ou AGARD doivent comporter la dénomination « STO », « RTO » ou « AGARD » selon le cas, suivi du numéro de série. Des informations analogues...rapports de la STO au fur et à mesure de leur publication, vous pouvez consulter notre site Web (http://www.sto.nato.int/) et vous abonner à ce service...le cas, suivie du numéro de série (par exemple AGARD-AG-315). Des informations analogues, telles que le titre et la date de publication sont

Issues Management Process Course # 38401

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

Binion, Ula Marie

The purpose of this training it to advise Issues Management Coordinators (IMCs) on the revised Contractor Assurance System (CAS) Issues Management (IM) process. Terminal Objectives: Understand the Laboratory’s IM process; Understand your role in the Laboratory’s IM process. Learning Objectives: Describe the IM process within the context of the CAS; Describe the importance of implementing an institutional IM process at LANL; Describe the process flow for the Laboratory’s IM process; Apply the definition of an issue; Use available resources to determine initial screening risk levels for issues; Describe the required major process steps for each risk level; Describe the personnelmore » responsibilities for IM process implementation; Access available resources to support IM process implementation.« less

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

USDA-ARS?s Scientific Manuscript database

The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization. | Office of Cancer Genomics

Cancer.gov

The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter.

Cas9, Cpf1 and C2c1/2/3―What's next?

PubMed Central

Yamamoto, Takashi; Sakuma, Tetsushi

2017-01-01

ABSTRACT Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012–2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond. PMID:28140746

Cell-type-specific genome editing with a microRNA-responsive CRISPR–Cas9 switch

PubMed Central

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J. C.; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut

2017-01-01

Abstract The CRISPR–Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR–Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. PMID:28525578

Cas9, Cpf1 and C2c1/2/3-What's next?

PubMed

Nakade, Shota; Yamamoto, Takashi; Sakuma, Tetsushi

2017-05-04

Since the rapid emergence of clustered regulatory interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system, developed as a genome engineering tool in 2012-2013, most researchers in the life science field have had a fixated interest in this fascinating technology. CRISPR-Cas9 is an RNA-guided DNA endonuclease system, which consists of Cas9 nuclease defining a few targeting base via protospacer adjacent motif complexed with easily customizable single guide RNA targeting around 20-bp genomic sequence. Although Streptococcus pyogenes Cas9 (SpCas9), one of the Cas9 proteins that applications in genome engineering were first demonstrated, still has wide usage because of its high nuclease activity and broad targeting range, there are several limitations such as large molecular weight and potential off-target effect. In this commentary, we describe various improvements and alternatives of CRISPR-Cas systems, including engineered Cas9 variants, Cas9 homologs, and novel Cas proteins other than Cas9. These variations enable flexible genome engineering with high efficiency and specificity, orthogonal genetic control at multiple gene loci, gene knockdown, or fluorescence imaging of transcripts mediated by RNA targeting, and beyond.

Corrective Action Decision Document/Closure Report for Corrective Action Unit 482: Area 15 U15a/e Muckpiles and Ponds Nevada Test Site

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

NSTec Environmental Restoration

This Corrective Action Decision Document /Closure Report (CADD/CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 482 U15a/e Muckpiles and Ponds. This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order agreed to by the State of Nevada, the U.S. Department of Energy, and the U.S. Department of Defense. Corrective Action Unit 482 is comprised of three Corrective Action Sites (CASs) and one adjacent area: CAS 15-06-01, U15e Muckpile; CAS 15-06-02, U15a Muckpile; CAS 15-38-01, Area 15 U15a/e Ponds; and Drainage below the U15a Muckpile. The purpose of thismore » CADD/CR is to provide justification and documentation supporting the recommendation for closure with no further corrective action, by placing use restrictions on the three CASs and the adjacent area of CAU 482. To support this recommendation, a corrective action investigation (CAI) was performed in September 2002. The purpose of the CAI was to fulfill the following data needs as defined during the Data Quality Objective (DQO) process: (1) Determine whether contaminants of concern (COCs) are present. (2) If COCs are present, determine their nature and extent. (3) Provide sufficient information and data to determine appropriate corrective actions. The CAU 482 dataset from the CAI was evaluated based on the data quality indicator parameters. This evaluation demonstrated the quality and acceptability of the dataset for use in fulfilling the DQO data needs. Analytes detected during the CAI were evaluated against final action levels (FALs) established in this document. Tier 2 FALS were determined for the hazardous constituents of total petroleum hydrocarbons (TPH)-diesel-range organics (DRO) and the radionuclides americium (Am)-241, cesium (Cs)-137, plutonium (Pu)-238, and Pu-239. The Tier 2 FALs were calculated for the radionuclides using site-specific information. The hazardous constituents of TPH-DRO were compared to the PALs defined in the CAIP, and because none of the preliminary action levels (PALs) were exceeded, the PALs became the FALs. The radionuclide FALs were calculated using the Residual Radioactive (RESRAD) code (version 6.21). The RESRAD calculation determined the activities of all radionuclides that together would sum to an exposure dose of 25 millirem per year to a site receptor (based on their relative abundances at each CAS). Based on the field investigation, the following contaminants were determined to be present at concentrations exceeding their corresponding FALs: (1) CAS 15-06-01 - None. (2) CAS 15-06-02 - Cs-137 and Pu-239. (3) CAS 15-38-01 - Am-241, Cs-137, Pu-238, and Pu-239. (4) Drainage below CAS 15-06-02 - Cs-137 and Pu-239. Based on the data and risk evaluations, the DQO data needs presented in the Corrective Action Investigation Plan were met, and the data accurately represent the radiological and chemical risk present at CAU 482. Based on the results of the CAI data evaluation, it was determined that closure in place with use restrictions is the appropriate corrective action for CAU 482 and that use restrictions will effectively control exposure to future land users. This is based on the fact that even though the FALs were exceeded in a few samples, this remote, controlled access site poses only limited risk overall to public health and the environment. Given the relatively low levels of contamination present, it would create a greater hazard to worker safety, public health, and the environment to remove the contamination, transport it, and bury it at another location. Therefore, DTRA provides the following recommendations: (1) Close COCs in place at CAS 15-06-02, CAS 15-38-01, and the drainage below CAS 15-06-02 with use restrictions. (2) No further action for CAU 482. (3) A Notice of Completion be issued to DTRA by the Nevada Division of Environmental Protection for closure of CAU 482. (4) Move CAU 482 from Appendix III to Appendix IV of the Federal Facility Agreement and Consent Order.« less

A ruler protein in a complex for antiviral defense determines the length of small interfering CRISPR RNAs.

PubMed

Hatoum-Aslan, Asma; Samai, Poulami; Maniv, Inbal; Jiang, Wenyan; Marraffini, Luciano A

2013-09-27

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation.

Cerium Oxide and Cerium Compounds

Integrated Risk Information System (IRIS)

EPA / 635 / R - 08 / 002F www.epa.gov / iris TOXICOLOGICAL REVIEW OF Cerium Oxide and Cerium Compounds ( CAS No . 1306 - 38 - 3 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) September 2009 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This

Methylene Diphenyl Diisocyanate (monomeric MDI) and polymeric MDI (PMDI)

Integrated Risk Information System (IRIS)

TOXICOLOGICAL REVIEW of METHYLENE DIPHENYL DIISOCYANATE ( MDI ) ( CAS No . 101 - 68 - 8 and 9016 - 87 - 9 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) February 1998 U.S . ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , DC TABLE OF CONTENTS TOXICOLOGICAL REV

Urea

Integrated Risk Information System (IRIS)

EPA / 635 / R - 10 / 005F www.epa.gov / iris TOXICOLOGICAL REVIEW OF UREA ( CAS No . 57 - 13 - 6 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) July 2011 U.S . Environmental Protection Agency Washington , DC ii DISCLAIMER This document has been reviewed in acc

Thallium (I), soluble salts

Integrated Risk Information System (IRIS)

EPA / 635 / R - 08 / 001F www.epa.gov / iris TOXICOLOGICAL REVIEW OF THALLIUM AND COMPOUNDS ( CAS No . 7440 - 28 - 0 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) September 2009 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document ha

Barium and Compounds

Integrated Risk Information System (IRIS)

EPA / 635 / R - 05 / 001 www.epa.gov / iris TOXICOLOGICAL REVIEW OF BARIUM AND COMPOUNDS ( CAS No . 7440 - 39 - 3 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) March 1998 Minor revisions January 1999 Reference dose revised June 2005 U.S . Environmental Protec

1,1,1-Trichloroethane

Integrated Risk Information System (IRIS)

EPA / 635 / R - 03 / 013 www.epa.gov / iris TOXICOLOGICAL REVIEW OF 1,1,1 - TRICHLOROETHANE ( CAS No . 71 - 55 - 6 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) August 2007 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This document has bee

Increased collagen maturity with sildenafil citrate: experimental high risk colonic anastomosis model.

PubMed

Cakir, Tebessum; Ozer, Ilter; Bostanci, Erdal Birol; Keklik, Tulay Timucin; Ercin, Ugur; Bilgihan, Ayse; Akoglu, Musa

2015-01-01

Inadequate healing and high anastomosis leak rates at rectal anastomosis may be due to lack of supportive serosal layer and technical difficulty of low anterior resections. Positive effects of sildenafil on wound healing were observed. The aim of this study was to simulate rectal anastomosis as a technical insufficient anastomosis and investigate the effects of sildenafil on anastomosis healing. Colonic anastomoses were carried out in 64 rats and randomized into four groups, CA-S, complete anastomoses without sildenafil (10 mg/kg for 5 days); CA+S, complete anastomoses with sildenafil; IA-S, incomplete anastomoses without sildenafil; IA+S, incomplete anastomoses with sildenafil. Half of the rats in every group were sacrificed on post-operative day (POD) 3, half of them sacrificed on POD 7. Tissues from the anastomoses were used for functional, histochemical, biochemical investigations. Sildenafil treatment resulted in increased bursting pressures in IA+S on POD 7 (p=0.010). Collagen maturity was higher in IA+S on POD 3 and POD 7, CA+S on POD 7 (p=0.010; p=0.010; p<0.007). Collagen content was higher in IA+S on POD 7 (p<0.001). Glutathione, hydroxyproline levels were similar. Malondialdehyde levels were lower in IA+S on POD 3 (p<0.001). Epithelization score was higher in IA+S on POD 7 (p=0.007). Inflammation score was higher in CA-S group on POD 3 and POD 7 (p<0.001; p<0.001). Neutrophil score was lower in CA+S on POD 3 (p=0.005). An increase in collagen content, maturity, and epithelization, a decrease in neutrophil infiltration, oxidative stress and better mechanical strength were observed with the administration of sildenafil. Copyright © 2014 Surgical Associates Ltd. Published by Elsevier Ltd. All rights reserved.

Determination of the number of ψ(3686) events at BESIII

NASA Astrophysics Data System (ADS)

Ablikim, M.; Achasov, M. N.; Ai, X. C.; Ambrose, D. J.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Baldini Ferroli, R.; Ban, Y.; Bennett, J. V.; Bertani, M.; Bian, J. M.; Boger, E.; Bondarenko, O.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Chu, X. K.; Chu, Y. P.; Cronin-Hennessy, D.; Dai, H. L.; Dai, J. P.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Du, S. X.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, Y.; Fava, L.; Feldbauer, F.; Feng, C. Q.; Fu, C. D.; Gao, Q.; Gao, Y.; Goetzen, K.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, Y. P.; Han, Y. L.; Harris, F. A.; He, K. L.; He, M.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, H. M.; Hu, T.; Huang, G. S.; Huang, J. S.; Huang, L.; Huang, X. T.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. L.; Jiang, X. S.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Kloss, B.; Kopf, B.; Kornicer, M.; Kupsc, A.; Kühn, W.; Lai, W.; Lange, J. S.; Lara, M.; Larin, P.; Li, C. H.; Li, Cheng; Li, D. M.; Li, F.; Li, G.; Li, H. B.; Li, J. C.; Li, Kang; Li, Ke; Li, Lei; Li, P. R.; Li, Q. J.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, X. R.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B. J.; Liu, C. X.; Liu, F. H.; Liu, Fang.; Liu, Feng.; Liu, H. B.; Liu, H. M.; Liu, Huihui.; Liu, J.; Liu, J. P.; Liu, K.; Liu, K. Y.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqiang.; Liu, Zhiqing.; Loehner, H.; Lou, X. C.; Lu, H. J.; Lu, H. L.; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lv, M.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, Q. M.; Ma, S.; Ma, T.; Ma, X. Y.; Maas, F. E.; Maggiora, M.; Mao, Y. J.; Mao, Z. P.; Messchendorp, J. G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Moriya, K.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Ping, J. L.; Ping, R. G.; Poling, R.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ripka, M.; Rong, G.; Sarantsev, A.; Schoenning, K.; Shan, W.; Shao, M.; Shen, C. P.; Shen, X. Y.; Sheng, H. Y.; Shepherd, M. R.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Toth, D.; Uman, I.; Varner, G. S.; Wang, B.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, Q. J.; Wang, W.; Wang, X. F.; Wang(Yadi, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. Y.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, W. B.; Yan, Y. H.; Yang, H. X.; Yang, Y.; Yang, Y. X.; Ye, H.; Ye, M.; Ye, M. H.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, Y.; Zafar, A. A.; Zeng, Y.; Zhang, B. X.; Zhang, B. Y.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, L.; Zhang, R.; Zhang, S. H.; Zhang, X. J.; Zhang, X. Y.; Zhang, Y. H.; Zhang, Yao.; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling.; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2018-02-01

The numbers of ψ(3686) events accumulated by the BESIII detector for the data taken during 2009 and 2012 are determined to be (107.0+/- 0.8)× {10}6 and (341.1+/- 2.1)× {10}6, respectively, by counting inclusive hadronic events, where the uncertainties are systematic and the statistical uncertainties are negligible. The number of events for the sample taken in 2009 is consistent with that of the previous measurement. The total number of ψ(3686) events for the two data taking periods is (448.1+/- 2.9)× {10}6. Supported by the Ministry of Science and Technology of China (2009CB825200), National Natural Science Foundation of China (NSFC) (11235011, 11322544, 11335008, 11425524, 11475207), the Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, the Collaborative Innovation Center for Particles and Interactions (CICPI), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (11179014), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (11179007, U1232201, U1532257, U1532258), Joint Funds of the National Natural Science Foundation of China (11079008), CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, National 1000 Talents Program of China, German Research Foundation DFG (Collaborative Research Center CRC 1044), Istituto Nazionale di Fisica Nucleare, Italy, Koninklijke Nederlandse Akademie van Wetenschappen (KNAW) (530-4CDP03), Ministry of Development of Turkey (DPT2006K-120470), National Natural Science Foundation of China (11205082), The Swedish Research Council, U. S. Department of Energy (DE-FG02-05ER41374, DE-SC-0010118, DE-SC-0010504), U.S. National Science Foundation, University of Groningen (RuG) and the Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0).

Carotid artery stenting in patients with symptomatic carotid stenosis: a single-center series.

PubMed

Kessler, I; Gory, B; Macian, F; Nakiri, G; Al-Khawaldeh, M; Riva, R; Boncoeur, M P; Mounayer, C

2013-03-01

Carotid angioplasty with stenting (CAS) in patients with carotid stenosis (CS) has become more restricted in France especially since the disclosure of such studies as EVA-3S and Stent-supported percutaneous angioplasty of the carotid artery versus endarterectomy (SPACE). This report is of a series of CS cases contraindicated for endarterectomy that underwent CAS at a French center of interventional neuroradiology. Fifty-five patients with symptomatic CS more than 60% consecutively submitted to CAS between September 2008 and February 2011. The primary endpoint was either death or stroke within 30 days of the procedure; a secondary goal was to identify any possible factors that might have influenced the success and outcome of the intervention. The overall periprocedural stroke/death rate at 30 days was 5.4% (three out of 55 patients), with three non-disabling strokes and no deaths. Twenty-seven patients (49.1%) were treated with a cerebral protection device (CPD). Stent placement was achieved in all cases. Open- and closed-cell stents were implanted in 40 (72.7%) and 15 procedures (27.3%), respectively. Neither the use of a CPD, the carotid stent cell design nor any anatomical or technical factors were associated with a lower risk of stroke or death within 30 days of CAS. CAS in symptomatic patients with CS contraindicated for endarterectomy in this selected French series proved feasible and safe, with acceptable levels of morbidity. Use of a CPD, type of stent (open- or closed-cell), and anatomical and technical factors had no influence on the success of the procedure or the outcome within 30 days of the operation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Measurements of the center-of-mass energies at BESIII via the di-muon process

NASA Astrophysics Data System (ADS)

Ablikim, M.; N. Achasov, M.; C. Ai, X.; Albayrak, O.; Albrecht, M.; J. Ambrose, D.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Baldini, Ferroli R.; Ban, Y.; Bennett, D. W.; Bennett, J. V.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, H. Y.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Cheng, H. P.; Chu, X. K.; Cibinetto, G.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Y. Deng, Z.; Denig, A.; Denysenko, I.; Destefanis, M.; De Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Du, S. X.; Duan, P. F.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, X.; Fang, Y.; Fava, L.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, X. L.; Gao, X. Y.; Gao, Y.; Gao, Z.; Garzia, I.; Goetzen, K.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, L. B.; Guo, Y.; Guo, Y. P.; Haddadi, Z.; Hafner, A.; Han, S.; Q. Hao, X. Q.; Harris, F. A.; He, K. L.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, C.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. M.; Huang, G. S.; Huang, J. S.; Huang, X. T.; Huang Y.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. W.; Jiang, X. S.; Jiang, X. Y.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Kiese, P.; Kliemt, R.; Kloss, B.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kühn, W.; Kupsc, A.; Lange, J. S.; Lara, M.; Larin, P.; Leng, C.; Li, C.; Cheng, Li; Li, D. M.; Li, F.; Li, F. Y.; Li, G.; Li, H. B.; Li, J. C.; Li, Jin; Li, K.; Li, K.; Li, Lei; Li, P. R.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. M.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, X.; Liu, B. J.; Liu, C. X.; Liu, D.; Liu, F. H.; Fang, Liu; Feng, Liu; Liu, H. B.; Liu, H. H.; Liu, H. H.; Liu, H. M.; Liu, J.; Liu, J. B.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, L. D.; Liu, P. L.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqing; Loehner, H.; Lou, X. C.; Lu, H. J; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, Q. M.; Ma, T.; Ma, X. N.; Ma, X. Y.; Maas, F. E.; Maggiora, M.; Mao, Y. Y.; Mao, Z. P.; Marcello, S.; Messchendorp, J. G.; Min, J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Moriya, K.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pan, Y.; Patteri, P.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Pettersson, J.; Ping, J. L.; Ping, R. G.; Poling, R.; Prasad, V.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, L. Q.; Qin, N.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ripka, M.; Rong, G.; Rosner, Ch.; Ruan, X. D.; Santoro, V.; Sarantsev, A. A.; Savrié, M.; Schoenning, B. K.; Schumann, S.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Tiemens, M.; Ullrich, M.; Uman, I.; Varner, G. S.; Wang, B.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, S. G.; Wang, W.; Wang, W. P.; Wang, X. F.; Wang, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. H.; Wang, Z. Y.; Weber, T.; Wei, D. H.; Wei, J. B.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, H.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. J.; Yang, H. X.; Yang, L.; Yang, Y.; Yang, Y. X.; Ye, M.; Ye, M. H.; Yin, J. H.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, W. L.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zallo, A.; Zeng, A. Y.; Zeng, Z.; Zhang, B. X.; Zhang, B. Y.; Zhang, C.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. N.; Zhang, Y. H.; Zhang, Y. T.; Zhang, Yu; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, S.; , S. H.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zotti, L.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2016-06-01

From 2011 to 2014, the BESIII experiment collected about 5 fb-1 data at center-of-mass energies around 4 GeV for the studies of the charmonium-like and higher excited charmonium states. By analyzing the di-muon process e+e- → γISR/FSRμ+μ-, the center-of-mass energies of the data samples are measured with a precision of 0.8 MeV. The center-of-mass energy is found to be stable for most of the time during data taking. Supported by National Key Basic Research Program of China (2015CB856700), National Natural Science Foundation of China (11125525, 11235011, 11322544, 11335008, 11425524, Y61137005C), Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, CAS Center for Excellence in Particle Physics (CCEPP), Collaborative Innovation Center for Particles and Interactions (CICPI), Joint Large-Scale Scientific Facility Funds of NSFC and CAS (11179007, U1232201, U1332201), CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, National 1000 Talents Program of China, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology, German Research Foundation DFG (Collaborative Research Center CRC-1044), Istituto Nazionale di Fisica Nucleare, Italy, Ministry of Development of Turkey (DPT2006K-120470), Russian Foundation for Basic Research (14-07-91152), Swedish Research Council, U. S. Department of Energy (DE-FG02-04ER41291, DE-FG02-05ER41374, DE-FG02-94ER40823, DESC0010118), U.S. National Science Foundation, University of Groningen (RuG) and Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0).

Measurement of the absolute branching fraction of D+ → K̅0 e+νe via K̅0 → π 0 π 0

NASA Astrophysics Data System (ADS)

Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Baldini Ferroli, R.; Ban, Y.; Bennett, D. W.; Bennett, J. V.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, H. Y.; Chen, J. C.; Chen, M. L.; Chen, S.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Cheng, H. P.; Chu, X. K.; Cibinetto, G.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; De Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Dou, Z. L.; Du, S. X.; Duan, P. F.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, X.; Fang, Y.; Farinelli, R.; Fava, L.; Fedorov, O.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, X. L.; Gao, X. Y.; Gao, Y.; Gao, Z.; Garzia, I.; Goetzen, K.; Gong, L.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, L. B.; Guo, R. P.; Guo, Y.; Guo, Y. P.; Haddadi, Z.; Hafner, A.; Han, S.; Hao, X. Q.; Harris, F. A.; He, K. L.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, C.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. S.; Huang, J. S.; Huang, X. T.; Huang, X. Z.; Huang, Y.; Huang, Z. L.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. W.; Jiang, X. S.; Jiang, X. Y.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Kiese, P.; Kliemt, R.; Kloss, B.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kupsc, A.; Kühn, W.; Lange, J. S.; Lara, M.; Larin, P.; Leng, C.; Li, C.; Li, Cheng; Li, D. M.; Li, F.; Li, F. Y.; Li, G.; Li, H. B.; Li, H. J.; Li, J. C.; Li, Jin; Li, K.; Li, K.; Li, Lei; Li, P. R.; Li, Q. Y.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, Y. B.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B.; Liu, B. J.; Liu, C. X.; Liu, D.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. H.; Liu, H. H.; Liu, H. M.; Liu, J.; Liu, J. B.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, L. D.; Liu, P. L.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqing; Loehner, H.; Lou, X. C.; Lü, H. J.; Lü, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lü, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, M. M.; Ma, Q. M.; Ma, T.; Ma, X. N.; Ma, X. Y.; Ma, Y. M.; Maas, F. E.; Maggiora, M.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; Messchendorp, J. G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pan, Y.; Patteri, P.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Pettersson, J.; Ping, J. L.; Ping, R. G.; Poling, R.; Prasad, V.; Qi, H. R.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, L. Q.; Qin, N.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ripka, M.; Rong, G.; Rosner, Ch.; Ruan, X. D.; Sarantsev, A.; Savrié, M.; Schoenning, K.; Schumann, S.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Shi, M.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, X. H.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Tiemens, M.; Ullrich, M.; Uman, I.; Varner, G. S.; Wang, B.; Wang, B. L.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, W.; Wang, W. P.; Wang, X. F.; Wang, Y.; Wang, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. H.; Wang, Z. Y.; Wang, Z. Y.; Weber, T.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, L. J.; Wu, Z.; Xia, L.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, H.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, J. J.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. J.; Yang, H. X.; Yang, L.; Yang, Y. X.; Ye, M.; Ye, M. H.; Yin, J. H.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, W. L.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zallo, A.; Zeng, Y.; Zeng, Z.; Zhang, B. X.; Zhang, B. Y.; Zhang, C.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J.; Zhang, J. J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, S. Q.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. H.; Zhang, Y. N.; Zhang, Y. T.; Zhang, Yu; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, S. H.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zotti, L.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2016-11-01

By analyzing 2.93 fb-1 data collected at the center-of-mass energy with the BESIII detector, we measure the absolute branching fraction of the semileptonic decay D+ → K̅0 e+νe to be ℬ(D + → K̅0 e+νe) = (8.59 ± 0.14 ± 0.21)% using , where the first uncertainty is statistical and the second systematic. Our result is consistent with previous measurements within uncertainties.. Supported by National Key Basic Research Program of China (2009CB825204, 2015CB856700), National Natural Science Foundation of China (NSFC) (10935007, 11125525, 11235011, 11305180, 11322544, 11335008, 11425524, 11475123), Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, CAS Center for Excellence in Particle Physics (CCEPP), Collaborative Innovation Center for Particles and Interactions (CICPI), Joint Large-Scale Scientific Facility Funds of NSFC and CAS (11179007, U1232201, U1332201, U1532101), CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, National 1000 Talents Program of China, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology, German Research Foundation DFG (Collaborative Research Center CRC-1044), Istituto Nazionale di Fisica Nucleare, Italy, Koninklijke Nederlandse Akademie van Wetenschappen (KNAW) (530-4CDP03), Ministry of Development of Turkey (DPT2006K-120470), National Natural Science Foundation of China (NSFC) (11405046, U1332103), Russian Foundation for Basic Research (14-07-91152), Swedish Resarch Council, U. S. Department of Energy (DE-FG02-04ER41291, DE-FG02-05ER41374, DE-SC0012069, DESC0010118), U.S. National Science Foundation, University of Groningen (RuG) and Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0).

Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

PubMed

Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

2017-01-12

Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9. Copyright © 2017 Elsevier Inc. All rights reserved.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

PubMed

Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

2015-03-12

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

PubMed

Kurihara, Takeshi; Fukuhara, Takasuke; Ono, Chikako; Yamamoto, Satomi; Uemura, Kentaro; Okamoto, Toru; Sugiyama, Masaya; Motooka, Daisuke; Nakamura, Shota; Ikawa, Masato; Mizokami, Masashi; Maehara, Yoshihiko; Matsuura, Yoshiharu

2017-07-21

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

Factor information retrieval system version 2. 0 (fire) (for microcomputers). Software

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

Not Available

FIRE Version 2.0 contains EPA's unique recommended criteria and toxic air emission estimation factors. FIRE consists of: (1) an EPA internal repository system that contains emission factor data identified and collected, and (2) an external distribution system that contains only EPA's recommended factors. The emission factors, compiled from a review of the literature, are identified by pollutant name, CAS number, process and emission source descriptions, SIC code, SCC, and control status. The factors are rated for quality using AP-42 rating criteria.

A-10 Thunderbolt II (Warthog) Systems Engineering Case Study

DTIC Science & Technology

2010-01-01

Visual Flight Rules (VFR) navigation aids. The “lean” package added Doppler Navigation for night and adverse weather, and a radar ranger and gun...and a big boost for the technology came in 1965 when the Air Force selected the TF39 engine to power the C-5 Galaxy heavy lift aircraft. Still, there...and Staff College, entitled to wear the Ranger Tab and has a real appreciation for the role of CAS in combat. Upon leaving active duty he served

Fragmentation of the CRISPR-Cas Type I-B signature protein Cas8b.

PubMed

Richter, Hagen; Rompf, Judith; Wiegel, Julia; Rau, Kristina; Randau, Lennart

2017-11-01

CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal structures of two tetrameric β-carbonic anhydrases from the filamentous ascomycete Sordaria macrospora.

PubMed

Lehneck, Ronny; Neumann, Piotr; Vullo, Daniela; Elleuche, Skander; Supuran, Claudiu T; Ficner, Ralf; Pöggeler, Stefanie

2014-04-01

Carbonic anhydrases (CAs) are metalloenzymes catalyzing the reversible hydration of carbon dioxide to bicarbonate (hydrogen carbonate) and protons. CAs have been identified in archaea, bacteria and eukaryotes and can be classified into five groups (α, β, γ, δ, ζ) that are unrelated in sequence and structure. The fungal β-class has only recently attracted attention. In the present study, we investigated the structure and function of the plant-like β-CA proteins CAS1 and CAS2 from the filamentous ascomycete Sordaria macrospora. We demonstrated that both proteins can substitute for the Saccharomyces cerevisiae β-CA Nce103 and exhibit an in vitro CO2 hydration activity (kcat /Km of CAS1: 1.30 × 10(6) m(-1) ·s(-1) ; CAS2: 1.21 × 10(6 ) m(-1) ·s(-1) ). To further investigate the structural properties of CAS1 and CAS2, we determined their crystal structures to a resolution of 2.7 Å and 1.8 Å, respectively. The oligomeric state of both proteins is tetrameric. With the exception of the active site composition, no further major differences have been found. In both enzymes, the Zn(2) (+) -ion is tetrahedrally coordinated; in CAS1 by Cys45, His101 and Cys104 and a water molecule and in CAS2 by the side chains of four residues (Cys56, His112, Cys115 and Asp58). Both CAs are only weakly inhibited by anions, making them good candidates for industrial applications. CAS1 and CAS2 bind by x-ray crystallography (View interaction) Structural data have been deposited in the Protein Data Bank database under accession numbers 4O1J for CAS1 and 4O1K for CAS2. © 2014 FEBS.

Conservation and variability in the structure and function of the Cas5d endoribonuclease in the CRISPR-mediated microbial immune system.

PubMed

Koo, Yoon; Ka, Donghyun; Kim, Eun-Jin; Suh, Nayoung; Bae, Euiyoung

2013-10-23

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form an RNA-mediated microbial immune system against invading foreign genetic elements. Cas5 proteins constitute one of the most prevalent Cas protein families in CRISPR-Cas systems and are predicted to have RNA recognition motif (RRM) domains. Cas5d is a subtype I-C-specific Cas5 protein that can be divided into two distinct subgroups, one of which has extra C-terminal residues while the other contains a longer insertion in the middle of its N-terminal RRM domain. Here, we report crystal structures of Cas5d from Streptococcus pyogenes and Xanthomonas oryzae, which respectively represent the two Cas5d subgroups. Despite a common domain architecture consisting of an N-terminal RRM domain and a C-terminal β-sheet domain, the structural differences between the two Cas5d proteins are highlighted by the presence of a unique extended helical region protruding from the N-terminal RRM domain of X. oryzae Cas5d. We also demonstrate that Cas5d proteins possess not only specific endoribonuclease activity for CRISPR RNAs but also nonspecific double-stranded DNA binding affinity. These findings suggest that Cas5d may play multiple roles in CRISPR-mediated immunity. Furthermore, the specific RNA processing was also observed between S. pyogenes Cas5d protein and X. oryzae CRISPR RNA and vice versa. This cross-species activity of Cas5d provides a special opportunity for elucidating conserved features of the CRISPR RNA processing event. Copyright © 2013 Elsevier Ltd. All rights reserved.

Domestic violence and mental health: a cross-sectional survey of women seeking help from domestic violence support services.

PubMed

Ferrari, Giulia; Agnew-Davies, Roxane; Bailey, Jayne; Howard, Louise; Howarth, Emma; Peters, Tim J; Sardinha, Lynnmarie; Feder, Gene

2014-01-01

Domestic violence and abuse (DVA) are associated with an increased risk of mental illness, but we know little about the mental health of female DVA survivors seeking support from domestic violence services. Domestic violence and abuse (DVA) are associated with an increased risk of mental illness, but we know little about the mental health of female DVA survivors seeking support from domestic violence services. Baseline data on 260 women enrolled in a randomized controlled trial of a psychological intervention for DVA survivors was analyzed. We report prevalence of and associations between mental health status and severity of abuse at the time of recruitment. We used logistic and normal regression models for binary and continuous outcomes, respectively. Mental health measures used were: Clinical Outcomes in Routine Evaluation-Outcome Measure (CORE-OM), Patient Health Questionnaire, Generalized Anxiety Disorder Assessment, and the Posttraumatic Diagnostic Scale (PDS) to measure posttraumatic stress disorder. The Composite Abuse Scale (CAS) measured abuse. Exposure to DVA was high, with a mean CAS score of 56 (SD 34). The mean CORE-OM score was 18 (SD 8) with 76% above the clinical threshold (95% confidence interval: 70-81%). Depression and anxiety levels were high, with means close to clinical thresholds, and all respondents recorded PTSD scores above the clinical threshold. Symptoms of mental illness increased stepwise with increasing severity of DVA. Exposure to DVA was high, with a mean CAS score of 56 (SD 34). The mean CORE-OM score was 18 (SD 8) with 76% above the clinical threshold (95% confidence interval: 70-81%). Depression and anxiety levels were high, with means close to clinical thresholds, and all respondents recorded PTSD scores above the clinical threshold. Symptoms of mental illness increased stepwise with increasing severity of DVA.

Spacer-length DNA intermediates are associated with Cas1 in cells undergoing primed CRISPR adaptation.

PubMed

Musharova, Olga; Klimuk, Evgeny; Datsenko, Kirill A; Metlitskaya, Anastasia; Logacheva, Maria; Semenova, Ekaterina; Severinov, Konstantin; Savitskaya, Ekaterina

2017-04-07

During primed CRISPR adaptation spacers are preferentially selected from DNA recognized by CRISPR interference machinery, which in the case of Type I CRISPR-Cas systems consists of CRISPR RNA (crRNA) bound effector Cascade complex that locates complementary targets, and Cas3 executor nuclease/helicase. A complex of Cas1 and Cas2 proteins is capable of inserting new spacers in the CRISPR array. Here, we show that in Escherichia coli cells undergoing primed adaptation, spacer-sized fragments of foreign DNA are associated with Cas1. Based on sensitivity to digestion with nucleases, the associated DNA is not in a standard double-stranded state. Spacer-sized fragments are cut from one strand of foreign DNA in Cas1- and Cas3-dependent manner. These fragments are generated from much longer S1-nuclease sensitive fragments of foreign DNA that require Cas3 for their production. We propose that in the course of CRISPR interference Cas3 generates fragments of foreign DNA that are recognized by the Cas1-Cas2 adaptation complex, which excises spacer-sized fragments and channels them for insertion into CRISPR array. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Transforming plant biology and breeding with CRISPR/Cas9, Cas12 and Cas13.

PubMed

Schindele, Patrick; Wolter, Felix; Puchta, Holger

2018-04-30

Currently, biology is revolutionized by ever growing applications of the CRISPR/Cas system. As discussed in this Review, new avenues have opened up for plant research and breeding by the use of the sequence-specific DNases Cas9 and Cas12 (formerly named Cpf1) and, more recently, the RNase Cas13 (formerly named C2c2). Although double strand break-induced gene editing based on error-prone nonhomologous end joining has been applied to obtain new traits, such as powdery mildew resistance in wheat or improved pathogen resistance and increased yield in tomato, improved technologies based on CRISPR/Cas for programmed change in plant genomes via homologous recombination have recently been developed. Cas9- and Cas12- mediated DNA binding is used to develop tools for many useful applications, such as transcriptional regulation or fluorescence-based imaging of specific chromosomal loci in plant genomes. Cas13 has recently been applied to degrade mRNAs and combat viral RNA replication. By the possibility to address multiple sequences with different guide RNAs and by the simultaneous use of different Cas proteins in a single cell, we should soon be able to achieve complex changes of plant metabolism in a controlled way. © 2018 Federation of European Biochemical Societies.

Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch.

PubMed

Hirosawa, Moe; Fujita, Yoshihiko; Parr, Callum J C; Hayashi, Karin; Kashida, Shunnichi; Hotta, Akitsu; Woltjen, Knut; Saito, Hirohide

2017-07-27

The CRISPR-Cas9 system is a powerful genome-editing tool useful in a variety of biotechnology and biomedical applications. Here we developed a synthetic RNA-based, microRNA (miRNA)-responsive CRISPR-Cas9 system (miR-Cas9 switch) in which the genome editing activity of Cas9 can be modulated through endogenous miRNA signatures in mammalian cells. We created miR-Cas9 switches by using a miRNA-complementary sequence in the 5΄-UTR of mRNA encoding Streptococcus pyogenes Cas9. The miR-21-Cas9 or miR-302-Cas9 switches selectively and efficiently responded to miR-21-5p in HeLa cells or miR-302a-5p in human induced pluripotent stem cells, and post-transcriptionally attenuated the Cas9 activity only in the target cells. Moreover, the miR-Cas9 switches could differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Our miR-Cas9 switch system provides a promising framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein.

PubMed

Yan, Winston X; Chong, Shaorong; Zhang, Huaibin; Makarova, Kira S; Koonin, Eugene V; Cheng, David R; Scott, David A

2018-04-19

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection. Copyright © 2018 Elsevier Inc. All rights reserved.

Annotation and Classification of CRISPR-Cas Systems

PubMed Central

Makarova, Kira S.; Koonin, Eugene V.

2018-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods. PMID:25981466

Annotation and Classification of CRISPR-Cas Systems.

PubMed

Makarova, Kira S; Koonin, Eugene V

2015-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

Genome Editing with CRISPR-Cas9: Can It Get Any Better?

PubMed

Haeussler, Maximilian; Concordet, Jean-Paul

2016-05-20

The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Changes in ecdysteroid levels and expression patterns of ecdysteroid-responsive factors and neuropeptide hormones during the embryogenesis of the blue crab, Callinectes sapidus.

PubMed

Techa, Sirinart; Alvarez, Javier V; Sook Chung, J

2015-04-01

Embryogenesis requires the involvement and coordination of multiple networks of various genes, according to a timeline governing development. Crustacean embryogenesis usually includes the first molt, a process that is known to be positively controlled by ecdysteroids. We determined the amounts of ecdysteroids, as well as other related factors: the ecdysone receptor (CasEcR), the retinoid X receptor (CasRXR), the molt-inhibiting hormone (CasMIH), and crustacean hyperglycemic hormone (CasCHH) during the ovarian and embryonic developments of Callinectes sapidus. In summary, the ovaries at stages 1-4 have expression levels of maternal CasEcR and CasRXR 10-50 times higher than levels seen in embryos at the yolk stage. This large difference in the amount of the these factors in C. sapidus ovaries suggests that these maternal ecdysteroid-responsive factors may be utilized at the initiation of embryogenesis. During embryogenesis, the changes in total ecdysteroids and levels of CasEcR and CasRXR expression are similar to those observed in juvenile molts. The full-length cDNA sequence of the C. sapidus BTB domain protein (CasBTBDP) initially isolated from Y-organ cDNA, contains only Broad-Complex, Tramtrack, and Bric a brac (BTB) domains. The levels of CasBTBDP are kept constant throughout embryogenesis. The expression profiles of CasMIH and CasCHH are similar to the titers of ecdysteroids. However, the timing of their appearance is followed by increases in CasEcRs and CasRXRs, implying that the expressions of these neuropeptides may be influenced by ecdysteroids. Moreover, the ecdysteroid profile during embryogenesis may track directly with the timing of organogenesis of Y-organs and their activity. Our work reports, for first time, the observed expression and changes of ecdysteroid-responsive factors, along with CasCHH and CasMIH, during embryogenesis in the crustacean C. sapidus. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of Various Nuclear Localization Signal-Fused Cas9 Proteins and Cas9 mRNA for Genome Editing in Zebrafish.

PubMed

Hu, Peinan; Zhao, Xueying; Zhang, Qinghua; Li, Weiming; Zu, Yao

2018-03-02

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been proven to be an efficient and precise genome editing technology in various organisms. However, the gene editing efficiencies of Cas9 proteins with a nuclear localization signal (NLS) fused to different termini and Cas9 mRNA have not been systematically compared. Here, we compared the ability of Cas9 proteins with NLS fused to the N-, C-, or both the N- and C-termini and N-NLS-Cas9-NLS-C mRNA to target two sites in the tyr gene and two sites in the gol gene related to pigmentation in zebrafish. Phenotypic analysis revealed that all types of Cas9 led to hypopigmentation in similar proportions of injected embryos. Genome analysis by T7 Endonuclease I (T7E1) assays demonstrated that all types of Cas9 similarly induced mutagenesis in four target sites. Sequencing results further confirmed that a high frequency of indels occurred in the target sites ( tyr1 > 66%, tyr2 > 73%, gol1 > 50%, and gol2 > 35%), as well as various types (more than six) of indel mutations observed in all four types of Cas9-injected embryos. Furthermore, all types of Cas9 showed efficient targeted mutagenesis on multiplex genome editing, resulting in multiple phenotypes simultaneously. Collectively, we conclude that various NLS-fused Cas9 proteins and Cas9 mRNAs have similar genome editing efficiencies on targeting single or multiple genes, suggesting that the efficiency of CRISPR/Cas9 genome editing is highly dependent on guide RNAs (gRNAs) and gene loci. These findings may help to simplify the selection of Cas9 for gene editing using the CRISPR/Cas9 system. Copyright © 2018 Hu et al.

Integrating complexity into data-driven multi-hazard supply chain network strategies

USGS Publications Warehouse

Long, Suzanna K.; Shoberg, Thomas G.; Ramachandran, Varun; Corns, Steven M.; Carlo, Hector J.

2013-01-01

Major strategies in the wake of a large-scale disaster have focused on short-term emergency response solutions. Few consider medium-to-long-term restoration strategies that reconnect urban areas to the national supply chain networks (SCN) and their supporting infrastructure. To re-establish this connectivity, the relationships within the SCN must be defined and formulated as a model of a complex adaptive system (CAS). A CAS model is a representation of a system that consists of large numbers of inter-connections, demonstrates non-linear behaviors and emergent properties, and responds to stimulus from its environment. CAS modeling is an effective method of managing complexities associated with SCN restoration after large-scale disasters. In order to populate the data space large data sets are required. Currently access to these data is hampered by proprietary restrictions. The aim of this paper is to identify the data required to build a SCN restoration model, look at the inherent problems associated with these data, and understand the complexity that arises due to integration of these data.

Corrective Action Decision Document/Closure Report for Corrective Action Unit 383: Area E-Tunnel Sites, Nevada Test Site

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

NSTec Environmental Restoration

This Corrective Action Decision Document/Closure Report (CADD/CR) was prepared by the Defense Threat Reduction Agency (DTRA) for Corrective Action Unit (CAU) 383, Area 12 E-Tunnel Sites, which is the joint responsibility of DTRA and the U.S. Department of Energy (DOE), National Nuclear Security Administration Nevada Site Office (NNSA/NSO). This CADD/CR is consistent with the requirements of the Federal Facility Agreement and Consent Order (FFACO) agreed to by the State of Nevada, the DOE, and the U.S. Department of Defense. Corrective Action Unit 383 is comprised of three Corrective Action Sites (CASs) and two adjacent areas: • CAS 12-06-06, Muckpile •more » CAS 12-25-02, Oil Spill • CAS 12-28-02, Radioactive Material • Drainage below the Muckpile • Ponds 1, 2, and 3 The purpose of this CADD/CR is to provide justification and documentation to support the recommendation for closure with no further corrective action, by placing use restrictions at the three CASs and two adjacent areas of CAU 383.« less

A complex adaptive systems perspective of health information technology implementation.

PubMed

Keshavjee, Karim; Kuziemsky, Craig; Vassanji, Karim; Ghany, Ahmad

2013-01-01

Implementing health information technology (HIT) is a challenge because of the complexity and multiple interactions that define HIT implementation. Much of the research on HIT implementation is descriptive in nature and has focused on distinct processes such as order entry or decision support. These studies fail to take into account the underlying complexity of the processes, people and settings that are typical of HIT implementations. Complex adaptive systems (CAS) is a promising field that could elucidate the complexity and non-linear interacting issues that are typical in HIT implementation. Initially we sought new models that would enable us to better understand the complex nature of HIT implementation, to proactively identify problem issues that could be a precursor to unintended consequences and to develop new models and new approaches to successful HIT implementations. Our investigation demonstrates that CAS does not provide prediction, but forces us to rethink our HIT implementation paradigms and question what we think we know. CAS provides new ways to conceptualize HIT implementation and suggests new approaches to increasing HIT implementation successes.

cis-1,2-Dichloroethylene

Integrated Risk Information System (IRIS)

EPA / 635 / R - 09 / 006 F www.epa.gov / iris TOXICOLOGICAL REVIEW OF cis - 1,2 - DICHLOROETHYLENE and trans - 1,2 - DICHLOROETHYLENE ( CAS Nos . cis : 156 - 59 - 2 ; trans : 156 - 60 - 5 ; mixture : 540 - 59 - 0 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS )

The CAS Classroom

ERIC Educational Resources Information Center

Garner, Sue

2004-01-01

The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

Complementary Information Derived from CRISPR Cas9 Mediated Gene Deletion and Suppression. | Office of Cancer Genomics

Cancer.gov

CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes.

Exploiting CRISPR/Cas systems for biotechnology

PubMed Central

Sampson, Timothy R.; Weiss, David S.

2015-01-01

The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. PMID:24323919

Exploiting CRISPR/Cas systems for biotechnology.

PubMed

Sampson, Timothy R; Weiss, David S

2014-01-01

The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. © 2014 WILEY Periodicals, Inc.

Evolution and classification of the CRISPR-Cas systems

PubMed Central

S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

2012-01-01

The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

PubMed

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9.

PubMed

Raper, Austin T; Stephenson, Anthony A; Suo, Zucai

2018-02-28

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

PubMed Central

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment. PMID:29503634

Disabling Cas9 by an anti-CRISPR DNA mimic.

PubMed

Shin, Jiyung; Jiang, Fuguo; Liu, Jun-Jie; Bray, Nicolas L; Rauch, Benjamin J; Baik, Seung Hyun; Nogales, Eva; Bondy-Denomy, Joseph; Corn, Jacob E; Doudna, Jennifer A

2017-07-01

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.

[Stoma use in the general surgery service of CHU Gabriel Touré].

PubMed

Traore, A; Diakite, I; Togo, A; Dembele, B T; Kante, L; Coulibaly, Y; Keita, M; Diango, D M; Diallo, A; Diallo, G

2010-01-01

Were to determine the frequency, to describe the clinical and therapeutic aspects digestive stoma. It was about six months an exploratory study from January 1st to June 30th, 2008 in the department of surgery general of the CHU Gabriel Touré. Were included in this study all the patients carrying a enter stoma or a colostomy, old of more than 15 ans. The digestive dents, the other types of stoma and the patients old of less than 15 years, were excluded. The results were analyzed by the software Epi information version 6.4 Fr, the tests of Khi 2 and Student with a threshold of significance for P < 0.05. We college 32 patients are 7.4% of all digestive surgical operations, 13.3% of the abdominal urgencies; composed of 26 men (81.25%) and 6 women (18.75%). The sex ratio at summer of 4.3. The average age was 44, 8 years with a standard deviation 8, 13 and the extremes varying between 16-80 years. Twenty and one (65.6%) sick were operated in urgency. We carried out 29 cases (90.6%) of final stoma, 3 cas (9.4%) side, 21 cas (65.6%) of colostomy, 9 cas (28.1%) of ileostomies. They were temporary in 25 cas (78.1%) and final 7 cas (21.9%). The volvulus of the sigmoid colonist with necroses 10 cas (31.3%), the peritonitis by typhus perforation ilea 9 cas (28.1%), occlusions on tumor of the left colonist 8 cas (25%), the traumatic perforations ileales 2 cas (6.3%), the digestive dents post appendicectomies 2 cas (6.3%) and the congenital megacolon 1 cas (3%) was the indications of the stoma. the operational continuations were simple in 21 cas (65.6%). The principal found complications were: coetaneous irritation 7 cas (21.8%), the prolapsed stomiale 4 cas (12.5%), the suppuration peristomial 3 cas (9.4%), the releasing of Stoma 3 cas (9.4%), the retraction of the stoma 3 cas (9.4%),the psychological disorders 3 cases (9.4%), the hemorrhage 2 cas (6.3), necroses peristomial 2 cas (3.1%), septic shock 2 cas (6.3%), and 1 cas (3.1%) of evisceration, obstruction of the bowels, shock hypovolemic. The intermediate duration of hospitalization was of 37,5 jours with a standard deviation = 13.58 and extremes varying between 02-73 days. Death rate was of 9.4%. The assumption of responsibility of the stomies is difficult in the absence of stomatherapeutes, and of the high cost of the parenteral nutrition in our context .

CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome.

PubMed

Tajkarimi, Mehrdad; Wexler, Hannah M

2017-01-01

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation. Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis ( n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes. Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis -three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup.

Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats.

PubMed

Shao, Yanjiao; Wang, Liren; Guo, Nana; Wang, Shengfei; Yang, Lei; Li, Yajing; Wang, Mingsong; Yin, Shuming; Han, Honghui; Zeng, Li; Zhang, Ludi; Hui, Lijian; Ding, Qiurong; Zhang, Jiqin; Geng, Hongquan; Liu, Mingyao; Li, Dali

2018-05-04

Hereditary tyrosinemia type I (HTI) is a metabolic genetic disorder caused by mutation of fumarylacetoacetate hydrolase (FAH). Because of the accumulation of toxic metabolites, HTI causes severe liver cirrhosis, liver failure, and even hepatocellular carcinoma. HTI is an ideal model for gene therapy, and several strategies have been shown to ameliorate HTI symptoms in animal models. Although CRISPR/Cas9-mediated genome editing is able to correct the Fah mutation in mouse models, WT Cas9 induces numerous undesired mutations that have raised safety concerns for clinical applications. To develop a new method for gene correction with high fidelity, we generated a Fah mutant rat model to investigate whether Cas9 nickase (Cas9n)-mediated genome editing can efficiently correct the Fah First, we confirmed that Cas9n rarely induces indels in both on-target and off-target sites in cell lines. Using WT Cas9 as a positive control, we delivered Cas9n and the repair donor template/single guide (sg)RNA through adenoviral vectors into HTI rats. Analyses of the initial genome editing efficiency indicated that only WT Cas9 but not Cas9n causes indels at the on-target site in the liver tissue. After receiving either Cas9n or WT Cas9-mediated gene correction therapy, HTI rats gained weight steadily and survived. Fah-expressing hepatocytes occupied over 95% of the liver tissue 9 months after the treatment. Moreover, CRISPR/Cas9-mediated gene therapy prevented the progression of liver cirrhosis, a phenotype that could not be recapitulated in the HTI mouse model. These results strongly suggest that Cas9n-mediated genome editing is a valuable and safe gene therapy strategy for this genetic disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and functional insights into the interaction between the Cas family scaffolding protein p130Cas and the focal adhesion-associated protein paxillin

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

Zhang, Chi; Miller, Darcie J.; Guibao, Cristina D.

The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by whichmore » the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd ~4.2 μM) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.« less

Fusion of SpCas9 to E. coli Rec A protein enhances CRISPR-Cas9 mediated gene knockout in mammalian cells.

PubMed

Lin, Lin; Petersen, Trine Skov; Jensen, Kristopher Torp; Bolund, Lars; Kühn, Ralf; Luo, Yonglun

2017-04-10

Mammalian cells repair double-strand DNA breaks (DSB) by a range of different pathways following DSB induction by the engineered clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein Cas9. While CRISPR-Cas9 thus enables predesigned modifications of the genome, applications of CRISPR-Cas9-mediated genome-editing are frequently hampered by the unpredictable and varying pathways for DSB repair in mammalian cells. Here we present a strategy of fusing Cas9 to recombinant proteins for fine-tuning of the DSB repair preferences in mammalian cells. By fusing Streptococcus Pyogenes Cas9 (SpCas9) to the recombinant protein A (Rec A, NP_417179.1) from Escherichia coli, we create a recombinant Cas9 protein (rSpCas9) which enhances the generation of indel mutations at DSB sites in mammalian cells, increases the frequency of DSB repair by homology-directed single-strand annealing (SSA), and represses homology-directed gene conversion by approximately 33%. Our study thus proves for the first time that fusing SpCas9 to recombinant proteins can influence the balance between DSB repair pathways in mammalian cells. This approach may form the basis for further investigations of the applications of recombinant Cas9 proteins to fine-tuning DSB repair pathways in eukaryotic cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Assisting Students' Cognitive Strategies with the Use of CAS

ERIC Educational Resources Information Center

Sarvari, Csaba; Lavicza, Zsolt; Klincsik, Mihaly

2010-01-01

This paper examines various cognitive strategies applied while CAS (Computer Algebra System) are used in undergraduate-level engineering mathematics teaching and learning. We posed some questions in relation to such CAS use: What kind of tools can CAS offer to enhance different cognitive strategies of students? How can the use of CAS widen the…

Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

PubMed Central

Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

2015-01-01

Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: A brief overview.

PubMed

Karvelis, Tautvydas; Gasiunas, Giedrius; Siksnys, Virginijus

2017-05-15

Recently the Cas9, an RNA guided DNA endonuclease, emerged as a powerful tool for targeted genome manipulations. Cas9 protein can be reprogrammed to cleave, bind or nick any DNA target by simply changing crRNA sequence, however a short nucleotide sequence, termed PAM, is required to initiate crRNA hybridization to the DNA target. PAM sequence is recognized by Cas9 protein and must be determined experimentally for each Cas9 variant. Exploration of Cas9 orthologs could offer a diversity of PAM sequences and novel biochemical properties that may be beneficial for genome editing applications. Here we briefly review and compare Cas9 PAM identification assays that can be adopted for other PAM-dependent CRISPR-Cas systems. Copyright © 2017 Elsevier Inc. All rights reserved.

The presence of an insulin-like androgenic gland factor (IAG) and insulin-like peptide binding protein (ILPBP) in the ovary of the blue crab, Callinectes sapidus and their roles in ovarian development.

PubMed

Huang, Xiaoshuai; Ye, Haihui; Chung, J Sook

2017-08-01

Insulin-like androgenic gland factor (IAG) that is produced by the male androgenic gland (AG), plays a role in sexual differentiation and maintenance of male secondary sex characteristics in decapod crustaceans. With an earlier finding of IAG expression in a female Callinectes sapidus ovary, we aimed to examine a putative role of IAG during the ovarian development of this species. To this end, the full-length cDNA sequence of the ovarian CasIAG (termed CasIAG-ova) has been isolated. The predicted mature peptide sequence of CasIAG-ova is identical to that of the IAG from the AG, except in their signal peptide regions. The CasIAG-ova contains an alternative initiation codon (UUG) as the start codon, which suggests that the translational regulation of CasIAG-ova may differ from that of the IAG from AG. To define the function of CasIAG-ova, the expressions of CasIAG-ova as well as its putative binding protein, insulin-like peptide binding protein (ILPBP), are measured in the ovaries at various developmental stages obtained from different seasons. Season affects both CasIAG and ILPBP expression in the ovary. Overall, summer females at earlier ovarian stages contain high levels of CasIAG and ILPBP than spring or fall females. These findings indicate that CasIAG-ova and CasILPBP may be involved in the ovarian development. When comparing the levels of CasIAG and CasILPBP in the ovary, the latter are much higher (∼10-10000 fold) than the former. Expression patterns of CasILPBP differ from those of CasIAG-ova during ovarian development and by season, suggesting that ILPBP may have an additional role in ovarian development rather than a function of a putative binding protein of IAG. Copyright © 2017 Elsevier Inc. All rights reserved.

AFTI/F-16 Spin chute close-up

NASA Technical Reports Server (NTRS)

1982-01-01

A close-up photo of the spin chute mounted on the rear fuselage of the AFTI F-16, a safety device designed to prevent the loss of aircraft in spin conditions. Under some circumstances, pilots cannot recover from spins using normal controls. It these instances, the spin chute is deployed, thus 'breaking' the spin and enabling the pilot to recover. The spin chute is held in a metal cylinder attached to the AFTI F-16 by four tubes, a structure strong enough to withstand the shock of the spin chute opening. Unlike the air probe in the last photo, spin chutes are not standard equipment on research or prototype aircraft but are commonly attached expressly for actual spin tests. During the 1980s and 1990s, NASA and the U.S. Air Force participated in a joint program to integrate and demonstrate new avionics technologies to improve close air support capabilities in next-generation aircraft. The testbed aircraft, seen here in flight over the desert at NASA's Dryden Flight Research Center, Edwards, California, was called the Advanced Fighter Technology Integration (AFTI) F-16. The tests demonstrated technologies to improve navigation and the pilot's ability to find and destroy enemy ground targets day or night, including adverse weather. The aircraft--an F-16A Fighting Falcon (Serial #75-0750)--underwent numerous modifications. A relatively low-cost testbed, it evaluated the feasability of advanced, intergrated-sensor, avionics, and flight control technologies. During the first phase of the AFTI/F-16 program, which began in 1983, the aircraft demonstrated voice-actuated commands, helmet-mounted sights, flat turns, and selective fuselage pointing using forward-mounted canards and a triplex digital flight control computer system. The second phase of research, which began in the summer of 1991, demonstrated advanced technologies and capabilities to find and destroy ground targets day or night, and in adverse weather while using maneuverability and speed at low altitude. This phase was known as the close air support and battlefield air interdiction (CAS/BAI) phase. Finally, the aircraft was used to assess the Automatic Ground Collision Avoidance System (Auto - GCAS), a joint project with the Swedish Government. For these tests, the pilot flew the aircraft directly toward the ground, simulating a total loss of control. The GCAS was designed to take command in such emergencies and bring the aircraft back to level flight. The AFTI F-16 program ended at Dryden on November 4, 1997 after 15 years and over 700 research flights. The USAF continued to fly the aircraft until retiring it to the Air Force Museum on January 9, 2001.

[CRISPR/CAS9, the King of Genome Editing Tools].

PubMed

Bannikov, A V; Lavrov, A V

2017-01-01

The discovery of CRISPR/Cas9 brought a hope for having an efficient, reliable, and readily available tool for genome editing. CRISPR/Cas9 is certainly easy to use, while its efficiency and reliability remain the focus of studies. The review describes the general principles of the organization and function of Cas nucleases and a number of important issues to be considered while planning genome editing experiments with CRISPR/Cas9. The issues include evaluation of the efficiency and specificity for Cas9, sgRNA selection, Cas9 variants designed artificially, and use of homologous recombination and nonhomologous end joining in DNA editing.

A novel association between p130Cas and resistance to the chemotherapeutic drug adriamycin in human breast cancer cells.

PubMed

Ta, Huy Q; Thomas, Keena S; Schrecengost, Randy S; Bouton, Amy H

2008-11-01

Resistance to chemotherapy remains a major obstacle for the treatment of breast cancer. Understanding the molecular mechanism(s) of resistance is crucial for the development of new effective therapies to treat this disease. This study examines the putative role of p130(Cas) (Cas) in resistance to the cytotoxic agent Adriamycin. High expression of Cas in primary breast tumors is associated with the failure to respond to the antiestrogen tamoxifen and poor prognosis, highlighting the potential clinical importance of this molecule. Here, we show a novel association between Cas and resistance to Adriamycin. We show that Cas overexpression renders MCF-7 breast cancer cells less sensitive to the growth inhibitory and proapoptotic effects of Adriamycin. The catalytic activity of the nonreceptor tyrosine kinase c-Src, but not the epidermal growth factor receptor, is critical for Cas-mediated protection from Adriamycin-induced death. The phosphorylation of Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) is elevated in Cas-overexpressing cells treated with Adriamycin, whereas expression of the proapoptotic protein Bak is decreased. Conversely, Cas depletion in the more resistant T47D and MDA-MB-231 cell lines increases sensitivity to Adriamycin. Based on these data, we propose that Cas activates growth and survival pathways regulated by c-Src, Akt, and ERK1/2 that lead to the inhibition of mitochondrial-mediated apoptosis in the presence of Adriamycin. Because Cas is frequently expressed at high levels in breast cancers, these findings raise the possibility of resensitizing Cas-overexpressing tumors to chemotherapy through perturbation of Cas signaling pathways.

Baseline frequency of chromosomal aberrations and sister chromatid exchanges in peripheral blood lymphocytes of healthy individuals living in Turin (North-Western Italy): assessment of the effects of age, sex and GSTs gene polymorphisms on the levels of genomic damage.

PubMed

Santovito, Alfredo; Cervella, Piero; Delpero, Massimiliano

2016-05-01

The increased exposure to environmental pollutants has led to the awareness of the necessity for constant monitoring of human populations, especially those living in urban areas. This study evaluated the background levels of genomic damage in a sample of healthy subjects living in the urban area of Turin (Italy). The association between DNA damage with age, sex and GSTs polymorphisms was assessed. One hundred and one individuals were randomly sampled. Sister Chromatid Exchanges (SCEs) and Chromosomal Aberrations (CAs) assays, as well as genotyping of GSTT1 and GSTM1 genes, were performed. Mean values of SCEs and CAs were 5.137 ± 0.166 and 0.018 ± 0.002, respectively. Results showed age and gender associated with higher frequencies of these two cytogenetic markers. The eldest subjects (51-65 years) showed significantly higher levels of genomic damage than younger individuals. GSTs polymorphisms did not appear to significantly influence the frequencies of either markers. The CAs background frequency observed in this study is one of the highest reported among European populations. Turin is one of the most polluted cities in Europe in terms of air fine PM10 and ozone and the clastogenic potential of these pollutants may explain the high frequencies of chromosomal rearrangements reported here.

Contact Lenses Wettability In Vitro: Effect of Surface-Active Ingredients

PubMed Central

Lin, Meng C.; Svitova, Tatyana F.

2010-01-01

Purpose To investigate the release of surface-active agents (surfactants) from unworn soft contact lenses and their influence on the lens surface wettability in vitro. Methods Surface tension (ST) of blister pack solutions was measured by pendant-drop technique. STs at the air-aqueous interface and contact angles (CAs) of four conventional and seven silicone hydrogel (SiH) soft contact lenses (SCLs) were evaluated in a dynamic-cycling regime using a modified captive-bubble tensiometer-goniometer. Measurements were performed immediately after removal from blister packs, and after soaking in a glass vial filled with a surfactant-free solution, which was replaced daily for one week. Lens surface wettability was expressed as adhesion energy (AE) according to Young’s equation. Results STs of all blister pack solutions were lower than the reference ST of pure water (72.5 mN/m), indicating the presence of surfactants. When lenses were depleted of surfactants by soaking, the STs of all studied lenses and advancing CAs of selected lenses increased (p < 0.001). Receding CAs of all studied lenses were 12° ± 5° and were not affected by the presence of surfactants. For most of the conventional lenses, the surface wettability was largely dependent on surfactants, and reduced significantly after surfactant depletion. In contrast, most SiH lenses exhibited stable and self-sustained surface wettability in vitro. Conclusions The manufacturer-added surfactants affected wetting properties of all studied SCLs, although to different degrees. PMID:20400924

Use Cases for Dynamic Secure Wireless Networking in Coalition Environments (Cas d utilisation de r seau sans fil dynamique et s curis dans des environnements de coalition)

DTIC Science & Technology

2015-05-01

la dénomination « STO », « RTO » ou « AGARD » selon le cas, suivi du numéro de série. Des informations analogues, telles que le titre est la ...supplémentaire de nécessiter moins de déploiement et de ressources de gestion que les réseaux fixes. Néanmoins, les propriétés qui, précisément, rendent...ces réseaux séduisants de prime abord – support ouvert, topologie souple, règles d’adhésion dynamiques, algorithmes simples de formation du

Generalizing the extensibility of a dynamic geometry software

NASA Astrophysics Data System (ADS)

Herceg, Đorđe; Radaković, Davorka; Herceg, Dejana

2012-09-01

Plug-and-play visual components in a Dynamic Geometry Software (DGS) enable development of visually attractive, rich and highly interactive dynamic drawings. We are developing SLGeometry, a DGS that contains a custom programming language, a computer algebra system (CAS engine) and a graphics subsystem. The basic extensibility framework on SLGeometry supports dynamic addition of new functions from attribute annotated classes that implement runtime metadata registration in code. We present a general plug-in framework for dynamic importing of arbitrary Silverlight user interface (UI) controls into SLGeometry at runtime. The CAS engine maintains a metadata storage that describes each imported visual component and enables two-way communication between the expressions stored in the engine and the UI controls on the screen.

Derived Born cross sections of e+e‑ annihilation into open charm mesons from CLEO-c measurements

NASA Astrophysics Data System (ADS)

Dong, Xiang-Kun; Wang, Liang-Liang; Yuan, Chang-Zheng

2018-04-01

The exclusive Born cross sections of the production of D0, D+ and {{{D}}}{{s}}{{+}} mesons in e+e‑ annihilation at 13 energy points between 3.970 and 4.260 GeV are obtained by applying corrections for initial state radiation and vacuum polarization to the observed cross sections measured by the CLEO-c experiment. Both the statistical and the systematic uncertainties for the obtained Born cross sections are estimated. Supported in part by National Natural Science Foundation of China (NSFC) (11235011, 11475187, 11521505, U1632106), the Ministry of Science and Technology of China (2015CB856701), Key Research Program of Frontier Sciences, CAS, (QYZDJ-SSW-SLH011) and the CAS Center for Excellence in Particle Physics (CCEPP)

RIFM fragrance ingredient safety assessment, benzyl butyrate, CAS Registry Number 103-37-7.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current use conditions is supported by the existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data from the suitable read across analog benzyl acetate (CAS # 140-11-4) show that this material is not genotoxic nor does it have skin sensitization potential and also provided a MOE > 100 for the repeated dose, developmental and reproductive, and local respiratory toxicity endpoints. The phototoxicity/photoallergenicity endpoint was completed based on suitable UV spectra. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

2,2\\',4,4\\'-Tetrabromodiphenyl ether (BDE-47)

Integrated Risk Information System (IRIS)

EPA / 635 / R - 07 / 005F www.epa.gov / iris TOXICOLOGICAL REVIEW OF 2,2 ' , 4,4 ' - TETRABROMODIPHENYL ETHER ( BDE - 47 ) ( CAS No . 5436 - 43 - 1 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) June 2008 U.S . Environmental Protection Agency Was

Human Pulmonary Hyperpolarized 129Xe MRI: a Preliminary Study

NASA Astrophysics Data System (ADS)

Yang, Hao; Wang, Ke; Zhang, Hui-Ting; Xie, Jun-Shuai; Wu, Guang-Yao; Zhou, Xin

2018-05-01

Not Available Supported by the National Natural Science Foundation of China under Grant Nos 81227902 and 81625011, the National Key Research and Development Program of China under Grant No 2016YFC1304702, and the Key Research Program of Frontier Sciences of CAS (QYZDY-SSW-SLH018).

32 CFR 169.2 - Applicability and scope.

Code of Federal Regulations, 2014 CFR

2014-07-01

... by or reimbursed from appropriated funds, such as libraries, open messes, and other morale, welfare.... (g) Does not provide authority to enter into contracts. (h) Does not apply to the conduct of research and development, except for severable in-house CAs that support research and development, such as...

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy.

PubMed

Shibata, Mikihiro; Nishimasu, Hiroshi; Kodera, Noriyuki; Hirano, Seiichi; Ando, Toshio; Uchihashi, Takayuki; Nureki, Osamu

2017-11-10

The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9.

A Single-Chain Photoswitchable CRISPR-Cas9 Architecture for Light-Inducible Gene Editing and Transcription.

PubMed

Zhou, Xin X; Zou, Xinzhi; Chung, Hokyung K; Gao, Yuchen; Liu, Yanxia; Qi, Lei S; Lin, Michael Z

2018-02-16

Optical control of CRISPR-Cas9-derived proteins would be useful for restricting gene editing or transcriptional regulation to desired times and places. Optical control of Cas9 functions has been achieved with photouncageable unnatural amino acids or by using light-induced protein interactions to reconstitute Cas9-mediated functions from two polypeptides. However, these methods have only been applied to one Cas9 species and have not been used for optical control of different perturbations at two genes. Here, we use photodissociable dimeric fluorescent protein domains to engineer single-chain photoswitchable Cas9 (ps-Cas9) proteins in which the DNA-binding cleft is occluded at baseline and opened upon illumination. This design successfully controlled different species and functional variants of Cas9, mediated transcriptional activation more robustly than previous optogenetic methods, and enabled light-induced transcription of one gene and editing of another in the same cells. Thus, a single-chain photoswitchable architecture provides a general method to control a variety of Cas9-mediated functions.

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

PubMed Central

Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

Single step production of Cas9 mRNA for zygote injection.

PubMed

Redel, Bethany K; Beaton, Benjamin P; Spate, Lee D; Benne, Joshua A; Murphy, Stephanie L; O'Gorman, Chad W; Spate, Anna M; Prather, Randall S; Wells, Kevin D

2018-03-01

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

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

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Guide-bound structures of an RNA-targeting A-cleaving CRISPR–Cas13a enzyme

DOE PAGES

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; ...

2017-09-11

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR–Cas systems include the Cas13a enzyme, an RNA-activated RNase capable of crRNA processing and single-stranded RNA degradation upon target-transcript binding. Here we present the 2.0-Å resolution crystal structure of a crRNA-bound Lachnospiraceae bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target-RNA-specifying sequence in the protein interior explains the conformational gatingmore » of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked before target-RNA recognition, with implications for both bacterial immunity and diagnostic applications.« less

Production of Purified CasRNPs for Efficacious Genome Editing.

PubMed

Lingeman, Emily; Jeans, Chris; Corn, Jacob E

2017-10-02

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Physicochemical study of mixed systems composed by bovine caseinate and the galactomannan from Gleditsia amorphoides.

PubMed

López, Débora N; Galante, Micaela; Alvarez, Estela M; Risso, Patricia H; Boeris, Valeria

2017-10-01

Model systems formed by sodium caseinate (NaCAS) and espina corona gum (ECG) were studied. There was no evidence of attractive interactions between NaCAS and ECG macromolecules. Aqueous mixtures of NaCAS and ECG phase-separate segregatively over a wide range of concentrations. According to the images obtained by confocal laser scanning microscopy, NaCAS particles form larger protein aggregates when ECG is present in the system. An increase in the hydrodynamic diameter of NaCAS particles, as a result of ECG addition, was also observed by light scattering in diluted systems. A depletion-flocculation phenomenon, in which ECG is excluded from NaCAS surface, is proposed to occur in the concentrated mixed systems, resulting in NaCAS aggregation. ECG raises the viscosity of NaCAS dispersions without affecting the Newtonian flow behaviour of NaCAS. These results contribute to improve the knowledge of a barely-studied hydrocolloid which may be useful in the development of innovative food systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

Recent Advances in Genome Editing Using CRISPR/Cas9.

PubMed

Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

2016-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding.

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity.

PubMed

Seamon, Kyle J; Light, Yooli K; Saada, Edwin A; Schoeniger, Joseph S; Harmon, Brooke

2018-06-05

The RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate its utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.

New CRISPR-Cas systems from uncultivated microbes

NASA Astrophysics Data System (ADS)

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

2017-02-01

CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

New CRISPR–Cas systems from uncultivated microbes

DOE PAGES

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; ...

2016-12-22

We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNAmore » extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Lastly, interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

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

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

Versatile High-Throughput Fluorescence Assay for Monitoring Cas9 Activity

DOE PAGES

Seamon, Kyle Jeffrey; Light, Yooli Kim; Saada, Edwin A.; ...

2018-05-14

Here, the RNA-guided DNA nuclease Cas9 is now widely used for the targeted modification of genomes of human cells and various organisms. Despite the extensive use of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) systems for genome engineering and the rapid discovery and engineering of new CRISPR-associated nucleases, there are no high-throughput assays for measuring enzymatic activity. The current laboratory and future therapeutic uses of CRISPR technology have a significant risk of accidental exposure or clinical off-target effects, underscoring the need for therapeutically effective inhibitors of Cas9. Here, we develop a fluorescence assay for monitoring Cas9 nuclease activity and demonstrate itsmore » utility with S. pyogenes (Spy), S. aureus (Sau), and C. jejuni (Cje) Cas9. The assay was validated by quantitatively profiling the species specificity of published anti-CRISPR (Acr) proteins, confirming the reported inhibition of Spy Cas9 by AcrIIA4 and Cje Cas9 by AcrIIC1 and no inhibition of Sau Cas9 by either anti-CRISPR. To identify drug-like inhibitors, we performed a screen of 189 606 small molecules for inhibition of Spy Cas9. Of 437 hits (0.2% hit rate), six were confirmed as Cas9 inhibitors in a direct gel electrophoresis secondary assay. The high-throughput nature of this assay makes it broadly applicable for the discovery of additional Cas9 inhibitors or the characterization of Cas9 enzyme variants.« less

New CRISPR–Cas systems from uncultivated microbes

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

Burstein, David; Harrington, Lucas B.; Strutt, Steven C.

We present that CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNAmore » extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Lastly, interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.« less

Closure Report for Corrective Action Unit 408: Bomblet Target Area Tonopah Test Range (TTR), Nevada, Revision 0

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

Mark Krauss

2010-09-01

This Closure Report (CR) presents information supporting the closure of Corrective Action Unit (CAU) 408: Bomblet Target Area (TTR), Tonopah Test Range, Nevada. This CR complies with the requirements of the Federal Facility Agreement and Consent Order that was agreed to by the State of Nevada; U.S. Department of Energy (DOE), Environmental Management; U.S. Department of Defense; and DOE, Legacy Management. Corrective Action Unit 408 is located at the Tonopah Test Range, Nevada, and consists of Corrective Action Site (CAS) TA-55-002-TAB2, Bomblet Target Areas. This CAS includes the following seven target areas: • Mid Target • Flightline Bomblet Location •more » Strategic Air Command (SAC) Target Location 1 • SAC Target Location 2 • South Antelope Lake • Tomahawk Location 1 • Tomahawk Location 2 The purpose of this CR is to provide documentation supporting the completed corrective actions and data confirming that the closure objectives for the CAS within CAU 408 were met. To achieve this, the following actions were performed: • Review the current site conditions, including the concentration and extent of contamination. • Implement any corrective actions necessary to protect human health and the environment. • Properly dispose of corrective action and investigation wastes. • Document Notice of Completion and closure of CAU 408 issued by the Nevada Division of Environmental Protection. From July 2009 through August 2010, closure activities were performed as set forth in the Streamlined Approach for Environmental Restoration Plan for CAU 408: Bomblet Target Area, Tonopah Test Range (TTR), Nevada. The purposes of the activities as defined during the data quality objectives process were as follows: • Identify and remove munitions of explosive concern (MEC) associated with DOE activities. • Investigate potential disposal pit locations. • Remove depleted uranium-contaminated fragments and soil. • Determine whether contaminants of concern (COCs) are present. • If COCs are present, determine their nature and extent, implement appropriate corrective actions, and properly dispose of wastes. Analytes detected during the closure activities were evaluated against final action levels to determine COCs for CAU 408. Assessment of the data indicated COCs are not present at CAS TA-55-002-TAB2; therefore, no corrective action is necessary. No use restrictions are required to be placed on this CAU because the investigation showed no evidence of remaining soil contamination or remaining debris/waste upon completion of all investigation activities. The MEC was successfully removed and dispositioned as planned using current best available technologies. As MEC guidance and general MEC standards acknowledge that MEC response actions cannot determine with 100 percent certainty that all MEC and unexploded ordnance (UXO) are removed, the clean closure of CAU 408 will implement a best management practice of posting UXO hazard warning signs near the seven target areas. The signs will warn future land users of the potential for encountering residual UXO hazards. The DOE, National Nuclear Security Administration Nevada Site Office, provides the following recommendations: • A Notice of Completion to the DOE, National Nuclear Security Administration Nevada Site Office, is requested from the Nevada Division of Environmental Protection for closure of CAU 408. • Corrective Action Unit 408 should be moved from Appendix III to Appendix IV of the Federal Facility Agreement and Consent Order.« less

CRISPR-Cas Systems in Bacteroides fragilis, an Important Pathobiont in the Human Gut Microbiome

PubMed Central

Tajkarimi, Mehrdad; Wexler, Hannah M.

2017-01-01

Background: While CRISPR-Cas systems have been identified in bacteria from a wide variety of ecological niches, there are no studies to describe CRISPR-Cas elements in Bacteroides species, the most prevalent anaerobic bacteria in the lower intestinal tract. Microbes of the genus Bacteroides make up ~25% of the total gut microbiome. Bacteroides fragilis comprises only 2% of the total Bacteroides in the gut, yet causes of >70% of Bacteroides infections. The factors causing it to transition from benign resident of the gut microbiome to virulent pathogen are not well understood, but a combination of horizontal gene transfer (HGT) of virulence genes and differential transcription of endogenous genes are clearly involved. The CRISPR-Cas system is a multi-functional system described in prokaryotes that may be involved in control both of HGT and of gene regulation. Results: Clustered regularly interspaced short palindromic repeats (CRISPR) elements in all strains of B. fragilis (n = 109) with publically available genomes were identified. Three different CRISPR-Cas types, corresponding most closely to Type IB, Type IIIB, and Type IIC, were identified. Thirty-five strains had two CRISPR-Cas types, and three strains included all three CRISPR-Cas types in their respective genomes. The cas1 gene in the Type IIIB system encoded a reverse-transcriptase/Cas1 fusion protein rarely found in prokaryotes. We identified a short CRISPR (3 DR) with no associated cas genes present in most of the isolates; these CRISPRs were found immediately upstream of a hipA/hipB operon and we speculate that this element may be involved in regulation of this operon related to formation of persister cells during antimicrobial exposure. Also, blood isolates of B. fragilis did not have Type IIC CRISPR-Cas systems and had atypical Type IIIB CRISPR-Cas systems that were lacking adjacent cas genes. Conclusions: This is the first systematic report of CRISPR-Cas systems in a wide range of B. fragilis strains from a variety of sources. There are four apparent CRISPR-Cas systems in B. fragilis—three systems have adjacent cas genes. Understanding CRISPR/Cas function in B. fragilis will elucidate their role in gene expression, DNA repair and ability to survive exposure to antibiotics. Also, based on their unique CRISPR-Cas arrays, their phylogenetic clustering and their virulence potential, we are proposing that blood isolates of B. fragilis be viewed a separate subgroup. PMID:29218031

Duplex-assisted carotid artery stenting without administration of contrast medium for patients with chronic kidney disease or allergic reaction.

PubMed

Mizowaki, Takashi; Fujita, Atsushi; Imahori, Taichiro; Uyama, Atsushi; Inoue, Satoshi; Kohta, Masaaki; Hamaguchi, Hirotoshi; Sasayama, Takashi; Hosoda, Kohkichi; Kohmura, Eiji

2016-07-01

We aimed to investigate the safety and feasibility of duplex-assisted carotid artery stenting (CAS) without administration of contrast medium for the prevention of adverse reactions. Fifteen patients (9 % of all CASs) with severe carotid stenosis (≥70 %) associated with chronic kidney disease (CKD) (stage ≥3) or allergy to contrast medium underwent duplex-assisted CAS without administration of contrast medium over 4 years. The procedural success rate and perioperative complication rates were compared between the duplex-assisted CAS (n = 15) and conventional CAS (n = 153) groups. The technical success rate was 100 % in both groups. Combined stroke or death rates during the post-procedural period did not differ significantly between the duplex-assisted CAS group (0/15, 0 %) and conventional CAS group (4/153, 2.6 %). None of the 14 patients with CKD in the duplex-assisted CAS group experienced further deterioration of renal function. The mean surface radiation dose of participants in the duplex-assisted CAS group (n = 13, 312 ± 131 mGy) was significantly lower than that of the conventional CAS group (n = 31, 1036 ± 571 mGy) (p < 0.001). The mean duration of CAS procedure was not significantly different between the duplex-assisted CAS group (156 ± 39.7 min) and the conventional CAS group (156 ± 37.4 min). Duplex-assisted CAS without administration of contrast medium could be an alternative option in selected patients deemed to be at high risk for renal failure from nephrotoxic contrast medium or who have an allergy to contrast medium.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium

PubMed Central

Palanisamy, Arun P.; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D.; Kuppuswamy, Dhandapani

2017-01-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src’s adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24–48 h PO myocardium. Our studies indicate that c-Src’s adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. PMID:25976166

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

PubMed Central

Ohga, Rie; Ota, Satoshi; Kawahara, Atsuo

2015-01-01

The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an in vitro transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of tyrosinase (tyr) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of spns2 by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish. PMID:26010089

A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease.

PubMed

Ivanov, Yury V; Shariat, Nikki; Register, Karen B; Linz, Bodo; Rivera, Israel; Hu, Kai; Dudley, Edward G; Harvill, Eric T

2015-10-26

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Here we describe a novel Type II-C CRISPR and its associated genes-cas1, cas2, and cas9-in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies.

Successful transient expression of Cas9 and single guide RNA genes in Chlamydomonas reinhardtii.

PubMed

Jiang, Wenzhi; Brueggeman, Andrew J; Horken, Kempton M; Plucinak, Thomas M; Weeks, Donald P

2014-11-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a powerful and precise tool for targeted gene modification (e.g., gene knockout and gene replacement) in numerous eukaryotic organisms. Initial attempts to apply this technology to a model, the single-cell alga, Chlamydomonas reinhardtii, failed to yield cells containing edited genes. To determine if the Cas9 and single guide RNA (sgRNA) genes were functional in C. reinhardtii, we tested the ability of a codon-optimized Cas9 gene along with one of four different sgRNAs to cause targeted gene disruption during a 24-h period immediately following transformation. All three exogenously supplied gene targets as well as the endogenous FKB12 (rapamycin sensitivity) gene of C. reinhardtii displayed distinct Cas9/sgRNA-mediated target site modifications as determined by DNA sequencing of cloned PCR amplicons of the target site region. Success in transient expression of Cas9 and sgRNA genes contrasted with the recovery of only a single rapamycin-resistant colony bearing an appropriately modified FKB12 target site in 16 independent transformation experiments involving >10(9) cells. Failure to recover transformants with intact or expressed Cas9 genes following transformation with the Cas9 gene alone (or even with a gene encoding a Cas9 lacking nuclease activity) provided strong suggestive evidence for Cas9 toxicity when Cas9 is produced constitutively in C. reinhardtii. The present results provide compelling evidence that Cas9 and sgRNA genes function properly in C. reinhardtii to cause targeted gene modifications and point to the need for a focus on development of methods to properly stem Cas9 production and/or activity following gene editing. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Viral Diversity Threshold for Adaptive Immunity in Prokaryotes

PubMed Central

Weinberger, Ariel D.; Wolf, Yuri I.; Lobkovsky, Alexander E.; Gilmore, Michael S.; Koonin, Eugene V.

2012-01-01

ABSTRACT Bacteria and archaea face continual onslaughts of rapidly diversifying viruses and plasmids. Many prokaryotes maintain adaptive immune systems known as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). CRISPR-Cas systems are genomic sensors that serially acquire viral and plasmid DNA fragments (spacers) that are utilized to target and cleave matching viral and plasmid DNA in subsequent genomic invasions, offering critical immunological memory. Only 50% of sequenced bacteria possess CRISPR-Cas immunity, in contrast to over 90% of sequenced archaea. To probe why half of bacteria lack CRISPR-Cas immunity, we combined comparative genomics and mathematical modeling. Analysis of hundreds of diverse prokaryotic genomes shows that CRISPR-Cas systems are substantially more prevalent in thermophiles than in mesophiles. With sequenced bacteria disproportionately mesophilic and sequenced archaea mostly thermophilic, the presence of CRISPR-Cas appears to depend more on environmental temperature than on bacterial-archaeal taxonomy. Mutation rates are typically severalfold higher in mesophilic prokaryotes than in thermophilic prokaryotes. To quantitatively test whether accelerated viral mutation leads microbes to lose CRISPR-Cas systems, we developed a stochastic model of virus-CRISPR coevolution. The model competes CRISPR-Cas-positive (CRISPR-Cas+) prokaryotes against CRISPR-Cas-negative (CRISPR-Cas−) prokaryotes, continually weighing the antiviral benefits conferred by CRISPR-Cas immunity against its fitness costs. Tracking this cost-benefit analysis across parameter space reveals viral mutation rate thresholds beyond which CRISPR-Cas cannot provide sufficient immunity and is purged from host populations. These results offer a simple, testable viral diversity hypothesis to explain why mesophilic bacteria disproportionately lack CRISPR-Cas immunity. More generally, fundamental limits on the adaptability of biological sensors (Lamarckian evolution) are predicted. PMID:23221803

A Ruler Protein in a Complex for Antiviral Defense Determines the Length of Small Interfering CRISPR RNAs

PubMed Central

Hatoum-Aslan, Asma; Samai, Poulami; Maniv, Inbal; Jiang, Wenyan; Marraffini, Luciano A.

2013-01-01

Small RNAs undergo maturation events that precisely determine the length and structure required for their function. CRISPRs (clustered regularly interspaced short palindromic repeats) encode small RNAs (crRNAs) that together with CRISPR-associated (cas) genes constitute a sequence-specific prokaryotic immune system for anti-viral and anti-plasmid defense. crRNAs are subject to multiple processing events during their biogenesis, and little is known about the mechanism of the final maturation step. We show that in the Staphylococcus epidermidis type III CRISPR-Cas system, mature crRNAs are measured in a Cas10·Csm ribonucleoprotein complex to yield discrete lengths that differ by 6-nucleotide increments. We looked for mutants that impact this crRNA size pattern and found that an alanine substitution of a conserved aspartate residue of Csm3 eliminates the 6-nucleotide increments in the length of crRNAs. In vitro, recombinant Csm3 binds RNA molecules at multiple sites, producing gel-shift patterns that suggest that each protein binds 6 nucleotides of substrate. In vivo, changes in the levels of Csm3 modulate the crRNA size distribution without disrupting the 6-nucleotide periodicity. Our data support a model in which multiple Csm3 molecules within the Cas10·Csm complex bind the crRNA with a 6-nucleotide periodicity to function as a ruler that measures the extent of crRNA maturation. PMID:23935102

2,2\\',4,4\\',5-Pentabromodiphenyl ether (BDE-99)

Integrated Risk Information System (IRIS)

EPA / 635 / R - 07 / 006F www.epa.gov / iris TOXICOLOGICAL REVIEW OF 2,2 ' , 4,4 ' , 5 - PENTABROMODIPHENYL ETHER ( BDE - 99 ) ( CAS No . 60348 - 60 - 9 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) June 2008 U.S . Environmental Protection Agency Washington ,

Discrete Mathematics Course Supported by CAS MATHEMATICA

ERIC Educational Resources Information Center

Ivanov, O. A.; Ivanova, V. V.; Saltan, A. A.

2017-01-01

In this paper, we discuss examples of assignments for a course in discrete mathematics for undergraduate students majoring in business informatics. We consider several problems with computer-based solutions and discuss general strategies for using computers in teaching mathematics and its applications. In order to evaluate the effectiveness of our…

Architecture for Building Conversational Agents that Support Collaborative Learning

ERIC Educational Resources Information Center

Kumar, R.; Rose, C. P.

2011-01-01

Tutorial Dialog Systems that employ Conversational Agents (CAs) to deliver instructional content to learners in one-on-one tutoring settings have been shown to be effective in multiple learning domains by multiple research groups. Our work focuses on extending this successful learning technology to collaborative learning settings involving two or…

Automation Activities that Support C2 Agility to Mitigate Type 7 Risks

DTIC Science & Technology

2014-06-01

on business trip • Space ship runs into space junk What are the probabilities for these events in a 45-year career time frame? Event that...representation that information system understands State- Space Diagram Common Agility Space (CAS) A simple C2 organization representation

32 CFR 169a.2 - Applicability and scope.

Code of Federal Regulations, 2014 CFR

2014-07-01

... partially with DoD civilian personnel paid by or reimbursed from appropriated funds, such as libraries, open... apply to the conduct of research and development, except for severable in-house CAs that support research and development, such as those listed in appendix A to this part. (i) Does not justify conversion...

Comparison of anterior chamber depth measurements by 3-dimensional optical coherence tomography, partial coherence interferometry biometry, Scheimpflug rotating camera imaging, and ultrasound biomicroscopy.

PubMed

Nakakura, Shunsuke; Mori, Etsuko; Nagatomi, Nozomi; Tabuchi, Hitoshi; Kiuchi, Yoshiaki

2012-07-01

To evaluate the congruity of anterior chamber depth (ACD) measurements using 4 devices. Saneikai Tsukazaki Hospital, Himeji City, Japan. Comparative case series. In 1 eye of 42 healthy participants, the ACD was measured by 3-dimensional corneal and anterior segment optical coherence tomography (CAS-OCT), partial coherence interferometry (PCI), Scheimpflug imaging, and ultrasound biomicroscopy (UBM). The differences between the measurements were evaluated by 2-way analysis of variance and post hoc analysis. Agreement between the measurements was evaluated using Bland-Altman analysis. To evaluate the true ACD using PCI, the automatically calculated ACD minus the central corneal thickness measured by CAS-OCT was defined as PCI true. Two ACD measurements were also taken with CAS-OCT. The mean ACD was 3.72 mm ± 0.23 (SD) (PCI), 3.18 ± 0.23 mm (PCI true), 3.24 ± 0.25 mm (Scheimpflug), 3.03 ± 0.25 mm (UBM), 3.14 ± 0.24 mm (CAS-OCT auto), and 3.12 ± 0.24 mm (CAS-OCT manual). A significant difference was observed between PCI biometry, Scheimpflug imaging, and UBM measurements and the other methods. Post hoc analysis showed no significant differences between PCI true and CAS-OCT auto or between CAS-OCT auto and CAS-OCT manual. Strong correlations were observed between all measurements; however, Bland-Altman analysis showed good agreement only between PCI true and Scheimpflug imaging and between CAS-OCT auto and CAS OCT manual. The ACD measurements obtained from PCI biometry, Scheimpflug imaging, CAS-OCT, and UBM were significantly different and not interchangeable except for PCI true and CAS-OCT auto and CAS-OCT auto and CAS-OCT manual. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2012 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Cytosolic and Nuclear Delivery of CRISPR/Cas9-ribonucleoprotein for Gene Editing Using Arginine Functionalized Gold Nanoparticles.

PubMed

Mout, Rubul; Rotello, Vincent M

2017-10-20

In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources. Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90-95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users' need. Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al ., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al ., 2017). We report here only the generation of the delivery vehicle i.e. , the fabrication of Cas9En-RNP/ArgNPs nanoassembly.

Antifungal Efficacy of Caspofungin (MK-0991) in Experimental Pulmonary Aspergillosis in Persistently Neutropenic Rabbits: Pharmacokinetics, Drug Disposition, and Relationship to Galactomannan Antigenemia

PubMed Central

Petraitiene, Ruta; Petraitis, Vidmantas; Groll, Andreas H.; Sein, Tin; Schaufele, Robert L.; Francesconi, Andrea; Bacher, John; Avila, Nilo A.; Walsh, Thomas J.

2002-01-01

The antifungal efficacy, pharmacokinetics, and safety of caspofungin (CAS) were investigated in the treatment and prophylaxis of invasive pulmonary aspergillosis due to Aspergillus fumigatus in persistently neutropenic rabbits. Antifungal therapy consisted of 1, 3, or 6 mg of CAS/kg of body weight/day (CAS1, CAS3, and CAS6, respectively) or 1 mg of deoxycholate amphotericin B (AMB)/kg/day intravenously for 12 days starting 24 h after endotracheal inoculation. Prophylaxis (CAS1) was initiated 4 days before endotracheal inoculation. Rabbits treated with CAS had significant improvement in survival and reduction in organism-mediated pulmonary injury (OMPI) measured by pulmonary infarct score and total lung weight (P < 0.01). However, animals treated with CAS demonstrated a paradoxical trend toward increased residual fungal burden (log CFU per gram) and increased serum galactomannan antigen index (GMI) despite improved survival. Rabbits receiving prophylactic CAS1 also showed significant improvement in survival and reduction in OMPI (P < 0.01), but there was no effect on residual fungal burden. In vitro tetrazolium salt hyphal damage assays and histologic studies demonstrated that CAS had concentration- and dose-dependent effects on hyphal structural integrity. In parallel with a decline in GMI, AMB significantly reduced the pulmonary tissue burden of A. fumigatus (P ≤ 0.01). The CAS1, CAS3, and CAS6 dose regimens demonstrated dose-proportional exposure and maintained drug levels in plasma above the MIC for the entire 24-h dosing interval at doses that were ≥3 mg/kg/day. As serial galactomannan antigen levels may be used for therapeutic monitoring, one should be aware that profoundly neutropenic patients receiving echinocandins for aspergillosis might have persistent galactomannan antigenemia despite clinical improvement. CAS improved survival, reduced pulmonary injury, and caused dose-dependent hyphal damage but with no reduction in residual fungal burden or galactomannan antigenemia in persistently neutropenic rabbits with invasive pulmonary aspergillosis. PMID:11751105

Postantifungal effect of the combination of caspofungin with voriconazole and amphotericin B against clinical Candida krusei isolates.

PubMed

Oz, Yasemin; Kiremitci, Abdurrahman; Dag, Ilknur; Metintas, Selma; Kiraz, Nuri

2013-01-01

We evaluated the postantifungal effects (PAFEs) of caspofungin (CAS), voriconazole (VOR), amphotericin B (AmB), and the combinations of CAS + VOR and CAS + AmB against 30 clinical Candida krusei isolates at 0.25, 1 and 4 times the MIC of each individually and in the indicated combinations. Antifungals were removed after 1 hour and colony counts were performed at 0, 2, 6, 24, and 48 h. VOR did not display any measurable PAFE regardless of antifungal concentrations, while AmB and CAS exhibited dose-dependent PAFE. The most effective agent producing a prolonged PAFE in this study was CAS. Although the combination of CAS with VOR generated longer PAFEs at 0.25 and 1 times their respective MICs in comparison with CAS alone, this combination was indifferent rather than synergistic. However, the combination of CAS with AmB at 4 times their MICs exhibited the best performance, reducing the colony counts during the 48 h after removal of drugs and resulted in synergic interaction in respect to 20 (67%) isolates. Consequently, CAS has a prolonged PAFE in vitro against C. krusei isolates, and the combination of AmB + CAS may increase significantly the efficacy of CAS. Our data may be useful in optimizing dosing regimens for these agents and their combinations, although further studies are needed to explore the clinical usefulness of our results.

Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications.

PubMed

Liu, Chang; Zhang, Li; Liu, Hao; Cheng, Kun

2017-11-28

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The single guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease of the system acts as a pair of scissors to cleave the double strands of DNA. Since its discovery, CRISPR-Cas9 has become the most robust platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications. CRISPR-Cas9 can be applied to correct disease-causing gene mutations or engineer T cells for cancer immunotherapy. The first clinical trial using the CRISPR-Cas9 technology was conducted in 2016. Despite the great promise of the CRISPR-Cas9 technology, several challenges remain to be tackled before its successful applications for human patients. The greatest challenge is the safe and efficient delivery of the CRISPR-Cas9 genome-editing system to target cells in human body. In this review, we will introduce the molecular mechanism and different strategies to edit genes using the CRISPR-Cas9 system. We will then highlight the current systems that have been developed to deliver CRISPR-Cas9 in vitro and in vivo for various therapeutic purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

PubMed

Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

2016-07-15

CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.

Chemical and Biophysical Modulation of Cas9 for Tunable Genome Engineering.

PubMed

Nuñez, James K; Harrington, Lucas B; Doudna, Jennifer A

2016-03-18

The application of the CRISPR-Cas9 system for genome engineering has revolutionized the ability to interrogate genomes of mammalian cells. Programming the Cas9 endonuclease to induce DNA breaks at specified sites is achieved by simply modifying the sequence of its cognate guide RNA. Although Cas9-mediated genome editing has been shown to be highly specific, cleavage events at off-target sites have also been reported. Minimizing, and eventually abolishing, unwanted off-target cleavage remains a major goal of the CRISPR-Cas9 technology before its implementation for therapeutic use. Recent efforts have turned to chemical biology and biophysical approaches to engineer inducible genome editing systems for controlling Cas9 activity at the transcriptional and protein levels. Here, we review recent advancements to modulate Cas9-mediated genome editing by engineering split-Cas9 constructs, inteins, small molecules, protein-based dimerizing domains, and light-inducible systems.

Anisotropic formation mechanism and nanomechanics for the self-assembly process of cross-β peptides

NASA Astrophysics Data System (ADS)

Deng, Li; Zhao, Yurong; Zhou, Peng; Xu, Hai; Wang, Yanting

2017-12-01

Not Available Project supported by the National Basic Research Program of China (Grant No. 2013CB932804), the National Natural Science Foundation of China (Grant Nos. 11421063, 11647601, 11504431, and 21503275), the Scientific Research Foundation of China University of Petroleum (East China) for Young Scholar (Grant Y1304073). YantingWang also thanks the financial support through the CAS Biophysics Interdisciplinary Innovation Team Project (Grant No. 2060299).

Cas9 gRNA engineering for genome editing, activation and repression

DOE PAGES

Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; ...

2015-09-07

Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

Effects of Using a Computer Algebra System (CAS) on Junior College Students' Attitudes towards CAS and Achievement in Mathematics

ERIC Educational Resources Information Center

Leng, Ng Wee; Choo, Kwee Tiow; Soon, Lau Hock; Yi-Huak, Koh; Sun, Yap Yew

2005-01-01

This study examines the effects of using Texas Instruments' Voyage 200 calculator (V200), a graphing calculator with a built-in computer algebra system (CAS), on attitudes towards CAS and achievement in mathematics of junior college students (17 year olds). Students' attitudes towards CAS were examined using a 40-item Likert-type instrument…

Tuberculose multifocale chez les immunocompétents

PubMed Central

Rezgui, Amel; Fredj, Fatma Ben; Mzabi, Anis; Karmani, Monia; Laouani, Chadia

2016-01-01

La tuberculose multifocale est définie par la l'atteinte d'au moins deux sites extra-pulmonaires associée ou non à une atteinte pulmonaire. On se propose d’étudier les différentes caractéristiques cliniques et évolutives de la tuberculose multifocale à travers une étude rétrospective de 10 cas. Parmi 41 cas de tuberculose colligés entre 1999 et 2013. Dix patients avaient une tuberculose multifocale, soit 24% des patients. Il s'agissait de 9 femmes et 1 homme d’âge moyen à 50 ans (30-68 ans). Nos patients étaient tous correctement vaccinés par le BCG. Un bilan à la recherche d'une éventuelle immunodépression fait pour tous les patients était négatif. Il s'agissait d'une tuberculose ganglionnaire dans 7 cas, digestive dans 3 cas, péricardique dans 2 cas, ostéo-articulaire dans 2 cas, cérébrale dans 1 cas, urinaire dans 2 cas, uro-génitale dans 4 cas, surrénalienne dans 1 cas, cutanée dans 1 cas et musculaire dans 1 cas. Tous nos patients ont bénéficié d'un traitement antituberculeux pour une durée moyenne de 10 mois avec bonne évolution. La tuberculose multifocale est une des maladies à diagnostic difficile. Elle peut toucher les immunocompétents mais son pronostic est souvent bon. Un traitement anti-tuberculeux doit être instauré le plus rapidement possible pour éviter les séquelles. PMID:27583077

Recent Advances in Genome Editing Using CRISPR/Cas9

PubMed Central

Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

2016-01-01

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

Increased plasma xanthine oxidoreductase activity deteriorates coronary artery spasm.

PubMed

Watanabe, Ken; Shishido, Tetsuro; Otaki, Yoichiro; Watanabe, Tetsu; Sugai, Takayuki; Toshima, Taku; Takahashi, Tetsuya; Yokoyama, Miyuki; Kinoshita, Daisuke; Murase, Takayo; Nakamura, Takashi; Wanezaki, Masahiro; Tamura, Harutoshi; Nishiyama, Satoshi; Takahashi, Hiroki; Arimoto, Takanori; Yamauchi, So; Yamanaka, Tamon; Miyamoto, Takuya; Kubota, Isao; Watanabe, Masafumi

2018-06-23

Increased reactive oxygen species (ROS) contributes to the development of endothelial dysfunction, which is involved in coronary artery spasm (CAS). Xanthine oxidoreductase (XOR) plays a pivotal role in producing both uric acid and ROS. However, the association between plasma XOR activity and CAS has not been elucidated. The aim of this study was to investigate whether plasma XOR activity is associated with CAS. We measured XOR activity in 104 patients suspected for CAS, who presented without significant coronary artery stenosis and underwent intracoronary acetylcholine provocation tests. CAS was provoked in 44 patients and they had significantly higher XOR activity as compared with those without CAS. The patients were divided into three groups based on the XOR activity. The prevalence rate of CAS was increased with increasing XOR activity. A multivariate logistic regression analysis showed that the 3rd tertile group exhibited a higher incidence of CAS as compared with the 1st tertile group [odds ratio (OR) 6.9, P = 0.001) and the 2nd tertile group (OR 3.2, P = 0.033) after adjustment for conventional CAS risk factors, respectively. The C index was significantly improved by the addition of XOR activity to the baseline model based on CAS risk factors. Furthermore, the 3rd tertile group had the highest incidence of severe spasm defined as total obstruction, flow-limiting stenosis, diffuse spasm, multivessel spasm, and/or lethal arrhythmia. This is a first report to elucidate the association of plasma XOR activity with CAS. Increased plasma XOR activity is significantly associated with CAS.

Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria

PubMed Central

Cai, Fei; Axen, Seth D.; Kerfeld, Cheryl A.

2013-01-01

Members of the phylum Cyanobacteria inhabit ecologically diverse environments. However, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR associated genes), an extremely adaptable defense system, has not been surveyed in this phylum. We analyzed 126 cyanobacterial genomes and, surprisingly, found CRISPR-Cas in the majority except the marine subclade (Synechococcus and Prochlorococcus), in which cyanophages are a known force shaping their evolution. Multiple observations of CRISPR loci in the absence of cas1/cas2 genes may represent an early stage of losing a CRISPR-Cas locus. Our findings reveal the widespread distribution of their role in the phylum Cyanobacteria and provide a first step to systematically understanding CRISPR-Cas systems in cyanobacteria. PMID:23628889

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases

PubMed Central

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-01-01

Abstract Background Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. Findings We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Conclusion Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required. PMID:29635374

Genome-wide determination of on-target and off-target characteristics for RNA-guided DNA methylation by dCas9 methyltransferases.

PubMed

Lin, Lin; Liu, Yong; Xu, Fengping; Huang, Jinrong; Daugaard, Tina Fuglsang; Petersen, Trine Skov; Hansen, Bettina; Ye, Lingfei; Zhou, Qing; Fang, Fang; Yang, Ling; Li, Shengting; Fløe, Lasse; Jensen, Kristopher Torp; Shrock, Ellen; Chen, Fang; Yang, Huanming; Wang, Jian; Liu, Xin; Xu, Xun; Bolund, Lars; Nielsen, Anders Lade; Luo, Yonglun

2018-03-01

Fusion of DNA methyltransferase domains to the nuclease-deficient clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (dCas9) has been used for epigenome editing, but the specificities of these dCas9 methyltransferases have not been fully investigated. We generated CRISPR-guided DNA methyltransferases by fusing the catalytic domain of DNMT3A or DNMT3B to the C terminus of the dCas9 protein from Streptococcus pyogenes and validated its on-target and global off-target characteristics. Using targeted quantitative bisulfite pyrosequencing, we prove that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can efficiently methylate the CpG dinucleotides flanking its target sites at different genomic loci (uPA and TGFBR3) in human embryonic kidney cells (HEK293T). Furthermore, we conducted whole genome bisulfite sequencing (WGBS) to address the specificity of our dCas9 methyltransferases. WGBS revealed that although dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B did not cause global methylation changes, a substantial number (more than 1000) of the off-target differentially methylated regions (DMRs) were identified. The off-target DMRs, which were hypermethylated in cells expressing dCas9 methyltransferase and guide RNAs, were predominantly found in promoter regions, 5΄ untranslated regions, CpG islands, and DNase I hypersensitivity sites, whereas unexpected hypomethylated off-target DMRs were significantly enriched in repeated sequences. Through chromatin immunoprecipitation with massive parallel DNA sequencing analysis, we further revealed that these off-target DMRs were weakly correlated with dCas9 off-target binding sites. Using quantitative polymerase chain reaction, RNA sequencing, and fluorescence reporter cells, we also found that dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B can mediate transient inhibition of gene expression, which might be caused by dCas9-mediated de novo DNA methylation as well as interference with transcription. Our results prove that dCas9 methyltransferases cause efficient RNA-guided methylation of specific endogenous CpGs. However, there is significant off-target methylation indicating that further improvements of the specificity of CRISPR-dCas9 based DNA methylation modifiers are required.

Guide-bound structures of an RNA-targeting A-cleaving CRISPR-Cas13a enzyme

PubMed Central

Knott, Gavin J.; East-Seletsky, Alexandra; Cofsky, Joshua C.; Holton, James M.; Charles, Emeric; O’Connell, Mitchell R.; Doudna, Jennifer A.

2018-01-01

CRISPR adaptive immune systems protect bacteria from infections by deploying CRISPR RNA (crRNA)-guided enzymes to recognize and cut foreign nucleic acids. Type VI-A CRISPR-Cas systems include the Cas13a enzyme, an RNA-activated ribonuclease (RNase) capable of crRNA processing and single-stranded RNA degradation upon target transcript binding. Here we present the 2.0 Å resolution crystal structure of a crRNA-bound L. bacterium Cas13a (LbaCas13a), representing a recently discovered Cas13a enzyme subtype. This structure and accompanying biochemical experiments define for the first time the Cas13a catalytic residues that are directly responsible for crRNA maturation. In addition, the orientation of the foreign-derived target RNA-specifying sequence in the protein interior explains the conformational gating of Cas13a nuclease activation. These results describe how Cas13a enzymes generate functional crRNAs and how catalytic activity is blocked prior to target RNA recognition, with implications for both bacterial immunity and diagnostic applications. PMID:28892041

CRISPR/Cas9 delivery with one single adenoviral vector devoid of all viral genes.

PubMed

Ehrke-Schulz, Eric; Schiwon, Maren; Leitner, Theo; Dávid, Stephan; Bergmann, Thorsten; Liu, Jing; Ehrhardt, Anja

2017-12-07

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system revolutionized the field of gene editing but viral delivery of the CRISPR/Cas9 system has not been fully explored. Here we adapted clinically relevant high-capacity adenoviral vectors (HCAdV) devoid of all viral genes for the delivery of the CRISPR/Cas9 machinery using a single viral vector. We present a platform enabling fast transfer of the Cas9 gene and gRNA expression units into the HCAdV genome including the option to choose between constitutive or inducible Cas9 expression and gRNA multiplexing. Efficacy and versatility of this pipeline was exemplified by producing different CRISPR/Cas9-HCAdV targeting the human papillomavirus (HPV) 18 oncogene E6, the dystrophin gene causing Duchenne muscular dystrophy (DMD) and the HIV co-receptor C-C chemokine receptor type 5 (CCR5). All CRISPR/Cas9-HCAdV proved to be efficient to deliver the respective CRISPR/Cas9 expression units and to introduce the desired DNA double strand breaks at their intended target sites in immortalized and primary cells.

Hit and go CAS9 delivered through a lentiviral based self-limiting circuit.

PubMed

Petris, Gianluca; Casini, Antonio; Montagna, Claudia; Lorenzin, Francesca; Prandi, Davide; Romanel, Alessandro; Zasso, Jacopo; Conti, Luciano; Demichelis, Francesca; Cereseto, Anna

2017-05-22

In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.

The Reverse Transcriptases Associated with CRISPR-Cas Systems.

PubMed

Toro, Nicolás; Martínez-Abarca, Francisco; González-Delgado, Alejandro

2017-08-02

CRISPR (clustered regularly interspaced short palindromic repeats) and associated proteins (Cas) act as adaptive immune systems in bacteria and archaea. Some CRISPR-Cas systems have been found to be associated with putative reverse transcriptases (RT), and an RT-Cas1 fusion associated with a type III-B system has been shown to acquire RNA spacers in vivo. Nevertheless, the origin and evolutionary relationships of these RTs and associated CRISPR-Cas systems remain largely unknown. We performed a comprehensive phylogenetic analysis of these RTs and associated Cas1 proteins, and classified their CRISPR-Cas modules. These systems were found predominantly in bacteria, and their presence in archaea may be due to a horizontal gene transfer event. These RTs cluster into 12 major clades essentially restricted to particular phyla, suggesting host-dependent functioning. The RTs and associated Cas1 proteins may have largely coevolved. They are, therefore, subject to the same selection pressures, which may have led to coadaptation within particular protein complexes. Furthermore, our results indicate that the association of an RT with a CRISPR-Cas system has occurred on multiple occasions during evolution.

Production of genome-edited pluripotent stem cells and mice by CRISPR/Cas.

PubMed

Horii, Takuro; Hatada, Izuho

2016-01-01

Clustered regularly at interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) nucleases, so-called CRISPR/Cas, was recently developed as an epoch-making genome engineering technology. This system only requires Cas9 nuclease and single-guide RNA complementary to a target locus. CRISPR/Cas enables the generation of knockout cells and animals in a single step. This system can also be used to generate multiple mutations and knockin in a single step, which is not possible using other methods. In this review, we provide an overview of genome editing by CRISPR/Cas in pluripotent stem cells and mice.

Speech Inconsistency in Children With Childhood Apraxia of Speech, Language Impairment, and Speech Delay: Depends on the Stimuli.

PubMed

Iuzzini-Seigel, Jenya; Hogan, Tiffany P; Green, Jordan R

2017-05-24

The current research sought to determine (a) if speech inconsistency is a core feature of childhood apraxia of speech (CAS) or if it is driven by comorbid language impairment that affects a large subset of children with CAS and (b) if speech inconsistency is a sensitive and specific diagnostic marker that can differentiate between CAS and speech delay. Participants included 48 children ranging between 4;7 to 17;8 (years;months) with CAS (n = 10), CAS + language impairment (n = 10), speech delay (n = 10), language impairment (n = 9), or typical development (n = 9). Speech inconsistency was assessed at phonemic and token-to-token levels using a variety of stimuli. Children with CAS and CAS + language impairment performed equivalently on all inconsistency assessments. Children with language impairment evidenced high levels of speech inconsistency on the phrase "buy Bobby a puppy." Token-to-token inconsistency of monosyllabic words and the phrase "buy Bobby a puppy" was sensitive and specific in differentiating children with CAS and speech delay, whereas inconsistency calculated on other stimuli (e.g., multisyllabic words) was less efficacious in differentiating between these disorders. Speech inconsistency is a core feature of CAS and is efficacious in differentiating between children with CAS and speech delay; however, sensitivity and specificity are stimuli dependent.

Single nucleotide editing without DNA cleavage using CRISPR/Cas9-deaminase in the sea urchin embryo.

PubMed

Shevidi, Saba; Uchida, Alicia; Schudrowitz, Natalie; Wessel, Gary M; Yajima, Mamiko

2017-12-01

A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks. In this study, we demonstrate a modified CRISPR/Cas9 system fused to cytosine deaminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence. These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations. Developmental Dynamics 246:1036-1046, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Editing Citrus Genome via SaCas9/sgRNA System

PubMed Central

Jia, Hongge; Xu, Jin; Orbović, Vladimir; Zhang, Yunzeng; Wang, Nian

2017-01-01

SaCas9/sgRNA, derived from Staphylococcus aureus, is an alternative system for genome editing to Streptococcus pyogenes SpCas9/sgRNA. The smaller SaCas9 recognizes a different protospacer adjacent motif (PAM) sequence from SpCas9. SaCas9/sgRNA has been employed to edit the genomes of Arabidopsis, tobacco and rice. In this study, we aimed to test its potential in genome editing of citrus. Transient expression of SaCas9/sgRNA in Duncan grapefruit via Xcc-facilitated agroinfiltration showed it can successfully modify CsPDS and Cs2g12470. Subsequently, binary vector GFP-p1380N-SaCas9/35S-sgRNA1:AtU6-sgRNA2 was developed to edit two target sites of Cs7g03360 in transgenic Carrizo citrange. Twelve GFP-positive Carrizo transformants were successfully established, designated as #Cz1 to #Cz12. Based on targeted next generation sequencing results, the mutation rates for the two targets ranged from 15.55 to 39.13% for sgRNA1 and 49.01 to 79.67% for sgRNA2. Therefore, SaCas9/sgRNA can be used as an alternative tool to SpCas9/sgRNA for citrus genome editing. PMID:29312390

Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.

PubMed

Huang, Mengqi; Zhou, Xiaoming; Wang, Huiying; Xing, Da

2018-02-06

A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.

Predicted cycloartenol synthase protein from Kandelia obovata and Rhizophora stylosa using online software of Phyre2 and Swiss-model

NASA Astrophysics Data System (ADS)

Basyuni, M.; Sulistiyono, N.; Wati, R.; Sumardi; Oku, H.; Baba, S.; Sagami, H.

2018-03-01

Cloning of Kandelia obovata KcCAS gene (previously known as Kandelia candel) and Rhizophora stylosa RsCAS have already have been reported and encoded cycloartenol synthases. In this study, the predicted KcCAS and RsCAS protein were analyzed using online software of Phyre2 and Swiss-model. The protein modelling for KcCAS and RsCAS cycloartenol synthases was determined using Pyre2 had similar results with slightly different in sequence identity. By contrast, the Swiss-model for KcCAS slightly had higher sequence identity (47.31%) and Qmean (0.70) compared to RsCAS. No difference of ligands binding site which is considered as modulators for both cycloartenol synthases. The range of predicted protein derived from 91-757 amino acid residues with coverage sequence similarities 0.86, respectively from template model of lanosterol synthase from the human. Homology modelling revealed that 706 residues (93% of the amino acid sequence) had been modelled with 100.0% confidence by the single highest scoring template for both KcCAS and RsCAS using Phyre2. This coverage was more elevated than swiss-model predicted (86%). The present study suggested that both genes are responsible for the genesis of cycloartenol in these mangrove plants.

Characterization of three caspases and their pathogen-induced expression pattern in Portunus trituberculatus.

PubMed

Ren, Xianyun; Yu, Xuan; Gao, Baoquan; Liu, Ping; Li, Jian

2017-07-01

Caspases are a family of proteases involved in many important biological processes including apoptosis and inflammation. In this study, we analyzed the expression patterns and effects on immune response in various tissues of the edible crab Portunus trituberculatus. PtCas 2, PtCas 3 and PtCas 4 share overall sequence identities of 55.88%-74.86%, 8.47%-46.54% and 20.11%-50.87%, respectively, with their other crustacean species. PtCas 2, PtCas 3 and PtCas 4 have the same caspase domain and catalytic site found in known caspases. The expression levels of the three caspases differed between tissues. Following bacterial and viral infection, the expression levels of the three caspases reached a maximum level at 24 h post-infection (hpi) in case of bacteria, whereas it was 48 hpi in virus. Moreover, the WSSV, Vibrio alginolyticus or V. parahaemolyticus induced the activities of PtCas 2-4 in a time-dependent manner. These results indicate an involvement of caspases in bacterial and viral induced immune response and demonstrate for the first time that PtCas 2, PtCas 3 and PtCas 4 are essential for optimal response to bacterial and virus infection in crabs. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Relationship between Carotid Atherosclerosis, Inflammatory Cytokines, and Oxidative Stress in Middle-Aged and Elderly Hemodialysis Patients

PubMed Central

Ren, Hongqi; Zhou, Xuan; Luan, Zhiyong; Luo, Xiaomei; Han, Shujing; Cai, Qing; Rui, Wang; Li, Yan

2013-01-01

Objective. To identify the relationship between microinflammation, oxidative stress, and carotid arterial stiffness in hemodialysis patients. Methods. The CAS β and PWV obtained by ultrasound technology were used to assess carotid arterial stiffness. We divided the patients into either the CAS group or the non-CAS group based on the presence or absence of CAS. The parameters of ALB, Ca, P, TC, HDL, LDL, TG, glucose, creatinine, and hs-CRP levels were routinely tested in both groups of patients. The levels of TNF-α, IL-6, and 8-isoprostane F2α were measured by ELISA. Results. A total of 42 patients were enrolled in the CAS group and 20 patients were enrolled in the non-CAS group. No significant differences between the CAS group and the non-CAS group were observed with respect to age, dialysis duration, DBP, BUN, Cr, TC, TG, HDL, LDL, and Hb. However, SBP , pulse pressure, and 8-isoprostane levels of the CAS group were higher than those of the non-CAS group. The hs-CRP, TNF-α, and IL-6 levels were elevated in both groups but showed no significant differences. Conclusions. Maintenance of hemodialysis patients exhibits a microinflammatory state that may lead to atherosclerosis. The roles of hypertension and oxidative stress may be more important. PMID:24187620

Low rates of complications for carotid artery stenting are associated with a high clinician volume of carotid artery stenting and aortic endografting but not with a high volume of percutaneous coronary interventions.

PubMed

Modrall, J Gregory; Chung, Jayer; Kirkwood, Melissa L; Baig, M Shadman; Tsai, Shirling X; Timaran, Carlos H; Valentine, R James; Rosero, Eric B

2014-07-01

Prior studies have demonstrated improved clinical outcomes for surgeons with a high-volume experience with certain open vascular operations. A high-volume experience with carotid artery stenting (CAS) improves clinical outcomes. Moreover, it is not known whether experience with other endovascular procedures, including percutaneous coronary interventions (PCIs), is an adequate substitute for experience with CAS. The goal of this study was to quantify the effect of increasing clinician volume of CAS, endovascular aneurysm repair (EVAR), and thoracic endovascular aortic aneurysm repair (TEVAR), and PCI on the outcomes for CAS. The Nationwide Inpatient Sample was analyzed to identify patients undergoing CAS for the years 2005 to 2009. Clinicians were stratified into tertiles of low-volume, medium-volume, and high-volume groups by annual volume of CAS, EVAR/TEVAR, and PCI. Multiple logistic regression analyses were used to examine the relationship between clinician volume and a composite outcome of the in-hospital stroke and death rate after CAS. Between 2005 and 2009, 56,374 elective CAS procedures were performed nationwide, with a crude in-hospital stroke and death rate of 3.22%. A median of nine CAS procedures (interquartile range, 3-20) were performed annually per clinician. As expected, stroke and death rates for CAS decreased with increasing volume of CAS performed by a clinician (low-volume vs medium-volume vs high-volume: 4.43% vs 2.89% vs 2.27%; P = .0001). Similar patterns were noted between clinicians' volume of EVAR/TEVAR (low-volume vs medium-volume vs high-volume: 4.58% vs 3.18% vs 2.16%; P = .0023). In contrast, increasing PCI volume was not associated with decreased stroke and death rates after CAS (low-volume vs medium-volume vs high-volume: 2.99% vs 3.18% vs 3.55%; P = .35). After adjusting for patient and hospital characteristics, clinician volume of CAS (odds ratio [OR], 0.84; 95% confidence interval [CI], 0.74-0.94; P = .003) and EVAR/TEVAR (OR, 0.85; 95% CI, 0.75-0.97; P = .020) remained significant predictors of stroke and death after CAS, whereas increasing clinician volume of PCI was associated with significantly increasing likelihood of stroke or death after CAS (OR, 1.025; 95% CI, 1.004-1.047; P = .019). The stroke and death rate for CAS to treat carotid stenosis is inversely affected by the number of CAS and EVAR/TEVAR procedures performed by a clinician. In contrast, a high-volume experience with PCI is not associated with improved outcomes after CAS. Copyright © 2014 Society for Vascular Surgery. All rights reserved.

Socioeconomic Impact Assessment: Communications Industry. Phase I. Technical Appendix Description and Documentation of the Current State-of-the-Art. Volume II.

DTIC Science & Technology

1979-02-02

R2 = 1.8 nmi (10,940 ft). An analysis of a CAS employing range and range rate indicated that the form of the equation used in ANTC-117 was valid ...interrogations persecond. Preliminary analysis of flight data indicated the system is capable of tracking successfully through garbled situations...ATC simulation, Monte-Carlo simulation of 12 mid-airs and analysis of ARTS III data for ATC interaction. The results of the effort points to the need

The A-10 Thunderbolt as an Organic Army Asset

DTIC Science & Technology

1991-06-07

whether report isinterim, final, etc. If Statements on Technical applicable, enter indusive report dates (e.g. 10 Documents." Jun 87 - 30 Jun 88). DOE See...delivered in 1984.31 At present, nearly 650 A-10’s remain in the Air Force inventory. 3 2 As of 30 Septem- ber 1989, the average age of the 447 A-10’s in...designed solely as an armor-defeating CAS aircraft. The aircraft was built around the General Electric GAU-8/A "Avenger," 30 millimeter cannon. The cannon

Kinetics of the CRISPR-Cas9 effector complex assembly and the role of 3′-terminal segment of guide RNA

PubMed Central

Mekler, Vladimir; Minakhin, Leonid; Semenova, Ekaterina; Kuznedelov, Konstantin; Severinov, Konstantin

2016-01-01

CRISPR-Cas9 is widely applied for genome engineering in various organisms. The assembly of single guide RNA (sgRNA) with the Cas9 protein may limit the Cas9/sgRNA effector complex function. We developed a FRET-based assay for detection of CRISPR–Cas9 complex binding to its targets and used this assay to investigate the kinetics of Cas9 assembly with a set of structurally distinct sgRNAs. We find that Cas9 and isolated sgRNAs form the effector complex efficiently and rapidly. Yet, the assembly process is sensitive to the presence of moderate concentrations of non-specific RNA competitors, which considerably delay the Cas9/sgRNA complex formation, while not significantly affecting already formed complexes. This observation suggests that the rate of sgRNA loading into Cas9 in cells can be determined by competition between sgRNA and intracellular RNA molecules for the binding to Cas9. Non-specific RNAs exerted particularly large inhibitory effects on formation of Cas9 complexes with sgRNAs bearing shortened 3′-terminal segments. This result implies that the 3′-terminal segment confers sgRNA the ability to withstand competition from non-specific RNA and at least in part may explain the fact that use of sgRNAs truncated for the 3′-terminal stem loops leads to reduced activity during genomic editing. PMID:26945042

Pathophysiology and Medical Treatment of Carotid Artery Stenosis

PubMed Central

Prasad, Kailash

2015-01-01

Stroke is the third leading cause of mortality. Approximately 80 to 85% strokes are ischemic due to carotid artery stenosis (CAS). The prevalence of significant CAS is 7% in women and 9% in men. Severe asymptomatic CAS varies from 0 to 3.1%. Prevalence of symptomatic CAS is high in patients with peripheral arterial disease. CAS is due to atherosclerosis, the major risk factors for which include dyslipidemia, hypertension, diabetes, obesity, cigarette smoking, advanced glycation end products (AGEs) and its receptors (RAGE, soluble RAGE [sRAGE]), lack of exercise and C-reactive protein (CRP). This article discusses the basic mechanism of atherosclerosis and the mechanisms by which these risk factors induce atherosclerosis. The role of AGEs and its receptors in the development and progression of CAS has been discussed in detail. Lifestyle changes and medical treatment of CAS such as lifestyle changes, lipid-lowering agents, antihypertensive agents, antidiabetic drugs, anti-AGE therapy, measures to elevate soluble receptors of AGE (sRAGE, esRAGE). CRP-lowering agents have been discussed in detail. The drugs especially lipid-lowering agents, and antihypertensive and antidiabetic drugs suppress, regress, and slow the progression of CAS. The possible role of lowering the levels of AGEs and raising the levels of sRAGE in the treatment of CAS has been proposed. Lifestyle changes besides medical treatment have been stressed. Lifestyle changes and medical treatment not only would slow the progression of CAS but would also regress the CAS. PMID:26417183

Biophysical properties of intrinsically disordered p130Cas substrate domain--implication in mechanosensing.

PubMed

Hotta, Kinya; Ranganathan, Soumya; Liu, Ruchuan; Wu, Fei; Machiyama, Hiroaki; Gao, Rong; Hirata, Hiroaki; Soni, Neelesh; Ohe, Takashi; Hogue, Christopher W V; Madhusudhan, M S; Sawada, Yasuhiro

2014-04-01

Mechanical stretch-induced tyrosine phosphorylation in the proline-rich 306-residue substrate domain (CasSD) of p130Cas (or BCAR1) has eluded an experimentally validated structural understanding. Cellular p130Cas tyrosine phosphorylation is shown to function in areas without internal actomyosin contractility, sensing force at the leading edge of cell migration. Circular dichroism shows CasSD is intrinsically disordered with dominant polyproline type II conformations. Strongly conserved in placental mammals, the proline-rich sequence exhibits a pseudo-repeat unit with variation hotspots 2-9 residues before substrate tyrosine residues. Atomic-force microscopy pulling experiments show CasSD requires minimal extension force and exhibits infrequent, random regions of weak stability. Proteolysis, light scattering and ultracentrifugation results show that a monomeric intrinsically disordered form persists for CasSD in solution with an expanded hydrodynamic radius. All-atom 3D conformer sampling with the TraDES package yields ensembles in agreement with experiment when coil-biased sampling is used, matching the experimental radius of gyration. Increasing β-sampling propensities increases the number of prolate conformers. Combining the results, we conclude that CasSD has no stable compact structure and is unlikely to efficiently autoinhibit phosphorylation. Taking into consideration the structural propensity of CasSD and the fact that it is known to bind to LIM domains, we propose a model of how CasSD and LIM domain family of transcription factor proteins may function together to regulate phosphorylation of CasSD and effect machanosensing.

Nucleic Acid-Dependent Conformational Changes in CRISPR-Cas9 Revealed by Site-Directed Spin Labeling.

PubMed

Vazquez Reyes, Carolina; Tangprasertchai, Narin S; Yogesha, S D; Nguyen, Richard H; Zhang, Xiaojun; Rajan, Rakhi; Qin, Peter Z

2017-06-01

In a type II clustered regularly interspaced short palindromic repeats (CRISPR) system, RNAs that are encoded at the CRISPR locus complex with the CRISPR-associated (Cas) protein Cas9 to form an RNA-guided nuclease that cleaves double-stranded DNAs at specific sites. In recent years, the CRISPR-Cas9 system has been successfully adapted for genome engineering in a wide range of organisms. Studies have indicated that a series of conformational changes in Cas9, coordinated by the RNA and the target DNA, direct the protein into its active conformation, yet details on these conformational changes, as well as their roles in the mechanism of function of Cas9, remain to be elucidated. Here, nucleic acid-dependent conformational changes in Streptococcus pyogenes Cas9 (SpyCas9) were investigated using the method of site-directed spin labeling (SDSL). Single nitroxide spin labels were attached, one at a time, at one of the two native cysteine residues (Cys80 and Cys574) of SpyCas9, and the spin-labeled proteins were shown to maintain their function. X-band continuous-wave electron paramagnetic resonance spectra of the nitroxide attached at Cys80 revealed conformational changes of SpyCas9 that are consistent with a large-scale domain re-arrangement upon binding to its RNA partner. The results demonstrate the use of SDSL to monitor conformational changes in CRISPR-Cas9, which will provide key information for understanding the mechanism of CRISPR function.

A Biophysical Model of CRISPR/Cas9 Activity for Rational Design of Genome Editing and Gene Regulation

PubMed Central

Farasat, Iman; Salis, Howard M.

2016-01-01

The ability to precisely modify genomes and regulate specific genes will greatly accelerate several medical and engineering applications. The CRISPR/Cas9 (Type II) system binds and cuts DNA using guide RNAs, though the variables that control its on-target and off-target activity remain poorly characterized. Here, we develop and parameterize a system-wide biophysical model of Cas9-based genome editing and gene regulation to predict how changing guide RNA sequences, DNA superhelical densities, Cas9 and crRNA expression levels, organisms and growth conditions, and experimental conditions collectively control the dynamics of dCas9-based binding and Cas9-based cleavage at all DNA sites with both canonical and non-canonical PAMs. We combine statistical thermodynamics and kinetics to model Cas9:crRNA complex formation, diffusion, site selection, reversible R-loop formation, and cleavage, using large amounts of structural, biochemical, expression, and next-generation sequencing data to determine kinetic parameters and develop free energy models. Our results identify DNA supercoiling as a novel mechanism controlling Cas9 binding. Using the model, we predict Cas9 off-target binding frequencies across the lambdaphage and human genomes, and explain why Cas9’s off-target activity can be so high. With this improved understanding, we propose several rules for designing experiments for minimizing off-target activity. We also discuss the implications for engineering dCas9-based genetic circuits. PMID:26824432

Molecular Mechanisms of RNA-Targeting by Cas13-containing Type VI CRISPR-Cas Systems.

PubMed

O'Connell, Mitchell

2018-06-22

Prokaryotic adaptive immune systems use CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR associated (Cas) proteins for RNA-guided cleavage of foreign genetic elements. The focus of this review, Type VI CRISPR-Cas systems, include a single protein known as Cas13 (formerly C2c2), that when assembled with a crRNA forms a crRNA-guided RNA-targeting effector complex. Type VI CRISPR-Cas systems can be divided into four subtypes (A-D) based on Cas13 phylogeny. All Cas13 proteins studied to date possess two enzymatically distinct ribonuclease activities that are required for optimal interference. One RNase is responsible for pre-crRNA processing to form mature Type VI interference complexes, while the other RNase activity provided by the two HEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains, is required for degradation of target RNA during viral interference. In this review, I will compare and contrast what is known about the molecular architecture and behavior of Type VI (A-D) CRISPR-Cas13 interference complexes, how this allows them to carry out their RNA-targeting function, how Type VI accessory proteins are able to modulate Cas13 activity, and how together all of these features have led to the rapid development of a range of RNA-targeting applications. Throughout I will also discuss some of the outstanding questions regarding Cas13's molecular behavior, and its role in bacterial adaptive immunity and RNA-targeting applications. Copyright © 2018. Published by Elsevier Ltd.

48 CFR 30.201-1 - CAS applicability.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability. 30.201-1 Section 30.201-1 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS...

Towards ubiquitous access of computer-assisted surgery systems.

PubMed

Liu, Hui; Lufei, Hanping; Shi, Weishong; Chaudhary, Vipin

2006-01-01

Traditional stand-alone computer-assisted surgery (CAS) systems impede the ubiquitous and simultaneous access by multiple users. With advances in computing and networking technologies, ubiquitous access to CAS systems becomes possible and promising. Based on our preliminary work, CASMIL, a stand-alone CAS server developed at Wayne State University, we propose a novel mobile CAS system, UbiCAS, which allows surgeons to retrieve, review and interpret multimodal medical images, and to perform some critical neurosurgical procedures on heterogeneous devices from anywhere at anytime. Furthermore, various optimization techniques, including caching, prefetching, pseudo-streaming-model, and compression, are used to guarantee the QoS of the UbiCAS system. UbiCAS enables doctors at remote locations to actively participate remote surgeries, share patient information in real time before, during, and after the surgery.

Cas9 Variants Expand the Target Repertoire in Caenorhabditis elegans

PubMed Central

Bell, Ryan T.; Fu, Becky X. H.; Fire, Andrew Z.

2016-01-01

The proliferation of CRISPR/Cas9-based methods in Caenorhabditis elegans has enabled efficient genome editing and precise genomic tethering of Cas9 fusion proteins. Experimental designs using CRISPR/Cas9 are currently limited by the need for a protospacer adjacent motif (PAM) in the target with the sequence NGG. Here we report the characterization of two modified Cas9 proteins in C. elegans that recognize NGA and NGCG PAMs. We found that each variant could stimulate homologous recombination with a donor template at multiple loci and that PAM specificity was comparable to that of wild-type Cas9. To directly compare effectiveness, we used CRISPR/Cas9 genome editing to generate a set of assay strains with a common single-guide RNA (sgRNA) target sequence, but that differ in the juxtaposed PAM (NGG, NGA, or NGCG). In this controlled setting, we determined that the NGA PAM Cas9 variant can be as effective as wild-type Cas9. We similarly edited a genomic target to study the influence of the base following the NGA PAM. Using four strains with four NGAN PAMs differing only at the fourth position and adjacent to the same sgRNA target, we observed that efficient homologous replacement was attainable with any base in the fourth position, with an NGAG PAM being the most effective. In addition to demonstrating the utility of two Cas9 mutants in C. elegans and providing reagents that permit CRISPR/Cas9 experiments with fewer restrictions on potential targets, we established a means to benchmark the efficiency of different Cas9::PAM combinations that avoids variations owing to differences in the sgRNA sequence. PMID:26680661

Poumon du puisatier

PubMed Central

Elidrissi, Amal Moustarhfir; Zaghba, Nahid; Benjelloun, Hanane; Yassine, Najiba

2016-01-01

Le puisatier a pour profession le creusement et l'entretien des puits pour fournir de l'eau. Il est au contact de divers minerais, particulièrement la silice, particule qui présente un risque certain de développement des maladies pulmonaires connues sous le nom de silicose. Le but de notre travail est de préciser le profil épidémiologique, clinique, radiologique et évolutif des patients puisatiers silicotiques. C'est une étude rétrospective concernant 54 cas de puisatiers ayant une silicose, colligés au service des maladies respiratoires du CHU Ibn Rochd de Casablanca, de Mars 1997 à Janvier 2016. Tous les malades étaient des puisatiers, de sexe masculin, avec une moyenne d'âge de 50 ans. Le tabagisme était retrouvé dans 36 cas et un antécédent de tuberculose était noté dans huit cas. La radiographie thoracique retrouvait des grandes opacités dans 39 cas, des petites opacités dans 15 cas, et un épaississement des septats dans 11 cas. Ce tableau de silicose s'était compliqué d'une surinfection bactérienne dans 37% des cas, d' un pneumothorax dans 4% des cas et d'une tuberculose dans 20% des cas. La prise en charge thérapeutique était celle des complications. La déclaration de la maladie professionnelle et de l'indemnisation était faite. L'évolution était bonne dans 12 cas, stationnaire dans 17 cas et mauvaise dans 16 cas. La silicose est une pneumoconiose fréquente chez les puisatiers. Elle retentit sur la fonction respiratoire. Nous soulignons l'association fréquente de tuberculose et nous insistons sur la prévention qui reste le meilleur traitement. PMID:28292119

CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

PubMed

Koonin, Eugene V; Makarova, Kira S

2013-05-01

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments

PubMed Central

Pearson, Bruce M.; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H.M.

2015-01-01

CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli. PMID:26338188

Mobile Genetic Elements and Evolution of CRISPR-Cas Systems: All the Way There and Back

PubMed Central

Makarova, Kira S.

2017-01-01

Abstract The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems of bacterial and archaeal adaptive immunity show multifaceted evolutionary relationships with at least five classes of mobile genetic elements (MGE). First, the adaptation module of CRISPR-Cas that is responsible for the formation of the immune memory apparently evolved from a Casposon, a self-synthesizing transposon that employs the Cas1 protein as the integrase and might have brought additional cas genes to the emerging immunity loci. Second, a large subset of type III CRISPR-Cas systems recruited a reverse transcriptase from a Group II intron, providing for spacer acquisition from RNA. Third, effector nucleases of Class 2 CRISPR-Cas systems that are responsible for the recognition and cleavage of the target DNA were derived from transposon-encoded TnpB nucleases, most likely, on several independent occasions. Fourth, accessory nucleases in some variants of types I and III toxin and type VI effectors RNases appear to be ultimately derived from toxin nucleases of microbial toxin–antitoxin modules. Fifth, the opposite direction of evolution is manifested in the recruitment of CRISPR-Cas systems by a distinct family of Tn7-like transposons that probably exploit the capacity of CRISPR-Cas to recognize unique DNA sites to facilitate transposition as well as by bacteriophages that employ them to cope with host defense. Additionally, individual Cas proteins, such as the Cas4 nuclease, were recruited by bacteriophages and transposons. The two-sided evolutionary connection between CRISPR-Cas and MGE fits the “guns for hire” paradigm whereby homologous enzymatic machineries, in particular nucleases, are shuttled between MGE and defense systems and are used alternately as means of offense or defense. PMID:28985291

Calcium polysulphide, its applications and emerging risk of environmental pollution-a review article.

PubMed

Dahlawi, Saad Mohammed; Siddiqui, Samreen

2017-01-01

Easy availability, preparation technique, and economic value make calcium polysulphide (CaS x ) a very useful inorganic chemical for various field and industrial applications. In this article, disparate applications of CaS x solution have been reviewed to suggest potential and future consolidation. This article also encompasses the physiochemical properties and production of CaS x solution, with critical appraisal on research focusing on CaS x application in agriculture industries and removal of potentially toxic elements (PTEs) from the environment. The kinetics of CaS x , technical issues associated with optimization of its dosage and environmental fate is also discussed in detail. This study covers almost all of the peer-reviewed research that has been performed since 1914. Some of the critiques in this article include the lack of integration between the exposure effect and the efficiency of treatment method, effects of oxidizing environments on the long-term performance of CaS x solution, and kinetics of CaS x solution with the PTEs. The working model of CaS x with PTEs is still system dependent, and therefore cannot be used with other applications. The kinetics of CaS x is described in detail with various phase stoichiometric reactions. Environmental fate is discussed based on applications, government reports, peer-reviewed articles and kinetics of CaS x , which provides a clear picture of emerging contaminants in the environment in relation to the insect resistance and ecotoxicology. Real time, lab based research articles are needed to identify toxicity limits of CaS x in environment in order to describe its effective permissible limit in environmental system. This review article provides a risk assessment of environmental pollution by CaS x based on its physicochemical characteristic, stoichiometry, kinetics, field, and industrial applications.

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna.

PubMed

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko; Watanabe, Hajime

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna.

CRISPR/Cas-mediated knock-in via non-homologous end-joining in the crustacean Daphnia magna

PubMed Central

Kumagai, Hitoshi; Nakanishi, Takashi; Matsuura, Tomoaki; Kato, Yasuhiko

2017-01-01

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is widely used for mediating the knock-in of foreign DNA into the genomes of various organisms. Here, we report a process of CRISPR/Cas-mediated knock-in via non-homologous end joining by the direct injection of Cas9/gRNA ribonucleoproteins (RNPs) in the crustacean Daphnia magna, which is a model organism for studies on toxicology, ecology, and evolution. First, we confirmed the cleavage activity of Cas9 RNPs comprising purified Cas9 proteins and gRNAs in D. magna. We used a gRNA that targets exon 10 of the eyeless gene. Cas9 proteins were incubated with the gRNAs and the resulting Cas9 RNPs were injected into D. magna eggs, which led to a typical phenotype of the eyeless mutant, i.e., eye deformity. The somatic and heritable mutagenesis efficiencies were up to 96% and 40%, respectively. Second, we tested the CRISPR/Cas-mediated knock-in of a plasmid by the injection of Cas9 RNPs. The donor DNA plasmid harboring the fluorescent reporter gene was designed to contain the gRNA recognition site. The co-injection of Cas9 RNPs together with the donor DNAs resulted in generation of one founder animal that produced fluorescent progenies. This transgenic Daphnia had donor DNA at the targeted genomic site, which suggested the concurrent cleavage of the injected plasmid DNA and genomic DNA. Owing to its simplicity and ease of experimental design, we suggest that the CRISPR/Cas-mediated knock-in method represents a promising tool for studying functional genomics in D. magna. PMID:29045453

Corrective Action Investigation Plan for Corrective Action Unit 536: Area 3 Release Site, Nevada Test Site, Nevada (Rev. 0 / June 2003), Including Record of Technical Change No. 1

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

None

This Corrective Action Investigation Plan contains the U.S. Department of Energy, National Nuclear Security Administration Nevada Site Office's approach to collect the data necessary to evaluate corrective action alternatives (CAAs) appropriate for the closure of Corrective Action Unit (CAU) 536: Area 3 Release Site, Nevada Test Site, Nevada, under the Federal Facility Agreement and Consent Order. Corrective Action Unit 536 consists of a single Corrective Action Site (CAS): 03-44-02, Steam Jenny Discharge. The CAU 536 site is being investigated because existing information on the nature and extent of possible contamination is insufficient to evaluate and recommend corrective action alternatives formore » CAS 03-44-02. The additional information will be obtained by conducting a corrective action investigation (CAI) prior to evaluating CAAs and selecting the appropriate corrective action for this CAS. The results of this field investigation are to be used to support a defensible evaluation of corrective action alternatives in the corrective action decision document. Record of Technical Change No. 1 is dated 3-2004.« less

CRISPR/Cas9 genome editing in human pluripotent stem cells: Harnessing human genetics in a dish.

PubMed

González, Federico

2016-07-01

Because of their extraordinary differentiation potential, human pluripotent stem cells (hPSCs) can differentiate into virtually any cell type of the human body, providing a powerful platform not only for generating relevant cell types useful for cell replacement therapies, but also for modeling human development and disease. Expanding this potential, structures resembling human organs, termed organoids, have been recently obtained from hPSCs through tissue engineering. Organoids exhibit multiple cell types self-organizing into structures recapitulating in part the physiology and the cellular interactions observed in the organ in vivo, offering unprecedented opportunities for human disease modeling. To fulfill this promise, tissue engineering in hPSCs needs to be supported by robust and scalable genome editing technologies. With the advent of the CRISPR/Cas9 technology, manipulating the genome of hPSCs has now become an easy task, allowing modifying their genome with superior precision, speed, and throughput. Here we review current and potential applications of the CRISPR/Cas9 technology in hPSCs and how they contribute to establish hPSCs as a model of choice for studying human genetics. Developmental Dynamics 245:788-806, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

RIFM fragrance ingredient safety assessment, 1-(1,2,3,4-tetrahydro-4,4-dimethyl-1-naphthyl)propan-1-one, CAS Registry Number 74499-60-8.

PubMed

Api, A M; Belsito, D; Bhatia, S; Bruze, M; Calow, P; Dagli, M L; Dekant, W; Fryer, A D; Kromidas, L; La Cava, S; Lalko, J F; Lapczynski, A; Liebler, D C; Miyachi, Y; Politano, V T; Ritacco, G; Salvito, D; Schultz, T W; Shen, J; Sipes, I G; Wall, B; Wilcox, D K

2016-11-01

The use of this material under current use conditions is supported by the existing information. This material was evaluated for genotoxicity, repeated dose toxicity, developmental and reproductive toxicity, local respiratory toxicity, phototoxicity/photoallergenicity, skin sensitization, as well as environmental safety. Data from the target material and the suitable read across analog 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (CAS # 21145-77-7) show that this material is not genotoxic. Data from the suitable read across analog 6-acetyl-1,1,2,4,4,7-hexamethyltetraline (CAS # 21145-77-7) provided a MOE > 100 for the repeat dose and developmental toxicity endpoints. The reproductive and local respiratory toxicity endpoints were completed using the TTC (Threshold of Toxicological Concern) for a Cramer Class II material (0.009 mg/kg/day and 0.47 mg/day, respectively). Data on the target material showed that this material is below the non-reactive DST for skin sensitization and did not have the potential for phototoxicity or photoallergenicity. The environmental endpoint was completed as described in the RIFM Framework. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Kinase-Independent Function of c-Src Mediates p130Cas Phosphorylation at the Serine-639 Site in Pressure Overloaded Myocardium.

PubMed

Palanisamy, Arun P; Suryakumar, Geetha; Panneerselvam, Kavin; Willey, Christopher D; Kuppuswamy, Dhandapani

2015-12-01

Early work in pressure overloaded (PO) myocardium shows that integrins mediate focal adhesion complex formation by recruiting the adaptor protein p130Cas (Cas) and nonreceptor tyrosine kinase c-Src. To explore c-Src role in Cas-associated changes during PO, we used a feline right ventricular in vivo PO model and a three-dimensional (3D) collagen-embedded adult cardiomyocyte in vitro model that utilizes a Gly-Arg-Gly-Asp-Ser (RGD) peptide for integrin stimulation. Cas showed slow electrophoretic mobility (band-shifting), recruitment to the cytoskeleton, and tyrosine phosphorylation at 165, 249, and 410 sites in both 48 h PO myocardium and 1 h RGD-stimulated cardiomyocytes. Adenoviral mediated expression of kinase inactive (negative) c-Src mutant with intact scaffold domains (KN-Src) in cardiomyocytes did not block the RGD stimulated changes in Cas. Furthermore, expression of KN-Src or kinase active c-Src mutant with intact scaffold function (A-Src) in two-dimensionally (2D) cultured cardiomyocytes was sufficient to cause Cas band-shifting, although tyrosine phosphorylation required A-Src. These data indicate that c-Src's adaptor function, but not its kinase function, is required for a serine/threonine specific phosphorylation(s) responsible for Cas band-shifting. To explore this possibility, Chinese hamster ovary cells that stably express Cas were infected with either β-gal or KN-Src adenoviruses and used for Cas immunoprecipitation combined with mass spectrometry analysis. In the KN-Src expressing cells, Cas showed phosphorylation at the serine-639 (human numbering) site. A polyclonal antibody raised against phospho-serine-639 detected Cas phosphorylation in 24-48 h PO myocardium. Our studies indicate that c-Src's adaptor function mediates serine-639 phosphorylation of Cas during integrin activation in PO myocardium. © 2015 Wiley Periodicals, Inc.

Stroke/Death Rates Following Carotid Artery Stenting and Carotid Endarterectomy in Contemporary Administrative Dataset Registries: A Systematic Review.

PubMed

Paraskevas, K I; Kalmykov, E L; Naylor, A R

2016-01-01

Randomised trials have reported higher stroke/death rates after carotid artery stenting (CAS) versus carotid endarterectomy (CEA). Despite this, the 2011 American Heart Association (AHA) guidelines expanded CAS indications, partly because of the Carotid Revascularization Endarterectomy versus Stenting Trial, but also because of improving outcomes in industry sponsored CAS Registries. The aim of this systematic review was: (i) to compare stroke/death rates after CAS/CEA in contemporary dataset registries, (ii) to examine whether published stroke/death rates after CAS fall within AHA thresholds, and, (iii) to see if there had been a decline (over time) in procedural risk after CAS/CEA. PubMed/Medline, Embase, and Cochrane databases were systematically searched according to the recommendations of the PRISMA statement from January 1, 2008 until February 23, 2015 for administrative dataset registries reporting outcomes after both CEA and CAS. Twenty-one registries reported outcomes involving more than 1,500,000 procedures. Stroke/death after CAS was significantly higher than after CEA in 11/21 registries (52%) involving "average risk for CEA" asymptomatic patients and in 11/18 registries (61%) involving "average risk for CEA" symptomatic patients. In another five registries, CAS was associated with higher stroke/death rates than CEA for both symptomatic and asymptomatic patients, but formal statistical comparison was not reported. CAS was associated with stroke/death rates that exceeded risk thresholds recommended by the AHA in 9/21 registries (43%) involving "average risk for CEA" asymptomatic patients and in 13/18 registries (72%) involving "average risk for CEA" symptomatic patients. In 5/18 registries (28%), the procedural risk after CAS in "average risk" symptomatic patients exceeded 10%. Data from contemporary administrative dataset registries suggest that stroke/death rates following CAS remain significantly higher than after CEA and often exceed accepted AHA thresholds. There was no evidence of a sustained decline in procedural risk after CAS. Copyright © 2015 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Nucleosome breathing and remodeling constrain CRISPR-Cas9 function

PubMed Central

Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

2016-01-01

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. DOI: http://dx.doi.org/10.7554/eLife.13450.001 PMID:27130520

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications.

PubMed

Ma, Xingliang; Zhu, Qinlong; Chen, Yuanling; Liu, Yao-Guang

2016-07-06

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

CRISPR/Cas9 for cancer research and therapy.

PubMed

Zhan, Tianzuo; Rindtorff, Niklas; Betge, Johannes; Ebert, Matthias P; Boutros, Michael

2018-04-16

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

Köklü, Erkan, E-mail: drerkankoklu@gmail.com; Arslan, Şakir; Yüksel, İsa Öner

Carotid artery stenting (CAS) is a revascularization modality that is an alternative to carotid endarterectomy. The efficacy of CAS in primary and secondary prevention from ischemic stroke has been demonstrated in various trials. Acute thrombosis of CAS is a rare complication that can lead to dramatic and catastrophic consequences. We discuss a case of acute CAS thrombosis in a patient who had previously undergone successful CAS. CAS was performed in a 73-year-old man who had had dysarthria lasting 2 weeks with 95 % stenosis in his left internal carotid artery. An acute cerebrovascular event resulting in right-sided hemiplegia developed 24 h after themore » procedure. Computed tomographic carotid angiography revealed complete occlusion of the stent with thrombus. The cause of stent thrombosis was thought to be antiaggregant resistance to both acetylsalicylic acid and clopidogrel. The most important cause of acute CAS thrombosis is inadequate or ineffective antiaggregant therapy. Evaluating patients who are candidates for CAS for acetylsalicylic acid and clopidogrel resistance may preclude this complication.« less

Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

PubMed

Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

2015-10-07

In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mitral valve prolapse associated with celiac artery stenosis: a new ultrasonographic syndrome?

PubMed Central

Arcari, Luciano

2004-01-01

Background Celiac artery stenosis (CAS) may be caused by atherosclerotic degeneration or compression exerted by the arched ligament of the diaphragm. Mitral valve prolapse (MVP) is the most common valvular disorder. There are no reports on an association between CAS and MVP. Methods 1560 (41%) out of 3780 consecutive patients undergoing echocardiographic assessment of MVP, had Doppler sonography of the celiac tract to detect CAS. Results CAS was found in 57 (3.7%) subjects (23 males and 34 females) none of whom complained of symptoms related to visceral ischemia. MVP was observed in 47 (82.4%) subjects with and 118 (7.9%) without CAS (p < 0.001). The agreement between MVP and CAS was 39% (95% CI 32–49%). PSV (Peak Systolic Velocity) was the only predictor of CAS in MPV patients (OR 0.24, 95% CI 0.08–0.69) as selected in a multivariate logistic model. Conclusion CAS and MVP seem to be significantly associated in patients undergoing consecutive ultrasonographic screening. PMID:15588321

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

PubMed

Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

2016-06-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

CRISPR/Cas9 for genome editing: progress, implications and challenges.

PubMed

Zhang, Feng; Wen, Yan; Guo, Xiong

2014-09-15

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements, and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 comprises of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair process leads to desired insertions, deletions or substitutions at target sites. The specificity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif locating at downstream of target sequences. Here, we review the molecular mechanism, applications and challenges of CRISPR/Cas9-mediated genome editing and clinical therapeutic potential of CRISPR/Cas9 in future. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

PubMed Central

Rollie, Clare; Schneider, Stefanie; Brinkmann, Anna Sophie; Bolt, Edward L; White, Malcolm F

2015-01-01

The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 PMID:26284603

Ethical issues of CRISPR technology and gene editing through the lens of solidarity.

PubMed

Mulvihill, John J; Capps, Benjamin; Joly, Yann; Lysaght, Tamra; Zwart, Hub A E; Chadwick, Ruth

2017-06-01

The avalanche of commentaries on CRISPR-Cas9 technology, a bacterial immune system modified to recognize any short DNA sequence, cut it out, and insert a new one, has rekindled hopes for gene therapy and other applications and raised criticisms of engineering genes in future generations. This discussion draws on articles that emphasize ethics, identified partly through PubMed and Google, 2014-2016. CRISPR-Cas9 has taken the pace and prospects for genetic discovery and applications to a high level, stoking anticipation for somatic gene engineering to help patients. We support a moratorium on germ line manipulation. We place increased emphasis on the principle of solidarity and the public good. The genetic bases of some diseases are not thoroughly addressable with CRISPR-Cas9. We see no new ethical issues, compared with gene therapy and genetic engineering in general, apart from the explosive rate of findings. Other controversies include eugenics, patentability and unrealistic expectations of professionals and the public. Biggest issues are the void of research on human germ cell biology, the appropriate routes for oversight and transparency, and the scientific and ethical areas of reproductive medicine. The principle of genomic solidarity and priority on public good should be a lens for bringing clarity to CRISPR debates. The valid claim of genetic exceptionalism supports restraint on experimentation in human germ cells, given the trans-generational dangers and the knowledge gap in germ cell biology. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

UAS in the NAS Air Traffic Controller Acceptability Study-1: The Effects of Horizontal Miss Distances on Simulated UAS and Manned Aircraft Encounters

NASA Technical Reports Server (NTRS)

Ghatas, Rania W.; Comstock, James R., Jr.; Consiglio, Maria C.; Chamberlain, James P.; Hoffler, Keith D.

2015-01-01

This study examined air traffic controller acceptability ratings based on the effects of differing horizontal miss distances (HMDs) for encounters between UAS and manned aircraft. In a simulation of the Dallas/Fort Worth (DFW) East-side airspace, the CAS-1 experiment at NASA Langley Research Center enlisted fourteen recently retired DFW air traffic controllers to rate well-clear volumes based on differing HMDs that ranged from 0.5 NM to 3.0 NM. The controllers were tasked with rating these HMDs from "too small" to "too excessive" on a defined, 1-5, scale and whether these distances caused any disruptions to the controller and/or to the surrounding traffic flow. Results of the study indicated a clear favoring towards a particular HMD range. Controller workload was also measured. Data from this experiment and subsequent experiments will play a crucial role in the FAA's establishment of rules, regulations, and procedures to safely and efficiently integrate UAS into the NAS.

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity

PubMed Central

Basila, Megan; Kelley, Melissa L.

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing. PMID:29176845

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

PubMed

Basila, Megan; Kelley, Melissa L; Smith, Anja van Brabant

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

Population Activities Fund Agency (PAFA): the journey so far.

PubMed

1993-01-01

Mechanisms are needed funding Nigerian Federal efforts to implement the National Policy on Population for Development, which was approved by the Armed Forces Ruling Council in February, 1988. Subprojects of the Population Activities Fund Agency (PAFA) which were approves are: the integration of family planning (FP) into maternal and child health (MCH) the promotion of Fp through health education, tertiary centers for reproductive health, public enlightenment on population, population/family life education in primary schools, monitoring of National Population Project impact, and integration of population into planning and budgeting. The last obstacle to implementation of PAFA's activities is the signing into law the decree establishing PAFA as a parastatal. The passage is required for continued operations. The national Population for Development policy is unique in providing for quantitative targets, which has attracted the needed financial support of agencies such as the World Bank. As part of the National Population Policy, the National Population Program (NPP) is developing an effective strategy for securing funding and evaluation of subprojects that are designed and implemented b Collaborating Agencies (CAs), both private and public. NPP aims 1) to provide funds for qualified CAs through the Population Activities Fund (PAF) and Agency (PAFA); 2) to monitor PAFA, which manages PAf with the Department of Population Activities, and 3) to stimulate analysis of sociocultural constraints to fertility reduction and international comparisons, and to design innovative interventions through the Population Research Fund (PRF). PAFA funds implementing agencies at all government and nongovernment levels with approaches to population information and services. The goal of PAFA is to realize NPP objectives. The motto is "Towards an improved quality of life for every Nigerian." The mandate is to provide funding for the PAF and NPP, to monitor CAs, to provide assistance to CAs preparing Phase II subprojects, and to supervise and guide CAs on funding, procurement, and implementation.

The Nurse Professional Competence (NPC) Scale: Self-reported competence among nursing students on the point of graduation.

PubMed

Gardulf, Ann; Nilsson, Jan; Florin, Jan; Leksell, Janeth; Lepp, Margret; Lindholm, Christina; Nordström, Gun; Theander, Kersti; Wilde-Larsson, Bodil; Carlsson, Marianne; Johansson, Eva

2016-01-01

International organisations, e.g. WHO, stress the importance of competent registered nurses (RN) for the safety and quality of healthcare systems. Low competence among RNs has been shown to increase the morbidity and mortality of inpatients. To investigate self-reported competence among nursing students on the point of graduation (NSPGs), using the Nurse Professional Competence (NPC) Scale, and to relate the findings to background factors. The NPC Scale consists of 88 items within eight competence areas (CAs) and two overarching themes. Questions about socio-economic background and perceived overall quality of the degree programme were added. In total, 1086 NSPGs (mean age, 28.1 [20-56]years, 87.3% women) from 11 universities/university colleges participated. NSPGs reported significantly higher scores for Theme I "Patient-Related Nursing" than for Theme II "Organisation and Development of Nursing Care". Younger NSPGs (20-27years) reported significantly higher scores for the CAs "Medical and Technical Care" and "Documentation and Information Technology". Female NSPGs scored significantly higher for "Value-Based Nursing". Those who had taken the nursing care programme at upper secondary school before the Bachelor of Science in Nursing (BSN) programme scored significantly higher on "Nursing Care", "Medical and Technical Care", "Teaching/Learning and Support", "Legislation in Nursing and Safety Planning" and on Theme I. Working extra paid hours in healthcare alongside the BSN programme contributed to significantly higher self-reported scores for four CAs and both themes. Clinical courses within the BSN programme contributed to perceived competence to a significantly higher degree than theoretical courses (93.2% vs 87.5% of NSPGs). Mean scores reported by NSPGs were highest for the four CAs connected with patient-related nursing and lowest for CAs relating to organisation and development of nursing care. We conclude that the NPC Scale can be used to identify and measure aspects of self-reported competence among NSPGs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Measurement of the integrated Luminosities of cross-section scan data samples around the {\\rm{\\psi }}(3770) mass region

NASA Astrophysics Data System (ADS)

Ablikim, M.; Achasov, M. N.; Ahmed, S.; Albrecht, M.; Alekseev, M.; Amoroso, A.; An, F. F.; An, Q.; Bai, Y.; Bakina, O.; Baldini Ferroli, R.; Ban, Y.; Begzsuren, K.; Bennett, D. W.; Bennett, J. V.; Berger, N.; Bertani, M.; Bettoni, D.; Bianchi, F.; Boger, E.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chai, J.; Chang, J. F.; Chang, W. L.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, J. C.; Chen, M. L.; Chen, P. L.; Chen, S. J.; Chen, X. R.; Chen, Y. B.; Chu, X. K.; Cibinetto, G.; Cossio, F.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; De Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Dou, Z. L.; Du, S. X.; Duan, P. F.; Fang, J.; Fang, S. S.; Fang, Y.; Farinelli, R.; Fava, L.; Fegan, S.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, X. L.; Gao, Y.; Gao, Y. G.; Gao, Z.; Garillon, B.; Garzia, I.; Gilman, A.; Goetzen, K.; Gong, L.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, L. M.; Gu, M. H.; Gu, Y. T.; Guo, A. Q.; Guo, L. B.; Guo, R. P.; Guo, Y. P.; Guskov, A.; Haddadi, Z.; Han, S.; Hao, X. Q.; Harris, F. A.; He, K. L.; He, X. Q.; Heinsius, F. H.; Held, T.; Heng, Y. K.; Holtmann, T.; Hou, Z. L.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. S.; Huang, J. S.; Huang, X. T.; Huang, X. Z.; Huang, Z. L.; Hussain, T.; Ikegami Andersson, W.; Irshad, M.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, X. S.; Jiang, X. Y.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Jin, Y.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Khan, T.; Khoukaz, A.; Kiese, P.; Kliemt, R.; Koch, L.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kuemmel, M.; Kuessner, M.; Kupsc, A.; Kurth, M.; Kühn, W.; Lange, J. S.; Lara, M.; Larin, P.; Lavezzi, L.; Leiber, S.; Leithoff, H.; Li, C.; Li, Cheng; Li, D. M.; Li, F.; Li, F. Y.; Li, G.; Li, H. B.; Li, H. J.; Li, J. C.; Li, J. W.; Li, K. J.; Li, Kang; Li, Ke; Li, Lei; Li, P. L.; Li, P. R.; Li, Q. Y.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Liao, L. Z.; Libby, J.; Lin, C. X.; Lin, D. X.; Liu, B.; Liu, B. J.; Liu, C. X.; Liu, D.; Liu, D. Y.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. L.; Liu, H. M.; Liu, Huanhuan; Liu, Huihui; Liu, J. B.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, Ke; Liu, L. D.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqing; Long, Y. F.; Lou, X. C.; Lu, H. J.; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, X. L.; Lusso, S.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, M. M.; Ma, Q. M.; Ma, X. N.; Ma, X. Y.; Ma, Y. M.; Maas, F. E.; Maggiora, M.; Malik, Q. A.; Mangoni, A.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; Meng, Z. X.; Messchendorp, J. G.; Mezzadri, G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Morello, G.; Muchnoi, N. Yu; Muramatsu, H.; Mustafa, A.; Nakhoul, S.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pan, Y.; Papenbrock, M.; Patteri, P.; Pelizaeus, M.; Pellegrino, J.; Peng, H. P.; Peng, Z. Y.; Peters, K.; Pettersson, J.; Ping, J. L.; Ping, R. G.; Pitka, A.; Poling, R.; Prasad, V.; Qi, H. R.; Qi, M.; Qi, T. Y.; Qian, S.; Qiao, C. F.; Qin, N.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Richter, M.; Ripka, M.; Rolo, M.; Rong, G.; Rosner, Ch.; Ruan, X. D.; Sarantsev, A.; Savrié, M.; Schnier, C.; Schoenning, K.; Shan, W.; Shan, X. Y.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Shi, X.; Song, J. J.; Song, W. M.; Song, X. Y.; Sosio, S.; Sowa, C.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, L.; Sun, S. S.; Sun, X. H.; Sun, Y. J.; Sun, Y. K.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tan, Y. T.; Tang, C. J.; Tang, G. Y.; Tang, X.; Tapan, I.; Tiemens, M.; Tsednee, B.; Uman, I.; Varner, G. S.; Wang, B.; Wang, B. L.; Wang, C. W.; Wang, D.; Wang, D. Y.; Wang, Dan; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, Meng; Wang, P.; Wang, P. L.; Wang, W. P.; Wang, X. F.; Wang, Y.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. Y.; Wang, Zongyuan; Weber, T.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, L. J.; Wu, Z.; Xia, L.; Xia, X.; Xia, Y.; Xiao, D.; Xiao, Y. J.; Xiao, Z. J.; Xie, Y. G.; Xie, Y. H.; Xiong, X. A.; Xiu, Q. L.; Xu, G. F.; Xu, J. J.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, F.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. J.; Yang, H. X.; Yang, L.; Yang, S. L.; Yang, Y. H.; Yang, Y. X.; Yang, Yifan; Ye, M.; Ye, M. H.; Yin, J. H.; You, Z. Y.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zallo, A.; Zeng, Y.; Zeng, Z.; Zhang, B. X.; Zhang, B. Y.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, S. F.; Zhang, T. J.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. H.; Zhang, Y. T.; Zhang, Yang; Zhang, Yao; Zhang, Yu; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, Q.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, A. N.; Zhu, J.; Zhu, J.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, S. H.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2018-05-01

To investigate the nature of the {{\\psi }}(3770) resonance and to measure the cross section for {{{e}}}+{{{e}}}-\\to {{D}}\\bar{{{D}}}, a cross-section scan data sample, distributed among 41 center-of-mass energy points from 3.73 to 3.89 GeV, was taken with the BESIII detector operated at the BEPCII collider in the year 2010. By analyzing the large angle Bhabha scattering events, we measure the integrated luminosity of the data sample at each center-of-mass energy point. The total integrated luminosity of the data sample is 76.16+/- 0.04+/- 0.61 {pb}}-1, where the first uncertainty is statistical and the second systematic. Supported by National Key Basic Research Program of China (2015CB856700), National Natural Science Foundation of China (NSFC) (11235011, 11335008, 11425524, 11625523, 11635010), the Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, the CAS Center for Excellence in Particle Physics (CCEPP), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (U1332201, U1532257, U1532258), CAS Key Research Program of Frontier Sciences (QYZDJ-SSW-SLH003, QYZDJ-SSW-SLH040), 100 Talents Program of CAS, National 1000 Talents Program of China, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology, German Research Foundation DFG under Contracts Nos. Collaborative Research Center CRC 1044, FOR 2359, Istituto Nazionale di Fisica Nucleare, Italy, Koninklijke Nederlandse Akademie van Wetenschappen (KNAW) (530-4CDP03), Ministry of Development of Turkey (DPT2006K-120470), National Science and Technology fund, The Swedish Research Council, U. S. Department of Energy (DE-FG02-05ER41374, DE-SC-0010118, DE-SC-0010504, DE-SC-0012069), University of Groningen (RuG) and the Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt, WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0)

Determination of the number of J/ψ events with inclusive J/ψ decays

NASA Astrophysics Data System (ADS)

Ablikim, M.; Achasov, M. N.; Ai, X. C.; Albayrak, O.; Albrecht, M.; Ambrose, D. J.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; Baldini Ferroli, R.; Ban, Y.; Bennett, D. W.; Bennett, J. V.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Boyko, I.; Briere, R. A.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; Cetin, S. A.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, H. Y.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Cheng, H. P.; Chu, X. K.; Cibinetto, G.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; De Mori, F.; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Dou, Z. L.; Du, S. X.; Duan, P. F.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, X.; Fang, Y.; Farinelli, R.; Fava, L.; Fedorov, O.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, X. L.; Gao, X. Y.; Gao, Y.; Gao, Z.; Garzia, I.; Goetzen, K.; Gong, L.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, L. B.; Guo, Y.; Guo, Y. P.; Haddadi, Z.; Hafner, A.; Han, S.; Hao, X. Q.; Harris, F. A.; He, K. L.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, C.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. S.; Huang, J. S.; Huang, X. T.; Huang, Y.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. W.; Jiang, X. S.; Jiang, X. Y.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; Ke, B. C.; Kiese, P.; Kliemt, R.; Kloss, B.; Kolcu, O. B.; Kopf, B.; Kornicer, M.; Kupsc, A.; Kühn, W.; Lange, J. S.; Lara, M.; Larin, P.; Leng, C.; Li, C.; Li, Cheng; Li, D. M.; Li, F.; Li, F. Y.; Li, G.; Li, H. B.; Li, J. C.; Li, Jin; Li, K.; Li, K.; Li, Lei; Li, P. R.; Li, Q. Y.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; Lin, D. X.; Liu, B. J.; Liu, C. X.; Liu, D.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. H.; Liu, H. H.; Liu, H. M.; Liu, J.; Liu, J. B.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, L. D.; Liu, P. L.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqing; Loehner, H.; Lou, X. C.; Lu, H. J.; Lu, J. G.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, Q. M.; Ma, T.; Ma, X. N.; Ma, X. Y.; Ma, Y. M.; Maas, F. E.; Maggiora, M.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; Messchendorp, J. G.; Min, J.; Min, T. J.; Mitchell, R. E.; Mo, X. H.; Mo, Y. J.; Morales Morales, C.; Muchnoi, N. Yu.; Muramatsu, H.; Nefedov, Y.; Nerling, F.; Nikolaev, I. B.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Pan, Y.; Patteri, P.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Pettersson, J.; Ping, J. L.; Ping, R. G.; Poling, R.; Prasad, V.; Qi, H. R.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, L. Q.; Qin, N.; Qin, X. S.; Qin, Z. H.; Qiu, J. F.; Rashid, K. H.; Redmer, C. F.; Ripka, M.; Rong, G.; Rosner, Ch.; Ruan, X. D.; Santoro, V.; Sarantsev, A.; Savrié, M.; Schoenning, K.; Schumann, S.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, X.; Tapan, I.; Thorndike, E. H.; Tiemens, M.; Ullrich, M.; Uman, I.; Varner, G. S.; Wang, B.; Wang, B. L.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, W.; Wang, W. P.; Wang, X. F.; Wang, Y. D.; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. H.; Wang, Z. Y.; Weber, T.; Wei, D. H.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, H.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. J.; Yang, H. X.; Yang, L.; Yang, Y. X.; Ye, M.; Ye, M. H.; Yin, J. H.; Yu, B. X.; Yu, C. X.; Yu, J. S.; Yuan, C. Z.; Yuan, W. L.; Yuan, Y.; Yuncu, A.; Zafar, A. A.; Zallo, A.; Zeng, Y.; Zeng, Z.; Zhang, B. X.; Zhang, B. Y.; Zhang, C.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. H.; Zhang, Y. N.; Zhang, Y. T.; Zhang, Yu; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, S. H.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zotti, L.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2017-01-01

A measurement of the number of J/ψ events collected with the BESIII detector in 2009 and 2012 is performed using inclusive decays of the J/ψ. The number of J/ψ events taken in 2009 is recalculated to be (223.7 ± 1.4) × 106, which is in good agreement with the previous measurement, but with significantly improved precision due to improvements in the BESIII software. The number of J/ψ events taken in 2012 is determined to be (1086.9 ± 6.0) × 106. In total, the number of J/ψ events collected with the BESIII detector is measured to be (1310.6 ± 7.0) × 106, where the uncertainty is dominated by systematic effects and the statistical uncertainty is negligible. Supported by National Key Basic Research Program of China (2015CB856700), National Natural Science Foundation of China (NSFC) (10805053, 11125525, 11175188, 11235011, 11322544, 11335008, 11425524), Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, the CAS Center for Excellence in Particle Physics (CCEPP), Collaborative Innovation Center for Particles and Interactions (CICPI), Joint Large-Scale Scientific Facility Funds of NSFC and CAS (11179007, U1232201, U1232107, U1332201), CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology, German Research Foundation DFG (Collaborative Research Center CRC-1044), Istituto Nazionale di Fisica Nucleare, Italy; Ministry of Development of Turkey (DPT2006K-120470), Russian Foundation for Basic Research (14-07-91152), U. S. Department of Energy (DE-FG02-04ER41291, DE-FG02-05ER41374, DE-FG02-94ER40823, DESC0010118), U.S. National Science Foundation, University of Groningen (RuG) and the Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt; WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0)

CRISPR-Cas9-Edited Site Sequencing (CRES-Seq): An Efficient and High-Throughput Method for the Selection of CRISPR-Cas9-Edited Clones.

PubMed

Veeranagouda, Yaligara; Debono-Lagneaux, Delphine; Fournet, Hamida; Thill, Gilbert; Didier, Michel

2018-01-16

The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells). Here we described a next-generation sequencing method called CRISPR-Cas9 Edited Site Sequencing (CRES-Seq) for the efficient and high-throughput screening of CRISPR-Cas9-edited clones. CRES-Seq facilitates the precise genotyping up to 96 CRISPR-Cas9-edited sites (CRES) in a single MiniSeq (Illumina) run with an approximate sequencing cost of $6/clone. CRES-Seq is particularly useful when multiple genes are simultaneously targeted by CRISPR-Cas9, and also for screening of clones generated from multiallelic genes/polyploid cells. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

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

Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi

Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing,more » Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.« less

Physicochemical characterization of native and modified sodium caseinate- Vitamin A complexes.

PubMed

Gupta, Chitra; Arora, Sumit; Syama, M A; Sharma, Apurva

2018-04-01

Native and modified sodium caseinate- Vitamin A complexes {Sodium caseinate- Vit A complex by stirring (NaCas-VA ST), succinylated sodium caseinate- Vit A complex by stirring (SNaCas-VA ST), reassembled sodium caseinate- Vit A complex (RNaCas-VA) and reassembled succinylated sodium caseinate- Vit A complex (RSNaCas-VA)} were prepared and characterized for their physicochemical characteristics e.g. particle size, zeta potential, turbidity analysis and tryptophan intensities which confirmed structural modification of both native (NaCas-VA ST) and modified (SNaCas-VA ST, RNaCas-VA and RSNaCas- VA) proteins upon complex formation with vitamin A. Binding of vitamin A to milk protein reduced the turbidity caused by vitamin A, however, the particle size and zeta potential of milk protein increased after complexation. Microstructure details of NaCas (spray dried) showed uniform spherical structure, however, other milk proteins and milk protein- Vit A complexes (freeze dried) showed broken glass and flaky structures. Tiny particles were observed on the surface of reassembled protein and reassembled protein- Vit A complexes. Binding of vitamin A to milk protein did not have an influence on the electrophoretic mobility and elution profile (RP-HPLC). Copyright © 2018 Elsevier Ltd. All rights reserved.

48 CFR 9903.201-2 - Types of CAS coverage.

Code of Federal Regulations, 2010 CFR

2010-10-01

.... 9903.201-2 Section 9903.201-2 Federal Acquisition Regulations System COST ACCOUNTING STANDARDS BOARD... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full... net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

PubMed

Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

2018-01-22

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

PubMed

Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

2018-05-09

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

Non-viral delivery systems for CRISPR/Cas9-based genome editing: Challenges and opportunities.

PubMed

Li, Ling; Hu, Shuo; Chen, Xiaoyuan

2018-07-01

In recent years, CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) genome editing systems have become one of the most robust platforms in basic biomedical research and therapeutic applications. To date, efficient in vivo delivery of the CRISPR/Cas9 system to the targeted cells remains a challenge. Although viral vectors have been widely used in the delivery of the CRISPR/Cas9 system in vitro and in vivo, their fundamental shortcomings, such as the risk of carcinogenesis, limited insertion size, immune responses and difficulty in large-scale production, severely limit their further applications. Alternative non-viral delivery systems for CRISPR/Cas9 are urgently needed. With the rapid development of non-viral vectors, lipid- or polymer-based nanocarriers have shown great potential for CRISPR/Cas9 delivery. In this review, we analyze the pros and cons of delivering CRISPR/Cas9 systems in the form of plasmid, mRNA, or protein and then discuss the limitations and challenges of CRISPR/Cas9-based genome editing. Furthermore, current non-viral vectors that have been applied for CRISPR/Cas9 delivery in vitro and in vivo are outlined in details. Finally, critical obstacles for non-viral delivery of CRISPR/Cas9 system are highlighted and promising strategies to overcome these barriers are proposed. Published by Elsevier Ltd.

Efficient genome editing by FACS enrichment of paired D10A Cas9 nickases coupled with fluorescent proteins.

PubMed

Gopalappa, Ramu; Song, Myungjae; Chandrasekaran, Arun Pandian; Das, Soumyadip; Haq, Saba; Koh, Hyun Chul; Ramakrishna, Suresh

2018-05-31

Targeted genome editing by clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) raised concerns over off-target effects. The use of double-nicking strategy using paired Cas9 nickase has been developed to minimize off-target effects. However, it was reported that the efficiency of paired nickases were comparable or lower than that of either corresponding nuclease alone. Recently, we conducted a systematic comparison of the efficiencies of several paired Cas9 with their corresponding Cas9 nucleases and showed that paired D10A Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption. However, sometimes the designed paired Cas9 nickases exhibited significantly lower mutation frequencies than nucleases, hampering the generation of cells containing paired Cas9 nickase-induced mutations. Here we implemented IRES peptide-conjugation of fluorescent protein to Cas9 nickase and subjected for fluorescence-activated cell sorting. The sorted cell populations are highly enriched with cells containing paired Cas9 nickase-induced mutations, by a factor of up to 40-fold as compared with the unsorted population. Furthermore, gene-disrupted single cell clones using paired nickases followed by FACS sorting strategy were generated highly efficiently, without compromising with its low off-target effects. We envision that our fluorescent protein coupled paired nickase-mediated gene disruption, facilitating efficient and highly specific genome editing in medical research.

Recruitment of CRISPR-Cas systems by Tn7-like transposons.

PubMed

Peters, Joseph E; Makarova, Kira S; Shmakov, Sergey; Koonin, Eugene V

2017-08-29

A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f , cas7f , and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas-containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.

Molecular identification and characterization of clustered regularly interspaced short palindromic repeats (CRISPRs) in a urease-positive thermophilic Campylobacter sp. (UPTC).

PubMed

Tasaki, E; Hirayama, J; Tazumi, A; Hayashi, K; Hara, Y; Ueno, H; Moore, J E; Millar, B C; Matsuda, M

2012-02-01

Novel clustered regularly-interspaced short palindromic repeats (CRISPRs) locus [7,500 base pairs (bp) in length] occurred in the urease-positive thermophilic Campylobacter (UPTC) Japanese isolate, CF89-12. The 7,500 bp gene loci consisted of the 5'-methylaminomethyl-2-thiouridylate methyltransferase gene, putative (P) CRISPR associated (p-Cas), putative open reading frames, Cas1 and Cas2, leader sequence region (146 bp), 12 CRISPRs consensus sequence repeats (each 36 bp) separated by a non-repetitive unique spacer region of similar length (26-31 bp) and the phosphatidyl glycerophosphatase A gene. When the CRISPRs loci in the UPTC CF89-12 and five C. jejuni isolates were compared with one another, these six isolates contained p-Cas, Cas1 and Cas2 within the loci. Four to 12 CRISPRs consensus sequence repeats separated by a non-repetitive unique spacer region occurred in six isolates and the nucleotide sequences of those repeats gave approximately 92-100% similarity with each other. However, no sequence similarity occurred in the unique spacer regions among these isolates. The putative σ(70) transcriptional promoter and the hypothetical ρ-independent terminator structures for the CRISPRs and Cas were detected. No in vivo transcription of p-Cas, Cas1 and Cas2 was confirmed in the UPTC cells.

Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

PubMed

Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

2015-10-01

The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

Not all predicted CRISPR-Cas systems are equal: isolated cas genes and classes of CRISPR like elements.

PubMed

Zhang, Quan; Ye, Yuzhen

2017-02-06

The CRISPR-Cas systems in prokaryotes are RNA-guided immune systems that target and deactivate foreign nucleic acids. A typical CRISPR-Cas system consists of a CRISPR array of repeat and spacer units, and a locus of cas genes. The CRISPR and the cas locus are often located next to each other in the genomes. However, there is no quantitative estimate of the co-location. In addition, ad-hoc studies have shown that some non-CRISPR genomic elements contain repeat-spacer-like structures and are mistaken as CRISPRs. Using available genome sequences, we observed that a significant number of genomes have isolated cas loci and/or CRISPRs. We found that 11%, 22% and 28% of the type I, II and III cas loci are isolated (without CRISPRs in the same genomes at all or with CRISPRs distant in the genomes), respectively. We identified a large number of genomic elements that superficially reassemble CRISPRs but don't contain diverse spacers and have no companion cas genes. We called these elements false-CRISPRs and further classified them into groups, including tandem repeats and Staphylococcus aureus repeat (STAR)-like elements. This is the first systematic study to collect and characterize false-CRISPR elements. We demonstrated that false-CRISPRs could be used to reduce the false annotation of CRISPRs, therefore showing them to be useful for improving the annotation of CRISPR-Cas systems.

Synergistic effect of casein glycomacropeptide on sodium caseinate foaming properties.

PubMed

Morales, R; Martinez, M J; Pilosof, A M R

2017-11-01

Several strategies to improve the interfacial properties and foaming properties of proteins may be developed; among them, the use of mixtures of biopolymers that exhibit synergistic interactions. The aim of the present work was to evaluate the effect of casein glycomacropeptide (CMP) on foaming and surface properties of sodium caseinate (NaCas) and to establish the role of protein interactions in the aqueous phase. To this end particles size, interfacial and foaming properties of CMP, NaCas and NaCas-CMP mixtures at pH 5.5 and 7 were determined. At both pH, the interaction between CMP and NaCas induced a decrease in the aggregation state of NaCas. Single CMP foams showed the highest and NaCas the lowest foam overrun (FO) and the mixture exhibited intermediate values. CMP foam quickly drained. The drainage profile of mixed foams was closer to NaCas foams; at pH 5.5, mixed foams drained even slower than NaCas foam, exhibiting a synergistic performance. Additionally, a strong synergism was observed on the collapse of mixed foams at pH 5.5. Finally, a model to explain the synergistic effect observed on foaming properties in CMP-NaCas mixtures has been proposed; the reduced aggregation state of NaCas in the presence of CMP, made it more efficient for foam stabilization. Copyright © 2017 Elsevier B.V. All rights reserved.

2,2\\',3,3\\',4,4\\',5,5\\',6,6\\'-Decabromodiphenyl ether (BDE-209)

Integrated Risk Information System (IRIS)

EPA / 635 / R - 07 / 008F www.epa.gov / iris TOXICOLOGICAL REVIEW OF DECABROMODIPHENYL ETHER ( BDE - 209 ) ( CAS No . 1163 - 19 - 5 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) June 2008 U.S . Environmental Protection Agency Washington , DC DISCLAIMER This d

Antiviral Goes Viral: Harnessing CRISPR/Cas9 to Combat Viruses in Humans.

PubMed

Soppe, Jasper Adriaan; Lebbink, Robert Jan

2017-10-01

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are RNA-guided sequence-specific prokaryotic antiviral immune systems. In prokaryotes, small RNA molecules guide Cas effector endonucleases to invading foreign genetic elements in a sequence-dependent manner, resulting in DNA cleavage by the endonuclease upon target binding. A rewired CRISPR/Cas9 system can be used for targeted and precise genome editing in eukaryotic cells. CRISPR/Cas has also been harnessed to target human pathogenic viruses as a potential new antiviral strategy. Here, we review recent CRISPR/Cas9-based approaches to combat specific human viruses in humans and discuss challenges that need to be overcome before CRISPR/Cas9 may be used in the clinic as an antiviral strategy. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Fast Algorithm to Compute Conical Pockets in Proteins. Application to the Structural Characterization of γ-Carbonic Anhydrases.

PubMed

Petitjean, Michel

2017-10-01

Some major proteins families, such as carbonic anhydrases (CAs), have a conical cavity at the active site. No algorithm was available to compute conical cavities, so we needed to design one. The fast algorithm we designed let us show on a set of 717 CAs extracted from the PDB database that γ-CAs are characterized by active site cavity cone angles significantly larger than those of α-CAs and β-CAs: the generatrix-axis angles are greater than 60° for the γ-CAs while they are smaller than 50° for the other CAs. Free binaries of the CONICA software implementing the algorithm are available through a software repository at http://petitjeanmichel.free.fr/itoweb.petitjean.freeware.html. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Short-Term Results of Carotid Endarterectomy and Stenting After the Introduction of Carotid Magnetic Resonance Imaging: A Single-Institution Retrospective Study.

PubMed

Fukumitsu, Ryu; Yoshida, Kazumichi; Kurosaki, Yoshitaka; Torihashi, Koichi; Sadamasa, Nobutake; Koyanagi, Masaomi; Narumi, Osamu; Sato, Tsukasa; Chin, Masaki; Handa, Akira; Yamagata, Sen; Miyamoto, Susumu

2017-05-01

Although carotid artery stenting (CAS) has been gaining popularity as an alternative to carotid endarterectomy (CEA), perioperative stroke rate following contemporary CAS remains significantly higher than stroke rate after CEA. The purpose of this study was to assess perioperative (within 30 days) therapeutic results in patients with carotid stenosis (CS) after introduction of preoperative carotid magnetic resonance imaging plaque evaluation in a single center performing both CEA and CAS. Based on prospectively collected data for patients with CS who were scheduled for carotid revascularization, retrospective analysis was conducted of 295 consecutive patients with CS. An intervention was selected after consideration of periprocedural risks for both CEA and CAS. Concerning risk factors for CAS, results of magnetic resonance imaging plaque evaluation were emphasized with a view toward reducing embolic complications. CAS was performed in 114 patients, and CEA was performed in 181 patients. Comparing baseline characteristics of the 295 patients, age, T1 signal intensity of plaque, symptomatic CS, urgent intervention, and diabetes mellitus differed significantly between CAS and CEA groups. Among patients who underwent CAS, new hyperintense lesions on diffusion-weighted imaging were confirmed in 47 patients. New hyperintense lesions on diffusion-weighted imaging were recognized in 21.4% of patients who underwent CEA (n = 39), significantly less frequent than in patients who underwent CAS. The overall short-term outcome of CEA and CAS is acceptable. Preoperative carotid magnetic resonance imaging evaluation of plaque might contribute to low rates of ischemic complications in CAS. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigation of binding phenomenon of NSP3 and p130Cas mutants and their effect on cell signalling.

PubMed

Balu K; Rajendran, Vidya; Sethumadhavan, Rao; Purohit, Rituraj

2013-11-01

Members of the novel SH2-containing protein (NSP3) and Crk-associated substrate (p130Cas) protein families form a multi-domain signalling platforms that mediate cell signalling process. We analysed the damaging consequences of three mutations, each from NSP3 (NSP3(L469R), NSP3(L623E), NSP3(R627E)) and p130Cas (p130Cas(F794R), p130Cas(L787E), p130Cas(D797R)) protein with respect to their native biological partners. Mutations depicted notable loss in interaction affinity towards their corresponding biological partners. NSP3(L469R) and p130Cas(D797R) mutations were predicted as most prominent in docking analysis. Molecular dynamics (MD) studies were conducted to evaluate structural consequences of most prominent mutation in NSP3 and p130Cas obtained from the docking analysis. MD analysis confirmed that mutation in NSP3(L469R) and p130Cas(D797R) showed significant structural deviation, changes in conformations and increased flexibility, which in turn affected the binding affinity with their biological partners. Moreover, the root mean square fluctuation has indicated a rise in fluctuation of residues involved in moderate interaction acquired between the NSP3 and p130Cas. It has significantly affected the binding interaction in mutant complexes. The results obtained in this work present a detailed overview of molecular mechanisms involved in the loss of cell signalling associated with NSP3 and p130Cas protein.

A comparison of clinical features of coronary artery spasm with and without thyrotoxicosis.

PubMed

Lee, Soo Youn; Yu, Cheol Woong; Choi, Young Jin; Choi, Rak Kyeong; Park, Jin Sik; Lee, Hyun Jong; Kim, Je Sang; Jang, Ho Jun; Jang, Duck Hyun; Chae, Myung Joon; Shim, Won Heum; Ro, Young Moo

2014-03-01

Several reports have suggested that thyrotoxicosis may induce severe coronary artery spasm (CAS). However, there are few data regarding the differences in clinical characteristics of CAS with and without thyrotoxicosis. The aim of our study is to compare the clinical features of CAS with and without thyrotoxicosis. We evaluated 430 consecutive patients with CAS [patients with thyrotoxicosis (N=32, group I) and those without (N=398, group II)] at a single institute between January 2001 and June 2011. We compared clinical presentations, angiographic findings, and adverse outcomes (a composite outcome of cardiac death, myocardial infarction, or rehospitalization due to cardiac cause) of both groups. There was higher incidence of acute myocardial infarction at initial presentation in group I (15.6 vs. 5.8%, P=0.04). CAS with thyrotoxicosis was more diffuse (59.4 vs. 39.3%, P=0.03), more medically intractable (9.4 vs. 0%, P=0.001), and more frequently involved the left main vessel (25.0 vs.0.8%, P=0.001) than CAS without thyrotoxicosis. During the follow-up period (median 43 months), there were no significant differences between the two groups in terms of the risk of adverse outcomes (hazard ratio for CAS with thyrotoxicosis, 1.029; 95% confidence interval, 0.347-3.054). Clinical and angiographic presentations of CAS with thyrotoxicosis were more severe than CAS without thyrotoxicosis, but clinical outcomes were similar in both groups. Optimal vasodilator therapy is essential for the management of CAS with thyrotoxicosis. Thyroid function test should be mandatory for all patients with CAS.

Relationship between drug resistance and the clustered, regularly interspaced, short, palindromic repeat-associated protein genes cas1 and cas2 in Shigella from giant panda dung.

PubMed

Ren, Lu; Deng, Lin-Hua; Zhang, Ri-Peng; Wang, Cheng-Dong; Li, De-Sheng; Xi, Li-Xin; Chen, Zhen-Rong; Yang, Rui; Huang, Jie; Zeng, Yang-Ru; Wu, Hong-Lin; Cao, San-Jie; Wu, Rui; Huang, Yong; Yan, Qi-Gui

2017-02-01

To detect drug resistance in Shigella obtained from the dung of the giant panda, explore the factors leading to drug resistance in Shigella, understand the characteristics of clustered, regularly interspaced, short, palindromic repeats (CRISPR), and assess the relationship between CRISPR and drug resistance. We collected fresh feces from 27 healthy giant pandas in the Giant Panda Conservation base (Wolong, China). We identified the strains of Shigella in the samples by using nucleotide sequence analysis. Further, the Kirby-Bauer paper method was used to determine drug sensitivity of the Shigella strains. CRISPR-associated protein genes cas1 and cas2 in Shigella were detected by polymerase chain reaction (PCR), and the PCR products were sequenced and compared. We isolated and identified 17 strains of Shigella from 27 samples, including 14 strains of Shigella flexneri, 2 strains of Shigella sonnei, and 1 strain of Shigella dysenteriae. Further, drug resistance to cefazolin, imipenem, and amoxicillin-clavulanic acid was identified as a serious problem, as multidrug-resistant strains were detected. Further, cas1 and cas2 showed different degrees of point mutations. The CRISPR system widely exists in Shigella and shares homology with that in Escherichia coli. The cas1 and cas 2 mutations contribute to the different levels of resistance. Point mutations at sites 3176455, 3176590, and 3176465 in cas1 (a); sites 3176989, 3176992, and 3176995 in cas1 (b); sites 3176156 and 3176236 in cas2 may affect the resistance of bacteria, cause emergence of multidrug resistance, and increase the types of drug resistance.

Canonical-ensemble state-averaged complete active space self-consistent field (SA-CASSCF) strategy for problems with more diabatic than adiabatic states: Charge-bond resonance in monomethine cyanines

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

Olsen, Seth, E-mail: seth.olsen@uq.edu.au

2015-01-28

This paper reviews basic results from a theory of the a priori classical probabilities (weights) in state-averaged complete active space self-consistent field (SA-CASSCF) models. It addresses how the classical probabilities limit the invariance of the self-consistency condition to transformations of the complete active space configuration interaction (CAS-CI) problem. Such transformations are of interest for choosing representations of the SA-CASSCF solution that are diabatic with respect to some interaction. I achieve the known result that a SA-CASSCF can be self-consistently transformed only within degenerate subspaces of the CAS-CI ensemble density matrix. For uniformly distributed (“microcanonical”) SA-CASSCF ensembles, self-consistency is invariant tomore » any unitary CAS-CI transformation that acts locally on the ensemble support. Most SA-CASSCF applications in current literature are microcanonical. A problem with microcanonical SA-CASSCF models for problems with “more diabatic than adiabatic” states is described. The problem is that not all diabatic energies and couplings are self-consistently resolvable. A canonical-ensemble SA-CASSCF strategy is proposed to solve the problem. For canonical-ensemble SA-CASSCF, the equilibrated ensemble is a Boltzmann density matrix parametrized by its own CAS-CI Hamiltonian and a Lagrange multiplier acting as an inverse “temperature,” unrelated to the physical temperature. Like the convergence criterion for microcanonical-ensemble SA-CASSCF, the equilibration condition for canonical-ensemble SA-CASSCF is invariant to transformations that act locally on the ensemble CAS-CI density matrix. The advantage of a canonical-ensemble description is that more adiabatic states can be included in the support of the ensemble without running into convergence problems. The constraint on the dimensionality of the problem is relieved by the introduction of an energy constraint. The method is illustrated with a complete active space valence-bond (CASVB) analysis of the charge/bond resonance electronic structure of a monomethine cyanine: Michler’s hydrol blue. The diabatic CASVB representation is shown to vary weakly for “temperatures” corresponding to visible photon energies. Canonical-ensemble SA-CASSCF enables the resolution of energies and couplings for all covalent and ionic CASVB structures contributing to the SA-CASSCF ensemble. The CASVB solution describes resonance of charge- and bond-localized electronic structures interacting via bridge resonance superexchange. The resonance couplings can be separated into channels associated with either covalent charge delocalization or chemical bonding interactions, with the latter significantly stronger than the former.« less

Canonical-ensemble state-averaged complete active space self-consistent field (SA-CASSCF) strategy for problems with more diabatic than adiabatic states: charge-bond resonance in monomethine cyanines.

PubMed

Olsen, Seth

2015-01-28

This paper reviews basic results from a theory of the a priori classical probabilities (weights) in state-averaged complete active space self-consistent field (SA-CASSCF) models. It addresses how the classical probabilities limit the invariance of the self-consistency condition to transformations of the complete active space configuration interaction (CAS-CI) problem. Such transformations are of interest for choosing representations of the SA-CASSCF solution that are diabatic with respect to some interaction. I achieve the known result that a SA-CASSCF can be self-consistently transformed only within degenerate subspaces of the CAS-CI ensemble density matrix. For uniformly distributed ("microcanonical") SA-CASSCF ensembles, self-consistency is invariant to any unitary CAS-CI transformation that acts locally on the ensemble support. Most SA-CASSCF applications in current literature are microcanonical. A problem with microcanonical SA-CASSCF models for problems with "more diabatic than adiabatic" states is described. The problem is that not all diabatic energies and couplings are self-consistently resolvable. A canonical-ensemble SA-CASSCF strategy is proposed to solve the problem. For canonical-ensemble SA-CASSCF, the equilibrated ensemble is a Boltzmann density matrix parametrized by its own CAS-CI Hamiltonian and a Lagrange multiplier acting as an inverse "temperature," unrelated to the physical temperature. Like the convergence criterion for microcanonical-ensemble SA-CASSCF, the equilibration condition for canonical-ensemble SA-CASSCF is invariant to transformations that act locally on the ensemble CAS-CI density matrix. The advantage of a canonical-ensemble description is that more adiabatic states can be included in the support of the ensemble without running into convergence problems. The constraint on the dimensionality of the problem is relieved by the introduction of an energy constraint. The method is illustrated with a complete active space valence-bond (CASVB) analysis of the charge/bond resonance electronic structure of a monomethine cyanine: Michler's hydrol blue. The diabatic CASVB representation is shown to vary weakly for "temperatures" corresponding to visible photon energies. Canonical-ensemble SA-CASSCF enables the resolution of energies and couplings for all covalent and ionic CASVB structures contributing to the SA-CASSCF ensemble. The CASVB solution describes resonance of charge- and bond-localized electronic structures interacting via bridge resonance superexchange. The resonance couplings can be separated into channels associated with either covalent charge delocalization or chemical bonding interactions, with the latter significantly stronger than the former.

Effects of Computer Algebra System (CAS) with Metacognitive Training on Mathematical Reasoning.

ERIC Educational Resources Information Center

Kramarski, Bracha; Hirsch, Chaya

2003-01-01

Describes a study that investigated the differential effects of Computer Algebra Systems (CAS) and metacognitive training (META) on mathematical reasoning. Participants were 83 Israeli eighth-grade students. Results showed that CAS embedded within META significantly outperformed the META and CAS alone conditions, which in turn significantly…

48 CFR 9903.201-2 - Types of CAS coverage.

Code of Federal Regulations, 2012 CFR

2012-10-01

... later award of a CAS-covered contract. Full coverage applies to contractor business units that— (1) Receive a single CAS-covered contract award of $50 million or more; or (2) Received $50 million or more in net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

48 CFR 9903.201-2 - Types of CAS coverage.

Code of Federal Regulations, 2014 CFR

2014-10-01

... later award of a CAS-covered contract. Full coverage applies to contractor business units that— (1) Receive a single CAS-covered contract award of $50 million or more; or (2) Received $50 million or more in net CAS-covered awards during its preceding cost accounting period. (b) Modified coverage. (1...

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements1[OPEN

PubMed Central

Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; Ries, Amber; Böhmer, Maik; You, Lei; Xiao, Chuanlei; Engineer, Cawas B.; Boron, Walter F.; Schroeder, Julian I.

2015-01-01

Elevated carbon dioxide (CO2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO2-induced stomatal movements by catalytic transmission of the CO2 signal in guard cells. However, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO2-regulated stomatal movements remains unknown. Here, we express fluorescently tagged CAs in guard cells of ca1ca4 double-mutant plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO2 regulation of stomatal conductance. Mathematical modeling of cellular CO2 catalysis suggests that the dynamics of the intracellular HCO3− concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Moreover, modeling supports the notion that the intracellular HCO3− concentration dynamics in guard cells are a key mechanism in mediating CO2-regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified. PMID:26243620

CRISPR adaptive immune systems of Archaea

PubMed Central

Vestergaard, Gisle; Garrett, Roger A; Shah, Shiraz A

2014-01-01

CRISPR adaptive immune systems were analyzed for all available completed genomes of archaea, which included representatives of each of the main archaeal phyla. Initially, all proteins encoded within, and proximal to, CRISPR-cas loci were clustered and analyzed using a profile–profile approach. Then cas genes were assigned to gene cassettes and to functional modules for adaptation and interference. CRISPR systems were then classified primarily on the basis of their concatenated Cas protein sequences and gene synteny of the interference modules. With few exceptions, they could be assigned to the universal Type I or Type III systems. For Type I, subtypes I-A, I-B, and I-D dominate but the data support the division of subtype I-B into two subtypes, designated I-B and I-G. About 70% of the Type III systems fall into the universal subtypes III-A and III-B but the remainder, some of which are phyla-specific, diverge significantly in Cas protein sequences, and/or gene synteny, and they are classified separately. Furthermore, a few CRISPR systems that could not be assigned to Type I or Type III are categorized as variant systems. Criteria are presented for assigning newly sequenced archaeal CRISPR systems to the different subtypes. Several accessory proteins were identified that show a specific gene linkage, especially to Type III interference modules, and these may be cofunctional with the CRISPR systems. Evidence is presented for extensive exchange having occurred between adaptation and interference modules of different archaeal CRISPR systems, indicating the wide compatibility of the functionally diverse interference complexes with the relatively conserved adaptation modules. PMID:24531374

Expanding the phenotypic profile of Kleefstra syndrome: A female with low-average intelligence and childhood apraxia of speech.

PubMed

Samango-Sprouse, Carole; Lawson, Patrick; Sprouse, Courtney; Stapleton, Emily; Sadeghin, Teresa; Gropman, Andrea

2016-05-01

Kleefstra syndrome (KS) is a rare neurogenetic disorder most commonly caused by deletion in the 9q34.3 chromosomal region and is associated with intellectual disabilities, severe speech delay, and motor planning deficits. To our knowledge, this is the first patient (PQ, a 6-year-old female) with a 9q34.3 deletion who has near normal intelligence, and developmental dyspraxia with childhood apraxia of speech (CAS). At 6, the Wechsler Preschool and Primary Intelligence testing (WPPSI-III) revealed a Verbal IQ of 81 and Performance IQ of 79. The Beery Buktenica Test of Visual Motor Integration, 5th Edition (VMI) indicated severe visual motor deficits: VMI = 51; Visual Perception = 48; Motor Coordination < 45. On the Receptive One Word Picture Vocabulary Test-R (ROWPVT-R), she had standard scores of 96 and 99 in contrast to an Expressive One Word Picture Vocabulary-R (EOWPVT-R) standard scores of 73 and 82, revealing a discrepancy in vocabulary domains on both evaluations. Preschool Language Scale-4 (PLS-4) on PQ's first evaluation reveals a significant difference between auditory comprehension and expressive communication with standard scores of 78 and 57, respectively, further supporting the presence of CAS. This patient's near normal intelligence expands the phenotypic profile as well as the prognosis associated with KS. The identification of CAS in this patient provides a novel explanation for the previously reported speech delay and expressive language disorder. Further research is warranted on the impact of CAS on intelligence and behavioral outcome in KS. Therapeutic and prognostic implications are discussed. © 2016 Wiley Periodicals, Inc.

Distinct Cellular Locations of Carbonic Anhydrases Mediate Carbon Dioxide Control of Stomatal Movements

DOE PAGES

Hu, Honghong; Rappel, Wouter-Jan; Occhipinti, Rossana; ...

2015-09-28

Elevated carbon dioxide (CO 2) in leaves closes stomatal apertures. Research has shown key functions of the β-carbonic anhydrases (βCA1 and βCA4) in rapid CO 2-induced stomatal movements by catalytic transmission of the CO 2 signal in guard cells. But, the underlying mechanisms remain unclear, because initial studies indicate that these Arabidopsis (Arabidopsis thaliana) βCAs are targeted to distinct intracellular compartments upon expression in tobacco (Nicotiana benthamiana) cells. Which cellular location of these enzymes plays a key role in native guard cells in CO 2-regulated stomatal movements remains unknown. We express fluorescently tagged CAs in guard cells of ca1ca4 double-mutantmore » plants and show that the specific locations of βCA4 at the plasma membrane and βCA1 in native guard cell chloroplasts each can mediate rapid CO 2 control of stomatal movements. Localization and complementation analyses using a mammalian αCAII-yellow fluorescent protein in guard cells further show that cytoplasmic localization is also sufficient to restore CO 2 regulation of stomatal conductance. Mathematical modeling of cellular CO 2 catalysis suggests that the dynamics of the intracellular HCO 3 - concentration change in guard cells can be driven by plasma membrane and cytoplasmic localizations of CAs but not as clearly by chloroplast targeting. Therefore, modeling supports the notion that the intracellular HCO 3 - concentration dynamics in guard cells are a key mechanism in mediating CO 2 -regulated stomatal movements but that an additional chloroplast role of CAs exists that has yet to be identified.« less

Mitochondrial Genetic Diversity and Phylogeography of Mus musculus castaneus in Northern Punjab, Pakistan.

PubMed

Bibi, Shahnaz; Nadeem, Muhammad Sajid; Wiewel, Andrew Stephen; Beg, Mirza Azhar; Hameed, Khalid; Jabeen, Musarrat; Raja, Ghazala Kaukab

2017-12-01

Regions of Iran, Afghanistan, Pakistan and northwestern India have been proposed as the place of origin of Mus musculus castaneus. But despite the fact that Pakistan encompasses an important part of its range, M. m. castaneus populations in Pakistan have not been the subject of intensive genetic and biogeographic studies, except for a very small number of samples included in past studies. We studied genetic variation in M. m. castaneus (CAS) from northern Punjab Province, Pakistan, by using cytochrome b (Cytb) analysis in a sample of 98 individuals. Median-joining network revealed four well differentiated CAS sub-lineages coexisting within a small geographical region; these had previously been thought to have largely non-overlapping geographic distributions. Moreover, haplotypes from Pakistan occupied a central position in the network and all identified global haplotypes were also present in Pakistan. All identified CAS sub-lineages proved to be highly diverse on the basis of haplotype and nucleotide diversity indices. Tajima's D test and Fu's Fs tests of neutrality suggest recent population expansions in all sub-lineages. Expansion times were estimated as 21,760-134,930, 10,800-64,400 and 4950-30,665 ybp using substitution rates of 2.5%, 5% and 11%, respectively. Our results support the hypothesis that northern Punjab Province in Pakistan is the most likely source area for M. m. castaneus, and that the CAS sub-lineages in this region have undergone rapid population expansion events at different time periods, which appear to have benefitted from human-mediated transport, although one of them clearly predates the establishment of human settlements in this region.

Feasibility of a Centralized Clinical Trials Coverage Analysis: A Joint Initiative of the American Society of Clinical Oncology and the National Cancer Institute.

PubMed

Szczepanek, Connie M; Hurley, Patricia; Good, Marjorie J; Denicoff, Andrea; Willenberg, Kelly; Dawson, Casey; Kurbegov, Dax

2017-06-01

Clinical trial billing compliance is a challenge that is faced by overburdened clinical trials sites. The requirements place institutions and research sites at increased potential for financial risk. To reduce their risk, sites develop a coverage analysis (CA) before opening each trial. For multisite trials, this translates into system-wide redundancies, inconsistencies, trial delays, and potential costs to sites and patients. These factors exacerbate low accrual rates to cancer clinical trials. ASCO and the National Cancer Institute (NCI) collaborated to address this problem. An ASCO Research Community Forum working group proposed the concept of providing centrally developed CAs to research sites at protocol startup. The group collaborated with NCI and billing compliance experts to hold a symposium for key stakeholders to share knowledge, build skills, provide tools to conduct centralized CAs, and strategize about the next steps. Forty-eight attendees, who represented a range of stakeholders, participated in the symposium. As a result of this initiative, NCI directed the Cancer Trials Support Unit to convene a working group with NCI's National Clinical Trials Network (NCTN) and Community Oncology Research Program (NCORP) to develop tools and processes for generating CAs for their trials. A CA template with core elements was developed and is being adapted in a pilot project across NCTN Group and NCORP Research Bases. Centralized CAs for multisite trials-using standardized tools and templates-are feasible. They have the potential to reduce risk for patients and sites, forecast budget needs, and help decrease trial startup times that impede patient access and accrual to clinical trials.

Precision measurement of the integrated luminosity of the data taken by BESIII at center-of-mass energies between 3.810 GeV and 4.600 GeV

NASA Astrophysics Data System (ADS)

Ablikim, M.; N. Achasov, M.; Ai, X. C.; Albayrak, O.; Albrecht, M.; J. Ambrose, D.; Amoroso, A.; An, F. F.; An, Q.; Bai, J. Z.; R. Baldini, Ferroli; Ban, Y.; W. Bennett, D.; V. Bennett, J.; Bertani, M.; Bettoni, D.; Bian, J. M.; Bianchi, F.; Boger, E.; Bondarenko, O.; Boyko, I.; A. Briere, R.; Cai, H.; Cai, X.; Cakir, O.; Calcaterra, A.; Cao, G. F.; A. Cetin, S.; Chang, J. F.; Chelkov, G.; Chen, G.; Chen, H. S.; Chen, H. Y.; Chen, J. C.; Chen, M. L.; Chen, S. J.; Chen, X.; Chen, X. R.; Chen, Y. B.; Cheng, H. P.; Chu, X. K.; Cibinetto, G.; Cronin-Hennessy, D.; Dai, H. L.; Dai, J. P.; Dbeyssi, A.; Dedovich, D.; Deng, Z. Y.; Denig, A.; Denysenko, I.; Destefanis, M.; F. De, Mori; Ding, Y.; Dong, C.; Dong, J.; Dong, L. Y.; Dong, M. Y.; Du, S. X.; Duan, P. F.; Fan, J. Z.; Fang, J.; Fang, S. S.; Fang, X.; Fang, Y.; Fava, L.; Feldbauer, F.; Felici, G.; Feng, C. Q.; Fioravanti, E.; Fritsch, M.; Fu, C. D.; Gao, Q.; Gao, Y.; Gao, Z.; Garzia, I.; Geng, C.; Goetzen, K.; Gong, W. X.; Gradl, W.; Greco, M.; Gu, M. H.; Gu, Y. T.; Guan, Y. H.; Guo, A. Q.; Guo, L. B.; Guo, Y.; P. Guo, Y.; Haddadi, Z.; Hafner, A.; Han, S.; Han, Y. L.; Hao, X. Q.; A. Harris, F.; He, K. L.; He, Z. Y.; Held, T.; Heng, Y. K.; Hou, Z. L.; Hu, C.; Hu, H. M.; Hu, J. F.; Hu, T.; Hu, Y.; Huang, G. M.; Huang, G. S.; Huang, H. P.; Huang, J. S.; Huang, X. T.; Huang, Y.; Hussain, T.; Ji, Q.; Ji, Q. P.; Ji, X. B.; Ji, X. L.; Jiang, L. L.; Jiang, L. W.; Jiang, X. S.; Jiao, J. B.; Jiao, Z.; Jin, D. P.; Jin, S.; Johansson, T.; Julin, A.; Kalantar-Nayestanaki, N.; Kang, X. L.; Kang, X. S.; Kavatsyuk, M.; C. Ke, B.; Kliemt, R.; Kloss, B.; B. Kolcu, O.; Kopf, B.; Kornicer, M.; Kuehn, W.; Kupsc, A.; Lai, W.; S. Lange, J.; M., Lara; Larin, P.; Leng, C.; Li, C. H.; Li, Cheng; Li, D. M.; Li, F.; Li, G.; Li, H. B.; Li, J. C.; Li, Jin; Li, K.; Li, K.; Li, Lei; Li, P. R.; Li, T.; Li, W. D.; Li, W. G.; Li, X. L.; Li, X. M.; Li, X. N.; Li, X. Q.; Li, Z. B.; Liang, H.; Liang, Y. F.; Liang, Y. T.; Liao, G. R.; X. Lin(Lin, D.; Liu, B. J.; Liu, C. X.; Liu, F. H.; Liu, Fang; Liu, Feng; Liu, H. B.; Liu, H. H.; Liu, H. H.; Liu, H. M.; Liu, J.; Liu, J. P.; Liu, J. Y.; Liu, K.; Liu, K. Y.; Liu, L. D.; Liu, P. L.; Liu, Q.; Liu, S. B.; Liu, X.; Liu, X. X.; Liu, Y. B.; Liu, Z. A.; Liu, Zhiqiang; Zhiqing, Liu; Loehner, H.; Lou, X. C.; Lu, H. J.; Lu, J. G.; Lu, R. Q.; Lu, Y.; Lu, Y. P.; Luo, C. L.; Luo, M. X.; Luo, T.; Luo, X. L.; Lv, M.; Lyu, X. R.; Ma, F. C.; Ma, H. L.; Ma, L. L.; Ma, Q. M.; Ma, S.; Ma, T.; Ma, X. N.; Ma, X. Y.; E. Maas, F.; Maggiora, M.; A. Malik, Q.; Mao, Y. J.; Mao, Z. P.; Marcello, S.; G. Messchendorp, J.; Min, J.; Min, T. J.; E. Mitchell, R.; Mo, X. H.; Mo, Y. J.; C. Morales, Morales; Moriya, K.; Yu. Muchnoi, N.; Muramatsu, H.; Nefedov, Y.; Nerling, F.; B. Nikolaev, I.; Ning, Z.; Nisar, S.; Niu, S. L.; Niu, X. Y.; Olsen, S. L.; Ouyang, Q.; Pacetti, S.; Patteri, P.; Pelizaeus, M.; Peng, H. P.; Peters, K.; Ping, J. L.; Ping, R. G.; Poling, R.; Pu, Y. N.; Qi, M.; Qian, S.; Qiao, C. F.; Qin, L. Q.; Qin, N.; Qin, X. S.; Qin, Y.; Qin, Z. H.; Qiu, J. F.; H. Rashid, K.; F. Redmer, C.; Ren, H. L.; Ripka, M.; Rong, G.; Ruan, X. D.; Santoro, V.; Sarantsev, A.; Savrié, M.; Schoenning, K.; Schumann, S.; Shan, W.; Shao, M.; Shen, C. P.; Shen, P. X.; Shen, X. Y.; Sheng, H. Y.; Song, W. M.; Song, X. Y.; Sosio, S.; Spataro, S.; Sun, G. X.; Sun, J. F.; Sun, S. S.; Sun, Y. J.; Sun, Y. Z.; Sun, Z. J.; Sun, Z. T.; Tang, C. J.; Tang, X.; Tapan, I.; H. Thorndike, E.; Tiemens, M.; Toth, D.; Ullrich, M.; Uman, I.; S. Varner, G.; Wang, B.; Wang, B. L.; Wang, D.; Wang, D. Y.; Wang, K.; Wang, L. L.; Wang, L. S.; Wang, M.; Wang, P.; Wang, P. L.; Wang, Q. J.; Wang, S. G.; Wang, W.; Wang, X. F.; Yadi, Wang; Wang, Y. F.; Wang, Y. Q.; Wang, Z.; Wang, Z. G.; Wang, Z. H.; Wang, Z. Y.; Weber, T.; Wei, D. H.; Wei, J. B.; Weidenkaff, P.; Wen, S. P.; Wiedner, U.; Wolke, M.; Wu, L. H.; Wu, Z.; Xia, L. G.; Xia, Y.; Xiao, D.; Xiao, Z. J.; Xie, Y. G.; Xiu, Q. L.; Xu, G. F.; Xu, L.; Xu, Q. J.; Xu, Q. N.; Xu, X. P.; Yan, L.; Yan, W. B.; Yan, W. C.; Yan, Y. H.; Yang, H. X.; Yang, L.; Yang, Y.; Yang, Y. X.; Ye, H.; Ye, M.; Ye, M. H.; Yin, J. H.; Yu, B. X.; Yu, C. X.; Yu, H. W.; Yu, J. S.; Yuan, C. Z.; Yuan, W. L.; Yuan, Y.; Yuncu, A.; A. Zafar, A.; Zallo, A.; Zeng, Y.; Zhang, B. X.; Zhang, B. Y.; Zhang, C.; Zhang, C. C.; Zhang, D. H.; Zhang, H. H.; Zhang, H. Y.; Zhang, J. J.; Zhang, J. L.; Zhang, J. Q.; Zhang, J. W.; Zhang, J. Y.; Zhang, J. Z.; Zhang, K.; Zhang, L.; Zhang, S. H.; Zhang, X. Y.; Zhang, Y.; Zhang, Y. H.; Zhang, Y. T.; Zhang, Z. H.; Zhang, Z. P.; Zhang, Z. Y.; Zhao, G.; Zhao, J. W.; Zhao, J. Y.; Zhao, J. Z.; Zhao, Lei; Zhao, Ling; Zhao, M. G.; Zhao, Q.; Zhao, Q. W.; Zhao, S. J.; Zhao, T. C.; Zhao, Y. B.; Zhao, Z. G.; Zhemchugov, A.; Zheng, B.; Zheng, J. P.; Zheng, W. J.; Zheng, Y. H.; Zhong, B.; Zhou, L.; Zhou, Li; Zhou, X.; Zhou, X. K.; Zhou, X. R.; Zhou, X. Y.; Zhu, K.; Zhu, K. J.; Zhu, S.; Zhu, X. L.; Zhu, Y. C.; Zhu, Y. S.; Zhu, Z. A.; Zhuang, J.; Zotti, L.; Zou, B. S.; Zou, J. H.; BESIII Collaboration

2015-09-01

From December 2011 to May 2014, about 5 fb-1 of data were taken with the BESIII detector at center-of-mass energies between 3.810 GeV and 4.600 GeV to study the charmonium-like states and higher excited charmonium states. The time-integrated luminosity of the collected data sample is measured to a precision of 1% by analyzing events produced by the large-angle Bhabha scattering process. Supported by National Key Basic Research Program of China (2015CB856700), National Natural Science Foundation of China (NSFC) (11125525, 11235011, 11322544, 11335008, 11425524), Chinese Academy of Sciences (CAS) Large-Scale Scientific Facility Program, Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (11179007, U1232201, U1332201) CAS (KJCX2-YW-N29, KJCX2-YW-N45), 100 Talents Program of CAS, INPAC and Shanghai Key Laboratory for Particle Physics and Cosmology, German Research Foundation DFG (Collaborative Research Center CRC-1044), Istituto Nazionale di Fisica Nucleare, Italy; Ministry of Development of Turkey (DPT2006K-120470), Russian Foundation for Basic Research (14-07-91152), U.S. Department of Energy (DE-FG02-04ER41291, DE-FG02-05ER41374, DE-FG02-94ER40823, DESC0010118), U.S. National Science Foundation, University of Groningen (RuG) and the Helmholtzzentrum fuer Schwerionenforschung GmbH (GSI), Darmstadt and WCU Program of National Research Foundation of Korea (R32-2008-000-10155-0)

Central Anticholinergic Syndrome due to Hypoxia-Induced Bradycardia in a Child with Difficult Intubation Undergoing Complete Dental Restoration: A Case Report.

PubMed

Gharavifard, Mohamad; Razavi, Majid; Ghandehari Motlagh, Mehdi; Ziyaeifard, Mohsen

2014-09-01

Central anticholinergic syndrome (CAS) following general anesthesia (GA) is a well known syndrome in children and adults. Many cases of CAS have been previously reported in the literature. However, there are only two reports of post resuscitation CAS after administration of small doses of atropine. Hereby, we report a case of CAS in a child undergoing complete dental restoration under GA after receiving a small dose of atropine to reverse hypoxia induced bradycardia. Intraoperative events such as hypoxia or cardiac arrest may play a role as triggers for CAS. However, we cannot establish a causal relationship between the occurrence of CAS and such critical events.

Advances in therapeutic CRISPR/Cas9 genome editing.

PubMed

Savić, Nataša; Schwank, Gerald

2016-02-01

Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports. Copyright © 2016 Elsevier Inc. All rights reserved.

Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

PubMed Central

Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

2016-01-01

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

[Application of CRISPR/Cas9 mediated genome editing in farm animals].

PubMed

Xing, Yu-yun; Yang, Qiang; Ren, Jun

2016-03-01

CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) is an acquired immune system found in bacteria and archaea that fight against invasion of viruses or plasmids. CRISPR/Cas systems are currently classified into three main types: I, II and III, of which type II has relatively simple components. The CRISPR/Cas9 technology modified from type II CRISPR/Cas system has been developed as an efficient genome editing tool. Since the initial application of the CRISPR/Cas9 technology in mammals in 2013, the reports of this system for genomic editing has skyrocketed. Farm animals are not only economically important animals, but also ideal animal models for human diseases and biomedical studies. In this review, we summarize the applications of CRISPR/Cas9 in farm animals, briefly describe the off-target effects and the main solutions, and finally highlight the future perspectives of this technology.

The application of CRISPR-Cas9 genome editing tool in cancer immunotherapy.

PubMed

Wu, Hong-Yan; Cao, Chun-Yu

2018-03-22

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system was originally discovered in prokaryotes functioned as a part of the adaptive immune system. Because of its high efficiency and easy operability, CRISPR-Cas9 system has been developed to be a powerful and versatile gene editing tool shortly after its discovery. Given that multiple genetic alterations are the main factors that drive genesis and development of tumor, CRISPR-Cas9 system has been applied to correct cancer-causing gene mutations and deletions and to engineer immune cells, such as chimeric antigen receptor T (CAR T) cells, for cancer immunotherapeutic applications. Recently, CRISPR-Cas9-based CAR T-cell preparation has been an important breakthrough in antitumor therapy. Here, we summarize the mechanism, delivery and the application of CRISPR-Cas9 in gene editing, and discuss the challenges and future directions of CRISPR-Cas9 in cancer immunotherapy.

CP asymmetries in Strange Baryon Decays

NASA Astrophysics Data System (ADS)

Bigi, I. I.; Kang, Xian-Wei; Li, Hai-Bo

2018-01-01

While indirect and direct CP violation (CPV) has been established in the decays of strange and beauty mesons, no CPV has yet been found for baryons. There are different paths to finding CP asymmetry in the decays of strange baryons; they are all highly non-trivial. The HyperCP Collaboration has probed CPV in the decays of single Ξ and Λ [1]. We discuss future lessons from {{{e}}}+{{{e}}}- collisions at BESIII/BEPCII: probing decays of pairs of strange baryons, namely Λ, Σ and Ξ. Realistic goals are to learn about non-perturbative QCD. One can hope to find CPV in the decays of strange baryons; one can also dream of finding the impact of New Dynamics. We point out that an important new era will start with the BESIII/BEPCII data accumulated by the end of 2018. This also supports new ideas to trigger {{J}}/{{\\psi }}\\to \\bar{{{Λ }}}{{Λ }} at the LHCb collaboration. Supported by National Science Foundation (PHY-1520966), National Natural Science Foundation of China (11335009, 11125525), Joint Large-Scale Scientific Facility Funds of the NSFC and CAS (U1532257), the National Key Basic Research Program of China (2015CB856700), Key Research Program of Frontier Sciences, CAS, (QYZDJ-SSW-SLH003), XWK’s work is also supported by MOST (Taiwan) (104-2112-M-001-022)

41 CFR 102-33.95 - What is the process for budgeting to acquire commercial aviation services (CAS)?

Code of Federal Regulations, 2014 CFR

2014-01-01

... budgeting to acquire commercial aviation services (CAS)? 102-33.95 Section 102-33.95 Public Contracts and... budgeting to acquire commercial aviation services (CAS)? Except for leases and lease-purchases, for which... fund your commercial aviation services (CAS) hires out of your agency's operating budget. Contracting...

41 CFR 102-33.95 - What is the process for budgeting to acquire commercial aviation services (CAS)?

Code of Federal Regulations, 2012 CFR

2012-01-01

... budgeting to acquire commercial aviation services (CAS)? 102-33.95 Section 102-33.95 Public Contracts and... budgeting to acquire commercial aviation services (CAS)? Except for leases and lease-purchases, for which... fund your commercial aviation services (CAS) hires out of your agency's operating budget. Contracting...

41 CFR 102-33.95 - What is the process for budgeting to acquire commercial aviation services (CAS)?

Code of Federal Regulations, 2011 CFR

2011-01-01

... budgeting to acquire commercial aviation services (CAS)? 102-33.95 Section 102-33.95 Public Contracts and... budgeting to acquire commercial aviation services (CAS)? Except for leases and lease-purchases, for which... fund your commercial aviation services (CAS) hires out of your agency's operating budget. Contracting...

41 CFR 102-33.95 - What is the process for budgeting to acquire commercial aviation services (CAS)?

Code of Federal Regulations, 2013 CFR

2013-07-01

... budgeting to acquire commercial aviation services (CAS)? 102-33.95 Section 102-33.95 Public Contracts and... budgeting to acquire commercial aviation services (CAS)? Except for leases and lease-purchases, for which... fund your commercial aviation services (CAS) hires out of your agency's operating budget. Contracting...

CAS-Induced Difficulties in Learning Mathematics?

ERIC Educational Resources Information Center

Jankvist, Uffe Thomas; Misfeldt, Morten

2015-01-01

In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

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

Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle

Here we demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9.

PubMed

Sternberg, Samuel H; Redding, Sy; Jinek, Martin; Greene, Eric C; Doudna, Jennifer A

2014-03-06

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

SHP-2 inhibits tyrosine phosphorylation of Cas-L and regulates cell migration.

PubMed

Yo, Koji; Iwata, Satoshi; Hashizume, Yutaka; Kondo, Shunsuke; Nomura, Sayaka; Hosono, Osamu; Kawasaki, Hiroshi; Tanaka, Hirotoshi; Dang, Nam H; Morimoto, Chikao

2009-04-24

The Src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SHP-2, plays an important role in cell migration by interacting with various proteins. In this report, we demonstrated that SHP-2 inhibits tyrosine phosphorylation of Crk-associated substrate lymphocyte type (Cas-L), a docking protein which mediates cell migration, and found that SHP-2 negatively regulates migration of A549 lung adenocarcinoma cells induced by fibronectin (FN). We showed that overexpressed SHP-2 co-localizes with Cas-L at focal adhesions and that exogenous expression of SHP-2 abrogates cell migration mediated by Cas-L. SHP-2 inhibits tyrosine phosphorylation of Cas-L, and associates with Cas-L to form a complex in a tyrosine phosphorylation-dependent manner. Finally, immunoprecipitation experiments with deletion mutants revealed that both SH2 domains of SHP-2 are necessary for this association. These results suggest that SHP-2 regulates tyrosine phosphorylation of Cas-L, hence opposing the effect of kinases, and SHP-2 is a negative regulator of cell migration mediated by Cas-L.

Current status of potential applications of repurposed Cas9 for structural and functional genomics of plants.

PubMed

Seth, Kunal; Harish

2016-11-25

Redesigned Cas9 has emerged as a tool with various applications like gene editing, gene regulation, epigenetic modification and chromosomal imaging. Target specific single guide RNA (sgRNA) can be used with Cas9 for precise gene editing with high efficiency than previously known methods. Further, nuclease-deactivated Cas9 (dCas9) can be fused with activator or repressor for activation (CRISPRa) and repression (CRISPRi) of gene expression, respectively. dCas9 fused with epigenetic modifier like methylase or acetylase further expand the scope of this technique. Fluorescent probes can be tagged to dCas9 to visualize the chromosome. Due to its wide-spread application, simplicity, accessibility, efficacy and universality, this technique is expanding the structural and functional genomic studies of plant and developing CRISPR crops. The present review focuses on current status of using repurposed Cas9 system in these various areas, with major focus on application in plants. Major challenges, concerns and future directions of using this technique are discussed in brief. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9.

PubMed

Yang, Hui; Patel, Dinshaw J

2017-07-06

Prokaryotic CRISPR-Cas adaptive immune systems utilize sequence-specific RNA-guided endonucleases to defend against infection by viruses, bacteriophages, and mobile elements, while these foreign genetic elements evolve diverse anti-CRISPR proteins to overcome the CRISPR-Cas-mediated defense of the host. Recently, AcrIIA2 and AcrIIA4, encoded by Listeria monocytogene prophages, were shown to block the endonuclease activity of type II-A Streptococcus pyogene Cas9 (SpyCas9). We now report the crystal structure of AcrIIA4 in complex with single-guide RNA-bound SpyCas9, thereby establishing that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks target DNA access to key catalytic residues lining the RuvC pocket. These structural insights, validated by biochemical assays on key mutants, demonstrate that AcrIIA4 competitively occupies both PAM-interacting and non-target DNA strand cleavage catalytic pockets. Our studies provide insights into anti-CRISPR-mediated suppression mechanisms for inactivating SpyCas9, thereby broadening the applicability of CRISPR-Cas regulatory tools for genome editing. Published by Elsevier Inc.

DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

NASA Astrophysics Data System (ADS)

Sternberg, Samuel H.; Redding, Sy; Jinek, Martin; Greene, Eric C.; Doudna, Jennifer A.

2014-03-01

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Programmable RNA Cleavage and Recognition by a Natural CRISPR-Cas9 System from Neisseria meningitidis.

PubMed

Rousseau, Beth A; Hou, Zhonggang; Gramelspacher, Max J; Zhang, Yan

2018-03-01

The microbial CRISPR systems enable adaptive defense against mobile elements and also provide formidable tools for genome engineering. The Cas9 proteins are type II CRISPR-associated, RNA-guided DNA endonucleases that identify double-stranded DNA targets by sequence complementarity and protospacer adjacent motif (PAM) recognition. Here we report that the type II-C CRISPR-Cas9 from Neisseria meningitidis (Nme) is capable of programmable, RNA-guided, site-specific cleavage and recognition of single-stranded RNA targets and that this ribonuclease activity is independent of the PAM sequence. We define the mechanistic feature and specificity constraint for RNA cleavage by NmeCas9 and also show that nuclease null dNmeCas9 binds to RNA target complementary to CRISPR RNA. Finally, we demonstrate that NmeCas9-catalyzed RNA cleavage can be blocked by three families of type II-C anti-CRISPR proteins. These results fundamentally expand the targeting capacities of CRISPR-Cas9 and highlight the potential utility of NmeCas9 as a single platform to target both RNA and DNA. Copyright © 2018 Elsevier Inc. All rights reserved.

The effect of Mycobacterium tuberculosis CRISPR-associated Cas2 (Rv2816c) on stress response genes expression, morphology and macrophage survival of Mycobacterium smegmatis.

PubMed

Huang, Qinqin; Luo, Hongping; Liu, Minqiang; Zeng, Jie; Abdalla, Abualgasim Elgaili; Duan, Xiangke; Li, Qiming; Xie, Jianping

2016-06-01

Clustered regularly interspaced short palindromic repeats (CRISPR) are present in the genome of 40% bacteria and 90% archaea. CRISPR and accompanying Cas proteins constitute an adaptive immune system against disruptive mobile genetic elements. Two CRISPRs and 9 genes encoding CRISPR-associated proteins have been found in the genome of Mycobacterium tuberculosis. The CRISPR-associated Cas2 is an endoribonuclease required for the acquisition of new spacers. In this study, Cas2 encoded by Rv2816c was expressed in Mycobacterium smegmatis lacking CRISPR-Cas system and its role in stress responses of M. smegmatis in vitro and within macrophages was studied. We found that Cas2 mediated M. smegmatis stress response changes were associated with the altered expression of sigma factors which involved in mycobacterial stress response and virulence. We also found that Cas2 decreased the survival of M. smegmatis within macrophages. This study provides new insights on the role of Cas2. Copyright © 2015 Elsevier B.V. All rights reserved.

RNA targeting with CRISPR-Cas13.

PubMed

Abudayyeh, Omar O; Gootenberg, Jonathan S; Essletzbichler, Patrick; Han, Shuo; Joung, Julia; Belanto, Joseph J; Verdine, Vanessa; Cox, David B T; Kellner, Max J; Regev, Aviv; Lander, Eric S; Voytas, Daniel F; Ting, Alice Y; Zhang, Feng

2017-10-12

RNA has important and diverse roles in biology, but molecular tools to manipulate and measure it are limited. For example, RNA interference can efficiently knockdown RNAs, but it is prone to off-target effects, and visualizing RNAs typically relies on the introduction of exogenous tags. Here we demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR-Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. After initial screening of 15 orthologues, we identified Cas13a from Leptotrichia wadei (LwaCas13a) as the most effective in an interference assay in Escherichia coli. LwaCas13a can be heterologously expressed in mammalian and plant cells for targeted knockdown of either reporter or endogenous transcripts with comparable levels of knockdown as RNA interference and improved specificity. Catalytically inactive LwaCas13a maintains targeted RNA binding activity, which we leveraged for programmable tracking of transcripts in live cells. Our results establish CRISPR-Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.

CRISPRscan: designing highly efficient sgRNAs for CRISPR/Cas9 targeting in vivo

PubMed Central

Moreno-Mateos, Miguel A.; Vejnar, Charles E.; Beaudoin, Jean-Denis; Fernandez, Juan P.; Mis, Emily K.; Khokha, Mustafa K.; Giraldez, Antonio J.

2015-01-01

CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo. PMID:26322839

Analysis of microsatellite instability in CRISPR/Cas9 editing mice.

PubMed

Huo, Xueyun; Du, Yating; Lu, Jing; Guo, Meng; Li, Zhenkun; Zhang, Shuangyue; Li, Xiaohong; Chen, Zhenwen; Du, Xiaoyan

2017-03-01

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated (Cas) protein 9 system is a novel and powerful tool which is widely used for genome editing. CRISPR/Cas9 is RNA-guided and can lead to desired genomic modifications. However, whether the CRISPR/Cas9-mediated genome editing causes genomic alterations and genomic instability, such as microsatellite instability (MSI), is still unknown. Here we detected MSI in 21 CRISPR/Cas9 mouse strains using a panel of 42 microsatellite loci which were selected from our previous studies. Surprisingly, MSI occurrence was common in CRISPR/Cas9 modified genome, and most of the strains (19/21, 90.5%) examined showed MSI. Of 42 loci examined, 8 loci (8/42, 19.05%) exhibited MSI in the Cas9 editing mice. The Ttll9 (4/42, 9.5%) were the most unstable strains, and D10Mit3 and D10Mit198 (9/21, 42.9%) were considered to be the most "hot" loci in the Cas9 strains we tested. Through analyzing the mutation of microsatellite loci, we provide new insights into the genomic alterations of CRISPR/Cas9 models and it will help us for a better understanding of this powerful technology. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome.

PubMed

Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

2017-02-07

The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.

Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

PubMed Central

Zhen, Shuai; Takahashi, Yoichiro; Narita, Shunichi; Yang, Yi-Chen; Li, Xu

2017-01-01

The potent ability of CRISPR/Cas9 system to inhibit the expression of targeted gene is being exploited as a new class of therapeutics for a variety of diseases. However, the efficient and safe delivery of CRISPR/Cas9 into specific cell populations is still the principal challenge in the clinical development of CRISPR/Cas9 therapeutics. In this study, a flexible aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery and increased flexibility. Our chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand. Cationic liposomes were linked to aptamers by the post-insertion method and were used to deliver therapeutic CRISPR/Cas9 that target the survival gene, polo-like kinase 1, in tumor cells. We demonstrate that the aptamer-liposome-CRISPR/Cas9 chimeras had a significant cell-type binding specificity and a remarkable gene silencing effect in vitro. Furthermore, silencing promoted a conspicuous regression of prostate cancer in vivo. Importantly, the approach described here provides a universal means of cell type–specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs. PMID:28030843

Optimization of genome editing through CRISPR-Cas9 engineering.

PubMed

Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

2016-04-01

CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

PubMed

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

PubMed Central

Cebrian-Serrano, Alberto; Zha, Shijun; Hanssen, Lars; Biggs, Daniel; Preece, Christopher

2017-01-01

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply. PMID:28081254

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

PubMed Central

Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

2018-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

Age-dependent effects of carotid endarterectomy or stenting on cognitive performance.

PubMed

Wasser, Katrin; Hildebrandt, Helmut; Gröschel, Sonja; Stojanovic, Tomislav; Schmidt, Holger; Gröschel, Klaus; Pilgram-Pastor, Sara M; Knauth, Michael; Kastrup, Andreas

2012-11-01

Although evidence is accumulating that age modifies the risk of carotid angioplasty and stenting (CAS) versus endarterectomy (CEA) for patients with significant carotid stenosis, the impact of age on cognition after either CEA or CAS remains unclear. In this study, we analyzed the effects of age on cognitive performance after either CEA or CAS using a comprehensive neuropsychological test battery with parallel test forms and a control group to exclude a learning effect. The neuropsychological outcomes after revascularization were determined in 19 CAS and 27 CEA patients with severe carotid stenosis. The patients were subdivided according to their median age (<68 years and ≥68 years); 27 healthy subjects served as a control group. In all patients clinical examinations, MRI scans and a neuropsychological test battery that assessed four major cognitive domains were performed immediately before, within 72 h, and 3 months after CEA or CAS. While patients <68 years of age showed no significant cognitive alteration after either CEA or CAS, a significant cognitive decline was observed in patients ≥68 years in both treatment groups (p = 0.001). Notably, this cognitive deterioration persisted in patients after CEA, whereas it was only transient in patients treated with CAS. These results demonstrate an age-dependent effect of CEA and CAS on cognitive functions. In contrast to the recently observed increased clinical complication rates in older subjects after CAS compared with CEA, CEA appears to be associated with a greater, persistent decline in cognitive performance than CAS in this subgroup of patients.

Programmable RNA recognition and cleavage by CRISPR/Cas9

PubMed Central

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

2014-01-01

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302