Science.gov

Sample records for alarm system cas

  1. Fire alarm system improvement

    SciTech Connect

    Hodge, S.G.

    1994-10-01

    This document contains the Fire Alarm System Test Procedure for Building 234-5Z, 200-West Area on the Hanford Reservation, Richland, Washington. This Acceptance Test Procedure (ATP) has been prepared to demonstrate that the modifications to the Fire Protection systems function as required by project criteria. The ATP will test the Fire Alarm Control Panels, Flow Alarm Pressure Switch, Heat Detectors, Smoke Detectors, Flow Switches, Manual Pull Stations, and Gong/Door by Pass Switches.

  2. Alarm Notification System

    1995-03-12

    AN/EMS, the Alarm Notification Energy Management System, is used to monitor digital sensors in PETC buildings and to notify the safety/security operator by both a video and an audio system when a possibly hazardous condition arises.

  3. FIRE ALARM SYSTEM OUTDATED.

    ERIC Educational Resources Information Center

    CHANDLER, L.T.

    AN EFFICIENT FIRE ALARM SYSTEM SHOULD--(1) PROVIDE WARNING OF FIRES THAT START IN HIDDEN OR UNOCCUPIED LOCATIONS, (2) INDICATE WHERE THE FIRE IS, (3) GIVE ADVANCE WARNING TO FACULTY AND ADMINISTRATION SO THAT PANIC AND CONFUSION CAN BE AVOIDED AND ORDERLY EVACUATION OCCUR, (4) AUTOMATICALLY NOTIFY CITY FIRE HEADQUARTERS OF THE FIRE, (5) OPERATE BY…

  4. IMPEDANCE ALARM SYSTEM

    DOEpatents

    Cowen, R.G.

    1959-09-29

    A description is given of electric protective systems and burglar alarm systems of the capacitance type in which the approach of an intruder at a place to be protected varies the capacitance in an electric circuit and the change is thereafter communicated to a remote point to actuate an alarm. According to the invention, an astable transitor multi-vibrator has the amplitude at its output voltage controlled by a change in the sensing capacitance. The sensing capacitance is effectively connected between collector and base of one stage of the multivibrator circuit through the detector-to-monitor line. The output of the detector is a small d-c voltage across the detector-to-monitor line. This d- c voltage is amplified and monitored at the other end of the line, where an appropriate alarm is actuated if a sudden change in the voltage occurs. The present system has a high degree of sensitivity and is very difficult to defeat by known techniques.

  5. Functional relationship-based alarm processing system

    DOEpatents

    Corsberg, Daniel R.

    1989-01-01

    A functional relationship-based alarm processing system and method analyzes each alarm as it is activated and determines its relative importance with other currently activated alarms and signals in accordance with the functional relationships that the newly activated alarm has with other currently activated alarms. Once the initial level of importance of the alarm has been determined, that alarm is again evaluated if another related alarm is activated or deactivated. Thus, each alarm's importance is continuously updated as the state of the process changes during a scenario. Four hierarchical relationships are defined by this alarm filtering methodology: (1) level precursor (usually occurs when there are two alarm settings on the same parameter); (2) direct precursor (based on causal factors between two alarms); (3) required action (system response or action expected within a specified time following activation of an alarm or combination of alarms and process signals); and (4) blocking condition (alarms that are normally expected and are not considered important). The alarm processing system and method is sensitive to the dynamic nature of the process being monitored and is capable of changing the relative importance of each alarm as necessary.

  6. Functional relationship-based alarm processing system

    DOEpatents

    Corsberg, D.R.

    1988-04-22

    A functional relationship-based alarm processing system and method analyzes each alarm as it is activated and determines its relative importance with other currently activated alarms and signals in accordance with the functional relationships that the newly activated alarm has with other currently activated alarms. Once the initial level of importance of the alarm has been determined, that alarm is again evaluated if another related alarm is activated or deactivated. Thus, each alarm's importance is continuously updated as the state of the process changes during a scenario. Four hierarchical relationships are defined by this alarm filtering methodology: (1) level precursor (usually occurs when there are two alarm settings on the same parameter); (2) direct precursor (based on causal factors between two alarms); (3) required action (system response or action expected within a specified time following activation of an alarm or combination of alarms and process signals); and (4) blocking condition (alarms that are normally expected and are not considered important). The alarm processing system and method is sensitive to the dynamic nature of the process being monitored and is capable of changing the relative importance of each alarm as necessary. 12 figs.

  7. Intensive care alarm system

    NASA Technical Reports Server (NTRS)

    Christensen, J. L.; Herbert, A. L.

    1973-01-01

    Inductive loop has been added to commercially available call system fitted with earphone receiver. System transmits high frequency signals to nurse's receiver to announce patient's need for help without disturbing others.

  8. Personal Alarm System

    NASA Technical Reports Server (NTRS)

    1977-01-01

    Trouble in the classroom is an unpleasant fact of modern life. Space technology can't stop the trouble from occurring, but it can prevent it from spreading. In recognition of this, NASA and the Sacramento, Cal. Unified School District developed a personal security system based on space telemetry technology. The first application was for schools, but the simplicity and reliability of the system has made it more widely applicable. The heart of the system is an ultrasonic pen-size transmitter. It can be used by prison guards, teachers, or others such as the handicapped and the elderly.

  9. SUBSURFACE VISUAL ALARM SYSTEM ANALYSIS

    SciTech Connect

    D.W. Markman

    2001-08-06

    The ''Subsurface Fire Hazard Analysis'' (CRWMS M&O 1998, page 61), and the document, ''Title III Evaluation Report for the Surface and Subsurface Communication System'', (CRWMS M&O 1999a, pages 21 and 23), both indicate the installed communication system is adequate to support Exploratory Studies Facility (ESF) activities with the exception of the mine phone system for emergency notification purposes. They recommend the installation of a visual alarm system to supplement the page/party phone system The purpose of this analysis is to identify data communication highway design approaches, and provide justification for the selected or recommended alternatives for the data communication of the subsurface visual alarm system. This analysis is being prepared to document a basis for the design selection of the data communication method. This analysis will briefly describe existing data or voice communication or monitoring systems within the ESF, and look at how these may be revised or adapted to support the needed data highway of the subsurface visual alarm. system. The existing PLC communication system installed in subsurface is providing data communication for alcove No.5 ventilation fans, south portal ventilation fans, bulkhead doors and generator monitoring system. It is given that the data communication of the subsurface visual alarm system will be a digital based system. It is also given that it is most feasible to take advantage of existing systems and equipment and not consider an entirely new data communication system design and installation. The scope and primary objectives of this analysis are to: (1) Briefly review and describe existing available data communication highways or systems within the ESF. (2) Examine technical characteristics of an existing system to disqualify a design alternative is paramount in minimizing the number of and depth of a system review. (3) Apply general engineering design practices or criteria such as relative cost, and degree of

  10. The Best Ever Alarm System Toolkit

    SciTech Connect

    Kasemir, Kay; Chen, Xihui; Danilova, Katia

    2009-01-01

    Learning from our experience with the standard Experimental Physics and Industrial Control System (EPICS) alarm handler (ALH) as well as a similar intermediate approach based on script-generated operator screens, we developed the Best Ever Alarm System Toolkit (BEAST). It is based on Java and Eclipse on the Control System Studio (CSS) platform, using a relational database (RDB) to store the configuration and log actions. It employs a Java Message Service (JMS) for communication between the modular pieces of the toolkit, which include an Alarm Server to maintain the current alarm state, an arbitrary number of Alarm Client user interfaces (GUI), and tools to annunciate alarms or log alarm related actions. Web reports allow us to monitor the alarm system performance and spot deficiencies in the alarm configuration. The Alarm Client GUI not only gives the end users various ways to view alarms in tree and table, but also makes it easy to access the guidance information, the related operator displays and other CSS tools. It also allows online configuration to be simply modified from the GUI. Coupled with a good "alarm philosophy" on how to provide useful alarms, we can finally improve the configuration to achieve an effective alarm system.

  11. Video systems for alarm assessment

    SciTech Connect

    Greenwoll, D.A.; Matter, J.C. ); Ebel, P.E. )

    1991-09-01

    The purpose of this NUREG is to present technical information that should be useful to NRC licensees in designing closed-circuit television systems for video alarm assessment. There is a section on each of the major components in a video system: camera, lens, lighting, transmission, synchronization, switcher, monitor, and recorder. Each section includes information on component selection, procurement, installation, test, and maintenance. Considerations for system integration of the components are contained in each section. System emphasis is focused on perimeter intrusion detection and assessment systems. A glossary of video terms is included. 13 figs., 9 tabs.

  12. INEL central alarm monitoring and assessment system

    SciTech Connect

    Niper, E.D.

    1983-07-01

    This paper concerns the design and development of a centrally located security monitoring and assessment system for processing alarms at several remote facilities. The system provides both live and recorded CCTV assessment of alarmed areas. Computer controlled video disc recordings are made at the time the alarm is activated. Alarming areas are displayed on a color graphics monitor and an operator interacts through an overlying transparent touch panel. Computer-generated messages are also displayed to assist and inform the operator. A bidirectional, frequency-multiplexed cable system provides digital alarm information, video control commands, and several channels of video from each remote facility.

  13. Masters Thesis- Criticality Alarm System Design Guide with Accompanying Alarm System Development for the Radioisotope Production Laboratory in Richland, Washington

    SciTech Connect

    Greenfield, Bryce A.

    2009-12-01

    A detailed instructional manual was created to guide criticality safety engineers through the process of designing a criticality alarm system (CAS) for Department of Energy (DOE) hazard class 1 and 2 facilities. Regulatory and technical requirements were both addressed. A list of design tasks and technical subtasks are thoroughly analyzed to provide concise direction for how to complete the analysis. An example of the application of the design methodology, the Criticality Alarm System developed for the Radioisotope Production Laboratory (RPL) of Richland, Washington is also included. The analysis for RPL utilizes the Monte Carlo code MCNP5 for establishing detector coverage in the facility. Significant improvements to the existing CAS were made that increase the reliability, transparency, and coverage of the system.

  14. Advanced alarm systems: Display and processing issues

    SciTech Connect

    O`Hara, J.M.; Wachtel, J.; Perensky, J.

    1995-05-01

    This paper describes a research program sponsored by the US Nuclear Regulatory Commission to address the human factors engineering (HFE) deficiencies associated with nuclear power plant alarm systems. The overall objective of the study is to develop HFE review guidance for alarm systems. In support of this objective, human performance issues needing additional research were identified. Among the important issues were alarm processing strategies and alarm display techniques. This paper will discuss these issues and briefly describe our current research plan to address them.

  15. An improved criticality alarm system

    SciTech Connect

    Tyree, W.H.; Gilpin, H.E.; Balmer, D.K.; Vennitti, D.A.

    1991-12-31

    The Rocky Flats Plant near Golden, Colorado is the primary facility for the production of plutonium components used in the US arsenal of nuclear weapons. It is operated by EG&G under contract to the US Department of Energy (DOE). There are ten production buildings on plant site with neutron based criticality alarm systems. These systems have been in operation for the past seventeen years. Changes in the interpretation of A.N.S.I. standards and DOE orders have precipitated an evaluation of detector sensitivity and placement criteria. As a result of this evaluation, improvements in detector design and calibration have improved detector sensitivity by a factor of six. Testing performed on the design defined a minimum sensitivity as required by A.N.S.I. 8.3 and provided information for saturation and survivability for a fission event of up to 1 {times} 10{sup 17} fissions in 80 microseconds. A rigorous testing and calibration program has been developed and is in place. Neutron sensitivity is certified at a nearby reactor which is traceable to N.I.S.T.. 4 refs.

  16. Criticality alarm system for plutonium process operations

    SciTech Connect

    Tyree, W H; Urano, H

    1981-05-25

    A criticality alarm system using neutron detection is described. The system includes a neutron detection unit with an internal operational testing mode. The unit uses a Li/sup 6/F foil for the production of alpha particles from a thermal neturon flux, a thorium-230 alpha particle source for the continuous monitoring of the raidation detection circuits, and an internal clock which produces an alarmed condition if the detector amplifier circuit fails. This neutron detector unit is designed to replace the existing neutron detector in the criticality alarm systems at Rocky Flats. Coincidence circuits, interface system and audio signal generators used in the output of the alarm system are described. The system meets the criteria for the American National Standards and the USDOE Manual Chapter for detection, alarm, and signal output requirements.

  17. 46 CFR 113.43-3 - Alarm system.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Alarm system. 113.43-3 Section 113.43-3 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Steering Failure Alarm Systems § 113.43-3 Alarm system. (a) Each vessel must have a steering failure alarm system that actuates...

  18. Development of medical equipment alarm monitoring system.

    PubMed

    Yamashita, Yoshinori; Ogaito, Tatoku; Kasamatsu, Shingo

    2013-01-01

    In a hospital, we use a large number of medical equipment. In these use, I support the safe use by the alarm such as errors from medical equipment. There is the instrument notifying of alarm in communication, but there is the instrument by a sound and the light. For the medical safety management, confirmation of the alarm is important. We thought that stability was improved by integrating alarm from the instrument of the different type. Therefore, we thought that we integrated alarm from medical equipment. We decided to transmit an alarm signal from medical equipment by adding radio module program unit. The type of the radio used IEEE 802.15.4 (ZigBee) at a point of view of low power, International Standard, simple radio equipment. This system deals with only alarm information from medical equipment and does not handle the data. However, we understood that we were helpful very much even if it was only alarm information. We were able to in this way reduce the number of incidents. PMID:23920969

  19. INEL central alarm monitoring and assessment system

    SciTech Connect

    Niper, E.D.

    1983-01-01

    This paper concerns the design and development of a centrally located security monitoring and assessment system for processing alarms at several remote facilities. The system provides both live and recorded CCTV assessment of alarmed areas. Computer controlled video disc recordings are displayed on a color graphics monitor and an operator interacts through an overlying transparent touch panel. Computer generated messages are also displayed to assist and inform the operator. A bidirectional, frequency-multiplexed cable system provides digital alarm information, video control commands, and several channels of video from each remote facility.

  20. 46 CFR 161.002-12 - Manual fire alarm systems.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    .... (a) General. A manual fire alarm system shall consist of a power supply, a control unit on which are... the control unit and terminating at manual fire alarm boxes. Power failure alarm devices may be... alarm system control unit. The manual fire alarm system control unit shall be as specified for...

  1. HYBRID ALARM SYSTEMS: COMBINING SPATIAL ALARMS AND ALARM LISTS FOR OPTIMIZED CONTROL ROOM OPERATION

    SciTech Connect

    Ronald L. Boring; J.J. Persensky

    2012-07-01

    The US Department of Energy (DOE) is sponsoring research, development, and deployment on Light Water Reactor Sustainability (LWRS), in which the Idaho National Laboratory (INL) is working closely with nuclear utilities to develop technologies and solutions to help ensure the safe operational life extension of current nuclear power plants. One of the main areas of focus is control room modernization. Within control room modernization, alarm system upgrades present opportunities to meet the broader goals of the LWRS project in demonstrating the use and safety of the advanced instrumentation and control (I&C) technologies and the short-term and longer term objectives of the plant. In this paper, we review approaches for and human factors issues behind upgrading alarms in the main control room of nuclear power plants.

  2. Automated Information System (AIS) Alarm System

    SciTech Connect

    Hunteman, W.

    1997-05-01

    The Automated Information Alarm System is a joint effort between Los Alamos National Laboratory, Lawrence Livermore National Laboratory, and Sandia National Laboratory to demonstrate and implement, on a small-to-medium sized local area network, an automated system that detects and automatically responds to attacks that use readily available tools and methodologies. The Alarm System will sense or detect, assess, and respond to suspicious activities that may be detrimental to information on the network or to continued operation of the network. The responses will allow stopping, isolating, or ejecting the suspicious activities. The number of sensors, the sensitivity of the sensors, the assessment criteria, and the desired responses may be set by the using organization to meet their local security policies.

  3. Development of Advanced Alarm System for SMART

    SciTech Connect

    Jang, Gwi-sook; Seoung, Duk-hyun; Suh, Sang-moon; Lee, Jong-bok; Park, Geun-ok; Koo, In-soo

    2004-07-01

    A SMART-Alarm System (SMART-AS) is a new system being developed as part of the SMART (System-integrated Modular Advanced Reactor) project. The SMART-AS employs modern digital technology to implement the alarm functions of the SMART. The use of modern digital technology can provide advanced alarm processing in which new algorithms such as a signal validation, advanced alarm processing logic and other features are applied to improve the control room man-machine interfaces. This paper will describe the design process of the SMART-AS, improving the system reliability and availability using the reliability prediction tool, design strategies regarding the human performance topics associated with a computer-based SMART-AS and the results of the performance analysis using a prototype of the SMART-AS. (authors)

  4. Alarm Systems: Library Confounds Criminal Capers.

    ERIC Educational Resources Information Center

    Gjettum, Pamela

    1978-01-01

    Tells the story of a small town library faced with the problem of preventing nuisance burglaries of the type becoming more and more common. The problems of selecting the right type of alarm system are discussed. (JPF)

  5. 46 CFR 113.20-1 - Sprinkler alarm system.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Sprinkler alarm system. 113.20-1 Section 113.20-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Automatic Sprinkler Systems § 113.20-1 Sprinkler alarm system. Each sprinkler alarm system, including annunciator,...

  6. 46 CFR 113.43-3 - Alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Alarm system. 113.43-3 Section 113.43-3 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Steering Failure Alarm Systems § 113.43-3 Alarm system. (a) Each vessel must have...

  7. 46 CFR 113.43-3 - Alarm system.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Alarm system. 113.43-3 Section 113.43-3 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Steering Failure Alarm Systems § 113.43-3 Alarm system. (a) Each vessel must have...

  8. 46 CFR 113.43-3 - Alarm system.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Alarm system. 113.43-3 Section 113.43-3 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Steering Failure Alarm Systems § 113.43-3 Alarm system. (a) Each vessel must have...

  9. 46 CFR 113.43-3 - Alarm system.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Alarm system. 113.43-3 Section 113.43-3 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Steering Failure Alarm Systems § 113.43-3 Alarm system. (a) Each vessel must have...

  10. Alarm system management: evidence-based guidance encouraging direct measurement of informativeness to improve alarm response.

    PubMed

    Rayo, Michael F; Moffatt-Bruce, Susan D

    2015-04-01

    Although there are powerful incentives for creating alarm management programmes to reduce 'alarm fatigue', they do not provide guidance on how to reduce the likelihood that clinicians will disregard critical alarms. The literature cites numerous phenomena that contribute to alarm fatigue, although many of these, including total rate of alarms, are not supported in the literature as factors that directly impact alarm response. The contributor that is most frequently associated with alarm response is informativeness, which is defined as the proportion of total alarms that successfully conveys a specific event, and the extent to which it is a hazard. Informativeness is low across all healthcare applications, consistently ranging from 1% to 20%. Because of its likelihood and strong evidential support, informativeness should be evaluated before other contributors are considered. Methods for measuring informativeness and alarm response are discussed. Design directions for potential interventions, as well as design alternatives to traditional alarms, are also discussed. With the increased attention and investment in alarm system management that alarm interventions are currently receiving, initiatives that focus on informativeness and the other evidence-based measures identified will allow us to more effectively, efficiently and reliably redirect clinician attention, ultimately improving alarm response. PMID:25734193

  11. An Undergraduate Experiment in Alarm System Design.

    ERIC Educational Resources Information Center

    Martini, R. A.; And Others

    1988-01-01

    Describes an experiment involving data acquisition by a computer, digital signal transmission from the computer to a digital logic circuit and signal interpretation by this circuit. The system is being used at the Illinois Institute of Technology. Discusses the fundamental concepts involved. Demonstrates the alarm experiment as it is used in…

  12. Nuclear power plant alarm systems: Problems and issues

    SciTech Connect

    O'Hara, J.M.; Brown, W.S.

    1991-01-01

    Despite the incorporation of advanced technology into nuclear power plant alarm systems, human factors problems remain. This paper identifies to be addressed in order to allow advanced technology to be used effectively in the design of nuclear power plant alarm systems. The operator's use and processing of alarm system information will be considered. Based upon a review of alarm system research, issues related to general system design, alarm processing, display and control are discussed. It is concluded that the design of effective alarm systems depends on an understanding of the information processing capabilities and limitations of the operator. 39 refs.

  13. Criticality accident alarm system at the Fernald Environmental Management Project

    SciTech Connect

    Marble, R.C.; Brown, T.D.; Wooldridge, J.C.

    1994-12-31

    This paper describes the staus of the Fernald Environmental Management Project (FEMP) criticality alarm system. A new radiation detection alarm system was installed in 1990. The anunciation system, calibration and maintenance, and detector placement is described.

  14. Alarm Management System for the D/3 Distributed Control System

    1997-03-19

    As industrial processes continue to grow in size and complexity, the Distrubuted Control Systems that automate and monitor these processes expand in a like manner. This increase in control system complexity has resulted in ever increasing numbers of alarms presented to the operator. The challenge for today's control system designer is to find innovative ways to present alarm information to the operator such that despite the large number of alarms, the operator is able tomore » quickly assess the status of the plant and immediately respond to the most critical alarms in a timely manner. This software package, designed and developed for the Savannah River Site Replacement High Level Waste Evaporator/Waste Removal Distributed Control System installed on the H-Area Tank Farm, provides an alarm system which utilizes the annunciator (SKID) panel as a means of statusing the plant and providing single keystroke access to the display on which an alarm resides.« less

  15. Alarm Management System for the D/3 Distributed Control System

    SciTech Connect

    McEver, M.

    1997-03-19

    As industrial processes continue to grow in size and complexity, the Distrubuted Control Systems that automate and monitor these processes expand in a like manner. This increase in control system complexity has resulted in ever increasing numbers of alarms presented to the operator. The challenge for today's control system designer is to find innovative ways to present alarm information to the operator such that despite the large number of alarms, the operator is able to quickly assess the status of the plant and immediately respond to the most critical alarms in a timely manner. This software package, designed and developed for the Savannah River Site Replacement High Level Waste Evaporator/Waste Removal Distributed Control System installed on the H-Area Tank Farm, provides an alarm system which utilizes the annunciator (SKID) panel as a means of statusing the plant and providing single keystroke access to the display on which an alarm resides.

  16. 46 CFR 113.20-1 - Sprinkler alarm system.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Sprinkler alarm system. 113.20-1 Section 113.20-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Automatic Sprinkler Systems § 113.20-1 Sprinkler alarm system....

  17. 46 CFR 113.20-1 - Sprinkler alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Sprinkler alarm system. 113.20-1 Section 113.20-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Automatic Sprinkler Systems § 113.20-1 Sprinkler alarm system....

  18. 46 CFR 113.20-1 - Sprinkler alarm system.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Sprinkler alarm system. 113.20-1 Section 113.20-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Automatic Sprinkler Systems § 113.20-1 Sprinkler alarm system....

  19. 46 CFR 113.20-1 - Sprinkler alarm system.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Sprinkler alarm system. 113.20-1 Section 113.20-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Automatic Sprinkler Systems § 113.20-1 Sprinkler alarm system....

  20. 46 CFR 161.002-12 - Manual fire alarm systems.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 6 2013-10-01 2013-10-01 false Manual fire alarm systems. 161.002-12 Section 161.002-12...: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Fire-Protective Systems § 161.002-12 Manual fire alarm systems. (a) General. A manual fire alarm system shall consist of a power supply, a control unit on which...

  1. 46 CFR 161.002-12 - Manual fire alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 6 2010-10-01 2010-10-01 false Manual fire alarm systems. 161.002-12 Section 161.002-12...: SPECIFICATIONS AND APPROVAL ELECTRICAL EQUIPMENT Fire-Protective Systems § 161.002-12 Manual fire alarm systems. (a) General. A manual fire alarm system shall consist of a power supply, a control unit on which...

  2. 46 CFR 76.05-5 - Manual alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 3 2010-10-01 2010-10-01 false Manual alarm system. 76.05-5 Section 76.05-5 Shipping... Fire Detecting and Extinguishing Equipment, Where Required § 76.05-5 Manual alarm system. (a) An approved manual alarm system shall be installed in all areas, other than the main machinery spaces,...

  3. 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

  4. 30 CFR 57.4360 - Underground alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Underground alarm systems. 57.4360 Section 57... MINE SAFETY AND HEALTH SAFETY AND HEALTH STANDARDS-UNDERGROUND METAL AND NONMETAL MINES Fire Prevention and Control Firefighting Procedures/alarms/drills § 57.4360 Underground alarm systems. (a) Fire...

  5. Alarm system for a nuclear control complex

    DOEpatents

    Scarola, Kenneth; Jamison, David S.; Manazir, Richard M.; Rescorl, Robert L.; Harmon, Daryl L.

    1994-01-01

    An advanced control room complex for a nuclear power plant, including a discrete indicator and alarm system (72) which is nuclear qualified for rapid response to changes in plant parameters and a component control system (64) which together provide a discrete monitoring and control capability at a panel (14-22, 26, 28) in the control room (10). A separate data processing system (70), which need not be nuclear qualified, provides integrated and overview information to the control room and to each panel, through CRTs (84) and a large, overhead integrated process status overview board (24). The discrete indicator and alarm system (72) and the data processing system (70) receive inputs from common plant sensors and validate the sensor outputs to arrive at a representative value of the parameter for use by the operator during both normal and accident conditions, thereby avoiding the need for him to assimilate data from each sensor individually. The integrated process status board (24) is at the apex of an information hierarchy that extends through four levels and provides access at each panel to the full display hierarchy. The control room panels are preferably of a modular construction, permitting the definition of inputs and outputs, the man machine interface, and the plant specific algorithms, to proceed in parallel with the fabrication of the panels, the installation of the equipment and the generic testing thereof.

  6. Design of anti-burglar alarm systems

    NASA Astrophysics Data System (ADS)

    Şchiopu, Paul; Costea, Aurelian

    2015-02-01

    Security, as an important element that defines the quality of a system, represents the capacity of a system to preserve his own functional characteristics under pressure of external disruptive agents capable to represent danger for the system, for the environment of the system, and for the life of people inside the defined risk zone. The main goal of security is system stability. With ever new ideas, technology, procedures, actions and specialized institutions, integrated security services offer protection, surveillance and optimum conditions for system to function and to be used properly. Therefore, security represents the main quality parameter of all systems and processes, without it efficiency was not possible. Keyword list: Security; Anti-Burglar Alarm

  7. CAS

    SciTech Connect

    Martinez, B.; Pomeroy, G. )

    1989-12-02

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  8. 46 CFR 58.25-25 - Indicating and alarm systems.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Indicating and alarm systems. 58.25-25 Section 58.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-25 Indicating and alarm systems. (a) Indication of the rudder angle must be provided both...

  9. 46 CFR 58.25-25 - Indicating and alarm systems.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Indicating and alarm systems. 58.25-25 Section 58.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-25 Indicating and alarm systems. (a) Indication of the rudder angle must be provided both...

  10. 46 CFR 183.550 - General alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false General alarm systems. 183.550 Section 183.550 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS (UNDER 100 GROSS TONS) ELECTRICAL INSTALLATION Miscellaneous Systems and Requirements § 183.550 General alarm systems. All...

  11. 29 CFR 1910.165 - Employee alarm systems.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 5 2011-07-01 2011-07-01 false Employee alarm systems. 1910.165 Section 1910.165 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Fire Protection Other Fire Protection Systems § 1910.165 Employee alarm systems. (a) Scope...

  12. 29 CFR 1910.165 - Employee alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 5 2010-07-01 2010-07-01 false Employee alarm systems. 1910.165 Section 1910.165 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR OCCUPATIONAL SAFETY AND HEALTH STANDARDS Fire Protection Other Fire Protection Systems § 1910.165 Employee alarm systems. (a) Scope...

  13. From alarm systems to smart houses.

    PubMed

    Vlaskamp, F J

    1992-01-01

    The percentage of senior citizens in the Netherlands will rise in coming years. The expected percentage for the year 2010 of persons over age 65 in the total population is 15%. More persons over age 65 than ever before will continue to live in their own environment. Emergency response systems (ERS) can support independent living. The most common type of organization distributing ERS is a small, partly subsidized local alarm organization run by a social welfare office for the elderly. Government subsidy has been reduced in recent years which has motivated small organizations to join together into larger regional organizations in order to get a more solid financial base. On the other hand new semi-commercial and commercial organizations have come into being. These developments are part of the growing importance of home care, leading to more medical applications of ERS. User satisfaction with ERS is high. Portable triggers can enhance the effectiveness of the system. However, many users do not wear the portable trigger when feeling well. Future technical developments will result in multifunctionality of ERS-devices. In the long term the hardware of today will be integrated in a multimedia home terminal replacing the telephone. The portable trigger will remain the only specific hardware at home for ERS. PMID:10126436

  14. 29 CFR 1910.165 - Employee alarm systems.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Fire Protection Other Fire Protection Systems § 1910.165 Employee... alarms required on various fixed extinguishing systems or to supervisory alarms on fire suppression... in this section that pertain to maintenance, testing and inspection shall apply to all local...

  15. 29 CFR 1910.165 - Employee alarm systems.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Fire Protection Other Fire Protection Systems § 1910.165 Employee... alarms required on various fixed extinguishing systems or to supervisory alarms on fire suppression... in this section that pertain to maintenance, testing and inspection shall apply to all local...

  16. 29 CFR 1910.165 - Employee alarm systems.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... OCCUPATIONAL SAFETY AND HEALTH STANDARDS Fire Protection Other Fire Protection Systems § 1910.165 Employee... alarms required on various fixed extinguishing systems or to supervisory alarms on fire suppression... in this section that pertain to maintenance, testing and inspection shall apply to all local...

  17. 46 CFR 154.1842 - Cargo system: Controls and alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Cargo system: Controls and alarms. 154.1842 Section 154.1842 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Operations § 154.1842 Cargo system: Controls and alarms. The master...

  18. Use of pagers with an alarm escalation system to reduce cardiac monitor alarm signals.

    PubMed

    Cvach, Maria M; Frank, Robert J; Doyle, Pete; Stevens, Zeina Khouri

    2014-01-01

    Alarm fatigue desensitizes nurses to alarm signals and presents potential for patient harm. This project describes an innovative method of communicating cardiac monitor alarms to pagers using an alarm escalation algorithm. This innovation was tested on 2 surgical progressive care units over a 6-month period. There was a significant decrease in mean frequency and duration of high-priority monitor alarms and improvement in nurses' perception of alarm response time, using this method of alarm communication. PMID:23963169

  19. 33 CFR 127.201 - Sensing and alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... systems. (a) Fixed sensors must have audio and visual alarms in the control room and audio alarms nearby. (b) Fixed sensors that continuously monitor for LNG vapors must— (1) Be in each enclosed area where vapor or gas may accumulate; and (2) Meet Section 9-4 of NFPA 59A. (c) Fixed sensors that...

  20. 33 CFR 127.201 - Sensing and alarm systems.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... systems. (a) Fixed sensors must have audio and visual alarms in the control room and audio alarms nearby. (b) Fixed sensors that continuously monitor for LNG vapors must— (1) Be in each enclosed area where vapor or gas may accumulate; and (2) Meet Section 9-4 of NFPA 59A. (c) Fixed sensors that...

  1. 33 CFR 127.201 - Sensing and alarm systems.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... systems. (a) Fixed sensors must have audio and visual alarms in the control room and audio alarms nearby. (b) Fixed sensors that continuously monitor for LNG vapors must— (1) Be in each enclosed area where vapor or gas may accumulate; and (2) Meet Section 9-4 of NFPA 59A. (c) Fixed sensors that...

  2. 33 CFR 127.201 - Sensing and alarm systems.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... systems. (a) Fixed sensors must have audio and visual alarms in the control room and audio alarms nearby. (b) Fixed sensors that continuously monitor for LNG vapors must— (1) Be in each enclosed area where vapor or gas may accumulate; and (2) Meet Section 9-4 of NFPA 59A. (c) Fixed sensors that...

  3. 33 CFR 127.201 - Sensing and alarm systems.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... systems. (a) Fixed sensors must have audio and visual alarms in the control room and audio alarms nearby. (b) Fixed sensors that continuously monitor for LNG vapors must— (1) Be in each enclosed area where vapor or gas may accumulate; and (2) Meet Section 9-4 of NFPA 59A. (c) Fixed sensors that...

  4. Pressurized security barrier and alarm system

    DOEpatents

    Carver, D.W.

    1995-04-11

    A security barrier for placement across a passageway is made up of interconnected pressurized tubing made up in a grid pattern with openings too small to allow passage. The tubing is connected to a pressure switch, located away from the barrier site, which activates an alarm upon occurrence of a pressure drop. A reinforcing bar is located inside and along the length of the tubing so as to cause the tubing to rupture and set off the alarm upon an intruder`s making an attempt to crimp and seal off a portion of the tubing by application of a hydraulic tool. Radial and rectangular grid patterns are disclosed. 7 figures.

  5. Pressurized security barrier and alarm system

    DOEpatents

    Carver, Don W.

    1995-01-01

    A security barrier for placement across a passageway is made up of interconnected pressurized tubing made up in a grid pattern with openings too small to allow passage. The tubing is connected to a pressure switch, located away from the barrier site, which activates an alarm upon occurrence of a pressure drop. A reinforcing bar is located inside and along the length of the tubing so as to cause the tubing to rupture and set off the alarm upon an intruder's making an attempt to crimp and seal off a portion of the tubing by application of a hydraulic tool. Radial and rectangular grid patterns are disclosed.

  6. Second Line of Defense Virtual Private Network Guidance for Deployed and New CAS Systems

    SciTech Connect

    Singh, Surya V.; Thronas, Aaron I.

    2010-01-01

    This paper discusses the importance of remote access via virtual private network (VPN) for the Second Line of Defense (SLD) Central Alarm System (CAS) sites, the requirements for maintaining secure channels while using VPN and implementation requirements for current and future sites.

  7. Computational Human Performance Modeling For Alarm System Design

    SciTech Connect

    Jacques Hugo

    2012-07-01

    The introduction of new technologies like adaptive automation systems and advanced alarms processing and presentation techniques in nuclear power plants is already having an impact on the safety and effectiveness of plant operations and also the role of the control room operator. This impact is expected to escalate dramatically as more and more nuclear power utilities embark on upgrade projects in order to extend the lifetime of their plants. One of the most visible impacts in control rooms will be the need to replace aging alarm systems. Because most of these alarm systems use obsolete technologies, the methods, techniques and tools that were used to design the previous generation of alarm system designs are no longer effective and need to be updated. The same applies to the need to analyze and redefine operators’ alarm handling tasks. In the past, methods for analyzing human tasks and workload have relied on crude, paper-based methods that often lacked traceability. New approaches are needed to allow analysts to model and represent the new concepts of alarm operation and human-system interaction. State-of-the-art task simulation tools are now available that offer a cost-effective and efficient method for examining the effect of operator performance in different conditions and operational scenarios. A discrete event simulation system was used by human factors researchers at the Idaho National Laboratory to develop a generic alarm handling model to examine the effect of operator performance with simulated modern alarm system. It allowed analysts to evaluate alarm generation patterns as well as critical task times and human workload predicted by the system.

  8. Perimeter security alarm system based on fiber Bragg grating

    NASA Astrophysics Data System (ADS)

    Zhang, Cui; Wang, Lixin

    2010-11-01

    With the development of the society and economy and the improvement of living standards, people need more and more pressing security. Perimeter security alarm system is widely regarded as the first line of defense. A highly sensitive Fiber Bragg grating (FBG) vibration sensor based on the theory of the string vibration, combined with neural network adaptive dynamic programming algorithm for the perimeter security alarm system make the detection intelligently. Intelligent information processing unit identify the true cause of the vibration of the invasion or the natural environment by analyzing the frequency of vibration signals, energy, amplitude and duration. Compared with traditional perimeter security alarm systems, such as infrared perimeter security system and electric fence system, FBG perimeter security alarm system takes outdoor passive structures, free of electromagnetic interference, transmission distance through optical fiber can be as long as 20 km It is able to detect the location of event within short period of time (high-speed response, less than 3 second).This system can locate the fiber cable's breaking sites and alarm automatically if the cable were be cut. And the system can prevent effectively the false alarm from small animals, birds, strong wind, scattering things, snowfalls and vibration of sensor line itself. It can also be integrated into other security systems. This system can be widely used in variety fields such as military bases, nuclear sites, airports, warehouses, prisons, residence community etc. It will be a new force of perimeter security technology.

  9. Research on the fire alarming system of fiber grating

    NASA Astrophysics Data System (ADS)

    Qi, Yaobin

    2007-09-01

    The application of fiber grating sensing technology in fire alarming based on temperature detection has the advantages of high accuracy, high reliability and strong immunity from electronic and magnetic fields. It is especially advantageous to use this system in the petroleum and chemistry industry because it can provide an extraordinary safe means for the fire alarm. But due to the traditional optical Wavelength Division Multiplexing (WDM) technology is limited by the optic source bandwidth, the number of its multiplexing points is few. In this paper WDM technology will be developed mixing with Identified Bragg, which is called Identified and Wavelength Multiplexing, to build the Fiber Grating (FBG) fire alarm system integrated with computers. Some technologies applied in fire alarming system of fiber grating such as the transmission of test signals which pass through modulate and demodulate, the disposal of software system, the output of control signal and the strong ability of anti-disturbance have been studied and discussed.

  10. 5. DETAIL VIEW OF OLD, PUNCHTYPE MASTER FIRE ALARM SYSTEM, ...

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

    5. DETAIL VIEW OF OLD, PUNCH-TYPE MASTER FIRE ALARM SYSTEM, LOCATED ON S WALL OF ENGINE STORAGE ROOM; LOOKING S. (Ceronie and Ryan) - Watervliet Arsenal, Building No. 22, Westervelt Avenue & Buffington Street, Watervliet, Albany County, NY

  11. Onsite Portable Alarm System - Its Merit and Application

    NASA Astrophysics Data System (ADS)

    Saita, J.; Sato, T.; Nakamura, Y.

    2007-12-01

    Recently an existence of the earthquake early warning system (EEWS) becomes popular. In general, the EEWS will be installed in a fixed observation site and it may consist of several separated components such as a sensing portion, A/D converter, an information processing potion and so on. The processed information for warning may be transmitted to network via fixed communication line, and therefore this kind of alarm system is called as Network Alarm System. On the other hand, after the severe earthquake damage, it is very important to save the disaster victims immediately. These rescue staffs are also under the risk of aftershocks and need a local alarm not depending on the network, so this kind of alarm can be called as Onsite Alarm. But the common early warning system is too complex to set onsite temporary, and even if possible to install, the alarm is too late to receive at the epicentral area. However, the new generation earthquake early warning system FREQL can issue the P wave alarm by minimum 0.2 seconds after P wave detection. And FREQL is characterized as the unique all-in-one seismometer with power unit. At the time of the 2004 Niigata-Ken-Chuetsu earthquake, a land slide attacked a car just passing. A hyper rescue team of Tokyo Fire Department pulled the survivor, one baby, from the land slide area. During their activity the rescue team was exposed to the risk of secondary hazards caused by the aftershocks. It was clear that it is necessary to use a portable warning system to issue the onsite P wave alarm. Because FREQL was originally developed as portable equipment, Tokyo Fire Department asked us to modify it to the portable equipment with the loud sound and the light signal. In this moment, this portable FREQL has equipped in nation wide. When the hyper rescue team of Tokyo Fire Department was sent to Pakistan as a task force for rescue work of the 2005 Pakistan earthquake, the portable FREQL was used as important onsite portable warning system and P

  12. Hypoglycemia Early Alarm Systems Based On Multivariable Models

    PubMed Central

    Turksoy, Kamuran; Bayrak, Elif S; Quinn, Lauretta; Littlejohn, Elizabeth; Rollins, Derrick; Cinar, Ali

    2013-01-01

    Hypoglycemia is a major challenge of artificial pancreas systems and a source of concern for potential users and parents of young children with Type 1 diabetes (T1D). Early alarms to warn the potential of hypoglycemia are essential and should provide enough time to take action to avoid hypoglycemia. Many alarm systems proposed in the literature are based on interpretation of recent trends in glucose values. In the present study, subject-specific recursive linear time series models are introduced as a better alternative to capture glucose variations and predict future blood glucose concentrations. These models are then used in hypoglycemia early alarm systems that notify patients to take action to prevent hypoglycemia before it happens. The models developed and the hypoglycemia alarm system are tested retrospectively using T1D subject data. A Savitzky-Golay filter and a Kalman filter are used to reduce noise in patient data. The hypoglycemia alarm algorithm is developed by using predictions of future glucose concentrations from recursive models. The modeling algorithm enables the dynamic adaptation of models to inter-/intra-subject variation and glycemic disturbances and provides satisfactory glucose concentration prediction with relatively small error. The alarm systems demonstrate good performance in prediction of hypoglycemia and ultimately in prevention of its occurrence. PMID:24187436

  13. Nuclear-power-plant perimeter-intrusion alarm systems

    SciTech Connect

    Halsey, D.J.

    1982-04-01

    Timely intercept of an intruder requires the examination of perimeter barriers and sensors in terms of reliable detection, immediate assessment and prompt response provisions. Perimeter security equipment and operations must at the same time meet the requirements of the Code of Federal Regulations, 10 CFR 73.55 with some attention to the performance and testing figures of Nuclear Regulatory Guide 5.44, Revision 2, May 1980. A baseline system is defined which recommends a general approach to implementing perimeter security elements: barriers, lighting, intrusion detection, alarm assessment. The baseline approach emphasizes cost/effectiveness achieved by detector layering and logic processing of alarm signals to produce reliable alarms and low nuisance alarm rates. A cost benefit of layering along with video assessment is reduction in operating expense. The concept of layering is also shown to minimize testing costs where detectability performance as suggested by Regulatory Guide 5.44 is to be performed. Synthesis of the perimeter intrusion alarm system and limited testing of CCTV and Video Motion Detectors (VMD), were performed at E-Systems, Greenville Division, Greenville, Texas during 1981.

  14. A TWACS system alarm function for distribution automation

    SciTech Connect

    Mak, S.T.; Radford, D. )

    1994-04-01

    A new application designated as the System Alarm Function'' is made possible by using the Unsolicited Inbound'' capability recently developed for the Two Way Automatic Communication System (TWACS) System-10, which is already used by several electric utilities in the USA for demand side management, remote metering and distribution automation is reviewed here. The UNSOLICITED INBOUND'' algorithm development, the operational issues that are pertinent to the understanding of collisions between multiple alarms and the effects on the alarm response time'' are discussed in detail. This master-to-master relationship between remote transponders in the distribution network and the central control computer opens the door to multiple functional capabilities in the area of distribution automation and remote monitoring.

  15. Development of real time alarm/surveillance system

    SciTech Connect

    Kajiyoshi, M.; Uchida, S.; Suenaga, S.; Iwabuchi, M.; Fujimoto, T.; Yoshimura, A.

    1987-07-01

    PNC has carried out real time alarm/surveillance system as a part of its r and d programs on the physical protection systems for its nuclear facilities from 1984 to 1986. The guard operates a closed circuit television (CCTV) camera to see whether the alarms are caused from intruders or from accident in the existing physical protection systems. But it is very difficult to assess rapidly moving objects using such a TV system. Monitor images are used to be continuously recorded by VTR, but it does not seem to be very suitable or it takes quite a long time to play back. Aiming at more effective and reliable physical protection systems, a new alarm/surveillance system was developed, the system detects persons entering into the surveillance zone and provides effective means to confirm. The system detects moving persons in a double fenced zone using image processing technique. Detection information such as time, position, detected images are displayed, printed and recorded, so that cause of false alarms, if occurred can be found easily.

  16. Integrated alarm annunciation and entry control systems -- Survey results

    SciTech Connect

    Clever, J.J.; Arakaki, L.H.; Monaco, F.M.; Juarros, L.E.; Quintana, G.R.

    1993-10-01

    This report provides the results and analyses of a detailed survey undertaken in Summer 1993 to address integrated intrusion detection alarm annunciation and entry control system issues. This survey was undertaken as a first attempt toward beginning to answer questions about integrated systems and commercial capabilities to meet or partially meet US Department of Energy (DOE) site needs.

  17. Cost-Effective School Alarm Systems. Security Topics Series.

    ERIC Educational Resources Information Center

    Kaufer, Steve

    This document outlines considerations in the selection of a cost-effective school-alarm system. Steps in the planning process include: conducting a district needs assessment; gathering input from all staff levels; consulting technical expertise; and selecting a security system that can be integrated with other site needs. It further describes the…

  18. Alarms Philosophy

    SciTech Connect

    White, Karen S; Kasemir, Kay

    2009-01-01

    An effective alarm system consists of a mechanism to monitor control points and generate alarm notifications, tools for operators to view, hear, acknowledge and handle alarms and a good configuration. Despite the availability of numerous fully featured tools, accelerator alarm systems continue to be disappointing to operations, frequently to the point of alarms being permanently silenced or totally ignored. This is often due to configurations that produce an excessive number of alarms or fail to communicate the required operator response. Most accelerator controls systems do a good job of monitoring specified points and generating notifications when parameters exceed predefined limits. In some cases, improved tools can help, but more often, poor configuration is the root cause of ineffective alarm systems. A SNS, we have invested considerable effort in generating appropriate configurations using a rigorous set of rules based on best practices in the industrial process controls community. This paper will discuss our alarm configuration philosophy and operator response to our new system.

  19. 46 CFR 154.1842 - Cargo system: Controls and alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Cargo system: Controls and alarms. 154.1842 Section 154.1842 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Operations § 154.1842...

  20. 46 CFR 154.1842 - Cargo system: Controls and alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Cargo system: Controls and alarms. 154.1842 Section 154.1842 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Operations § 154.1842...

  1. 46 CFR 154.1842 - Cargo system: Controls and alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Cargo system: Controls and alarms. 154.1842 Section 154.1842 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Operations § 154.1842...

  2. 46 CFR 154.1842 - Cargo system: Controls and alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Cargo system: Controls and alarms. 154.1842 Section 154.1842 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Operations § 154.1842...

  3. 46 CFR 120.550 - General alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false General alarm systems. 120.550 Section 120.550 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) SMALL PASSENGER VESSELS CARRYING MORE THAN 150 PASSENGERS OR WITH OVERNIGHT ACCOMMODATIONS FOR MORE THAN 49 PASSENGERS ELECTRICAL INSTALLATION...

  4. Bibliography for nuclear criticality accident experience, alarm systems, and emergency management

    SciTech Connect

    Putman, V.L.

    1995-09-01

    The characteristics, detection, and emergency management of nuclear criticality accidents outside reactors has been an important component of criticality safety for as long as the need for this specialized safety discipline has been recognized. The general interest and importance of such topics receives special emphasis because of the potentially lethal, albeit highly localized, effects of criticality accidents and because of heightened public and regulatory concerns for any undesirable event in nuclear and radiological fields. This bibliography lists references which are potentially applicable to or interesting for criticality alarm, detection, and warning systems; criticality accident emergency management; and their associated programs. The lists are annotated to assist bibliography users in identifying applicable: industry and regulatory guidance and requirements, with historical development information and comments; criticality accident characteristics, consequences, experiences, and responses; hazard-, risk-, or safety-analysis criteria; CAS design and qualification criteria; CAS calibration, maintenance, repair, and testing criteria; experiences of CAS designers and maintainers; criticality accident emergency management (planning, preparedness, response, and recovery) requirements and guidance; criticality accident emergency management experience, plans, and techniques; methods and tools for analysis; and additional bibliographies.

  5. SeaQuest/E906 Shift Alarm System

    NASA Astrophysics Data System (ADS)

    Kitts, Noah

    2014-09-01

    SeaQuest, Fermilab E906, is a fixed target experiment that measures the Drell-Yan cross-section ratio of proton-proton to proton-deuterium collisions in order to extract the sea anti-quark structure of the proton. SeaQuest will extend the measurements made by E866/NuSea with greater precision at higher Bjorken-x. The continuously running experiment is always being monitored. Those on shift must keep track of all of the detector readouts in order to make sure the experiment is running correctly. As an experiment that is still in its early stages of running, an alarm system for people on shift is being created to provide warnings, such as a plot showing a detector's performance is sufficiently different to need attention. This plan involves python scripts that track live data. When the data shows a problem within the experiment, a corresponding alarm ID is sent to the MySQL database which then sets off an alarm. These alarms, which will alert the person on shift through both an audible and visual response, are important for ensuring that issues do not go unnoticed, and to help make sure the experiment is recording good data.

  6. Reducing SCADA System Nuisance Alarms in the Water Industry in Northern Ireland.

    PubMed

    O'Donoghue, Nigel; Phillips, Debra H; Nicell, Ciaran

    2015-08-01

    The advancement of telemetry control for the water industry has increased the difficulty of managing large volumes of nuisance alarms (i.e., alarms that do not require a response). The aim of this study was to identify and reduce the number of nuisance alarms that occur for Northern Ireland (NI) Water by carrying out alarm duration analysis to determine the appropriate length of persistence (an advanced alarm management tool) that could be applied. All data were extracted from TelemWeb (NI Water's telemetry monitoring system) and analyzed in Excel. Over a 6-week period, an average of 40 000 alarms occurred per week. The alarm duration analysis, which has never been implemented before by NI Water, found that an average of 57% of NI Water alarms had a duration of <5 minutes. Applying 5-minute persistence, therefore, could prevent an average 26 816 nuisance alarms per week. Most of these alarms were from wastewater assets. PMID:26237691

  7. Alarm timing, trust and driver expectation for forward collision warning systems.

    PubMed

    Abe, Genya; Richardson, John

    2006-09-01

    In order to improve road safety, automobile manufacturers are now developing Forward Collision Warning Systems (FCWS). However, there has been insufficient consideration of how drivers may respond to FCWS. This driving simulator study focused on alarm timing and its impact on driver response to alarm. The experimental investigation considered driver perception of alarm timings and its influence on trust at three driving speeds (40, 60 and 70 mile/h) and two time headways (1.7 and 2.2 s). The results showed that alarm effectiveness varied in response to driving conditions. Alarm promptness had a greater influence on ratings of trust than improvements in braking performance enabled by the alarm system. Moreover, alarms which were presented after braking actions had been initiated were viewed as late alarms. It is concluded that drivers typically expect alarms to be presented before they initiate braking actions and when this does not happen driver trust in the system is substantially decreased. PMID:16364231

  8. All-optical SOA latch fail-safe alarm system

    NASA Astrophysics Data System (ADS)

    McAulay, Alastair D.

    2004-11-01

    Emergency alarm systems, for example, that switch off critical processes in process plant, are vulnerable to deliberate or accidental sabotage through coupling of electromagnetic pulses (EMP) to wires and/or from sparks due to broken wires. A proposed system significantly reduces vulnerability by using a fast all-optical latch in conjunction with an optical sensor and optical fibers. Sparks cannot be created on breaking an optical beam and electromagnetic field transients have negligible effect on optical signals. The optical latch uses optical semiconductor amplifiers (SOAs) configured to form a flip-flop. The flip-flop latches after the occurrence of an intrusion that may be as short as a few nanoseconds, much faster than most environmental changes occur. Detection of an emergency or any break in connections causes the light to drop, triggering the alarm. Computer simulation shows that the all-optical latch is fast and effective.

  9. Ultra low frequency electromagnetic fire alarm system for underground mines

    SciTech Connect

    Not Available

    1991-01-01

    During an underground mine fire, air can be rapidly depleted of oxygen and contaminated with smoke and toxic fire gases. Any delay in warning miners could have disastrous consequences. Unfortunately, present mine fire alarm systems, such as stench, audible or visual alarms, telephones, and messengers, are often slow, unreliable, and limited in mine area coverage. Recent research by the U.S. Bureau of Mines has demonstrated that ultra-low-frequency electromagnetic signaling can be used for an underground mine fire alarm. In field tests of prototype equipment at five mines, electromagnetic signals from 630 to 2,000 Hz were transmitted through mine rock for distances as great as 1,645 m to an intrinsically safe receiver. The prototype system uses off-the-shelf components and state-of-the-art technology to ensure high reliability and low cost. When utilized, this technology would enable simultaneous and instantaneous warning of all underground personnel, regardless of their location or work activity, thereby increasing the likelihood of their successfully escaping a mine disaster. This paper presents the theoretical basis for through-the-rock ultra-low-frequency electromagnetic transmission, design of the prototype transmitter and receiver, and the results of in-mine tests of the prototype system.

  10. Web-based integrated alarm monitoring system in the ICU.

    PubMed

    Murakami, Akitsugu; Kinouchi, Yohsuke; Akutagawa, Masatake; Ohnishi, Yoshiaki; Kuroda, Yasuhiro

    2005-01-01

    A web-based monitoring system for the alarm of equipment has developed for the conventional environment of Intensive Care Unit (ICU). The system communicates with equipment using Data Collection Interface (DCI) that converts the protocol of the output of equipment from RS-232C to TCP/IP. The system creates a web-document that can be referred from any internet-connected personal computer in the hospital. Using the system, a staff can easily monitor the state of the patient and the equipment. If the system is installed in the ICU, monitoring and management for the equipment will be highly improved. PMID:17281636

  11. 46 CFR 154.1325 - Liquid level alarm system: All cargo tanks.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Liquid level alarm system: All cargo tanks. 154.1325... Equipment Instrumentation § 154.1325 Liquid level alarm system: All cargo tanks. Except as allowed under § 154.1330, each cargo tank must have a high liquid level alarm system that: (a) Is independent of...

  12. 46 CFR 154.1325 - Liquid level alarm system: All cargo tanks.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Liquid level alarm system: All cargo tanks. 154.1325... Equipment Instrumentation § 154.1325 Liquid level alarm system: All cargo tanks. Except as allowed under § 154.1330, each cargo tank must have a high liquid level alarm system that: (a) Is independent of...

  13. 46 CFR 154.1325 - Liquid level alarm system: All cargo tanks.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Liquid level alarm system: All cargo tanks. 154.1325... Equipment Instrumentation § 154.1325 Liquid level alarm system: All cargo tanks. Except as allowed under § 154.1330, each cargo tank must have a high liquid level alarm system that: (a) Is independent of...

  14. 46 CFR 154.1325 - Liquid level alarm system: All cargo tanks.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Liquid level alarm system: All cargo tanks. 154.1325... Equipment Instrumentation § 154.1325 Liquid level alarm system: All cargo tanks. Except as allowed under § 154.1330, each cargo tank must have a high liquid level alarm system that: (a) Is independent of...

  15. 33 CFR 149.665 - What are the requirements for a general alarm system?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...? Each pumping platform complex must have a general alarm system that: (a) Is capable of being manually activated by using alarm boxes; (b) Is audible in all parts of the pumping platform complex, except in...

  16. Substrate generation for endonucleases of CRISPR/cas systems.

    PubMed

    Zoephel, Judith; Dwarakanath, Srivatsa; Richter, Hagen; Plagens, André; Randau, Lennart

    2012-01-01

    The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) (1-3). Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems (4). The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster (5). The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs (6-8). These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence (9). A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity(10). Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA (11,12) . These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases (4). Here, we present methods to generate crRNAs and precursor-cRNAs for

  17. Orthos, an alarm system for the ALICE DAQ operations

    NASA Astrophysics Data System (ADS)

    Chapeland, Sylvain; Carena, Franco; Carena, Wisla; Chibante Barroso, Vasco; Costa, Filippo; Denes, Ervin; Divia, Roberto; Fuchs, Ulrich; Grigore, Alexandru; Simonetti, Giuseppe; Soos, Csaba; Telesca, Adriana; Vande Vyvre, Pierre; von Haller, Barthelemy

    2012-12-01

    ALICE (A Large Ion Collider Experiment) is the heavy-ion detector studying the physics of strongly interacting matter and the quark-gluon plasma at the CERN LHC (Large Hadron Collider). The DAQ (Data Acquisition System) facilities handle the data flow from the detectors electronics up to the mass storage. The DAQ system is based on a large farm of commodity hardware consisting of more than 600 devices (Linux PCs, storage, network switches), and controls hundreds of distributed hardware and software components interacting together. This paper presents Orthos, the alarm system used to detect, log, report, and follow-up abnormal situations on the DAQ machines at the experimental area. The main objective of this package is to integrate alarm detection and notification mechanisms with a full-featured issues tracker, in order to prioritize, assign, and fix system failures optimally. This tool relies on a database repository with a logic engine, SQL interfaces to inject or query metrics, and dynamic web pages for user interaction. We describe the system architecture, the technologies used for the implementation, and the integration with existing monitoring tools.

  18. Human factors engineering guidance for the review of advanced alarm systems

    SciTech Connect

    O`Hara, J.M.; Brown, W.S.; Higgins, J.C.; Stubler, W.F.

    1994-09-01

    This report provides guidance to support the review of the human factors aspects of advanced alarm system designs in nuclear power plants. The report is organized into three major sections. The first section describes the methodology and criteria that were used to develop the design review guidelines. Also included is a description of the scope, organization, and format of the guidelines. The second section provides a systematic review procedure in which important characteristics of the alarm system are identified, described, and evaluated. The third section provides the detailed review guidelines. The review guidelines are organized according to important characteristics of the alarm system including: alarm definition; alarm processing and reduction; alarm prioritization and availability; display; control; automated; dynamic, and modifiable characteristics; reliability, test, maintenance, and failure indication; alarm response procedures; and control-display integration and layout.

  19. Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

    PubMed

    Wilkinson, Max E; Nakatani, Yoshio; Staals, Raymond H J; Kieper, Sebastian N; Opel-Reading, Helen K; McKenzie, Rebecca E; Fineran, Peter C; Krause, Kurt L

    2016-04-15

    CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity. PMID:26929403

  20. Adaptation in CRISPR-Cas Systems.

    PubMed

    Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

    2016-03-17

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity. PMID:26949040

  1. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    PubMed Central

    2011-01-01

    Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to

  2. Recent Results on "Approximations to Optimal Alarm Systems for Anomaly Detection"

    NASA Technical Reports Server (NTRS)

    Martin, Rodney Alexander

    2009-01-01

    An optimal alarm system and its approximations may use Kalman filtering for univariate linear dynamic systems driven by Gaussian noise to provide a layer of predictive capability. Predicted Kalman filter future process values and a fixed critical threshold can be used to construct a candidate level-crossing event over a predetermined prediction window. An optimal alarm system can be designed to elicit the fewest false alarms for a fixed detection probability in this particular scenario.

  3. Development of an on-line expert system for integrated alarm processing in nuclear power plants

    SciTech Connect

    Kim, Han Gon; Choi, Seong Soo; Kang, Ki Sig; Chang, Soon Heung

    1994-12-31

    An on-line expert system, called AFDS (Alarm Filtering and Diagnostic System), has been developed to assist operators in effectively maintaining plant safety and to enhance plant availability using advanced computer technologies for alarm processing. The AFDS is designed to perform alarm filtering and overall plantwide diagnosis when an abnormal state occurs. in addition to these functions, it carries out alarm prognosis to provide the operator with prediction-based messages and to generate high-level alarms that can be used as another diagnostic information. The system is developed on a SUN SPARC 2 workstation, and its target domain is the alarm system in the main control room of Yonggwang units 1 and 2.

  4. 46 CFR 31.35-5 - Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc...

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 1 2010-10-01 2010-10-01 false Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.-TB/ALL. 31.35-5 Section 31.35-5 Shipping COAST GUARD, DEPARTMENT... Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.—TB/ALL. All...

  5. 46 CFR 31.35-5 - Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc...

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 1 2014-10-01 2014-10-01 false Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.-TB/ALL. 31.35-5 Section 31.35-5 Shipping COAST GUARD, DEPARTMENT... Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.—TB/ALL. All...

  6. 46 CFR 31.35-5 - Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc...

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 1 2013-10-01 2013-10-01 false Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.-TB/ALL. 31.35-5 Section 31.35-5 Shipping COAST GUARD, DEPARTMENT... Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.—TB/ALL. All...

  7. 46 CFR 31.35-5 - Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc...

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 1 2011-10-01 2011-10-01 false Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.-TB/ALL. 31.35-5 Section 31.35-5 Shipping COAST GUARD, DEPARTMENT... Communications; alarm systems, telephone and voice tube systems, engine telegraph systems, etc.—TB/ALL. All...

  8. 46 CFR 113.25-25 - General emergency alarm systems for manned ocean and coastwise barges.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false General emergency alarm systems for manned ocean and coastwise barges. 113.25-25 Section 113.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Systems § 113.25-25 General emergency alarm systems for manned ocean and coastwise barges. A manned...

  9. 46 CFR 113.25-25 - General emergency alarm systems for manned ocean and coastwise barges.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false General emergency alarm systems for manned ocean and coastwise barges. 113.25-25 Section 113.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Systems § 113.25-25 General emergency alarm systems for manned ocean and coastwise barges. A manned...

  10. 46 CFR 113.25-25 - General emergency alarm systems for manned ocean and coastwise barges.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false General emergency alarm systems for manned ocean and coastwise barges. 113.25-25 Section 113.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Systems § 113.25-25 General emergency alarm systems for manned ocean and coastwise barges. A manned...

  11. 46 CFR 113.25-25 - General emergency alarm systems for manned ocean and coastwise barges.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false General emergency alarm systems for manned ocean and coastwise barges. 113.25-25 Section 113.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Systems § 113.25-25 General emergency alarm systems for manned ocean and coastwise barges. A manned...

  12. 46 CFR 113.25-25 - General emergency alarm systems for manned ocean and coastwise barges.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false General emergency alarm systems for manned ocean and coastwise barges. 113.25-25 Section 113.25-25 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY... Systems § 113.25-25 General emergency alarm systems for manned ocean and coastwise barges. A manned...

  13. The research of highway traffic accident management and pre-alarm system

    NASA Astrophysics Data System (ADS)

    Xu, Jianping; Zhang, Tiejun; Wan, Jiaonan; Zhang, Juwen; Wang, Rui

    For the rigorous traffic safety issues resulting from rapid transportation development, as well as the more and more attention paid to the traffic accidents dynamic analysis and pre-alarm methods, combined with the practical needs of the highway safety management, this paper summarizes the experience of traffic safety pre-alarm research both in domestic and abroad, designs the frame of highway traffic accident management and pre-alarm system from the function and software engineering requirement, and refines kernel modules such as accident prone section judgement, traffic safety pre-alarm analysis and perfecting safety measures analysis, in order to guide the exploitation and application of the system.

  14. A Human Factors Perspective on Alarm System Research and Development 2000 to 2010

    SciTech Connect

    Curt Braun; John Grimes; Eric Shaver; Ronald Boring

    2011-09-01

    By definition, alarms serve to notify human operators of out-of-parameter conditions that could threaten equipment, the environment, product quality and, of course, human life. Given the complexities of industrial systems, human machine interfaces, and the human operator, the understanding of how alarms and humans can best work together to prevent disaster is continually developing. This review examines advances in alarm research and development from 2000 to 2010 and includes the writings of trade professionals, engineering and human factors researchers, and standards organizations with the goal of documenting advances in alarms system design, research, and implementation.

  15. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella. PMID:25479838

  16. Target specificity of the CRISPR-Cas9 system

    PubMed Central

    Wu, Xuebing; Kriz, Andrea J.; Sharp, Phillip A.

    2015-01-01

    The CRISPR-Cas9 system, naturally a defense mechanism in prokaryotes, has been repurposed as an RNA-guided DNA targeting platform. It has been widely used for genome editing and transcriptome modulation, and has shown great promise in correcting mutations in human genetic diseases. Off-target effects are a critical issue for all of these applications. Here we review the current status on the target specificity of the CRISPR-Cas9 system. PMID:25722925

  17. The Structural Biology of CRISPR-Cas Systems

    PubMed Central

    Jiang, Fuguo; Doudna, Jennifer A.

    2015-01-01

    Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ~30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding. PMID:25723899

  18. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

    PubMed

    Barrangou, Rodolphe; Marraffini, Luciano A

    2014-04-24

    Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

  19. CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity

    PubMed Central

    Barrangou, Rodolphe; Marraffini, Luciano A.

    2014-01-01

    Summary Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing, and can be repurposed for numerous DNA targeting applications including transcriptional control. PMID:24766887

  20. An updated evolutionary classification of CRISPR-Cas systems.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Alkhnbashi, Omer S; Costa, Fabrizio; Shah, Shiraz A; Saunders, Sita J; Barrangou, Rodolphe; Brouns, Stan J J; Charpentier, Emmanuelle; Haft, Daniel H; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J M; Terns, Rebecca M; Terns, Michael P; White, Malcolm F; Yakunin, Alexander F; Garrett, Roger A; van der Oost, John; Backofen, Rolf; Koonin, Eugene V

    2015-11-01

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. PMID:26411297

  1. 46 CFR 95.05-1 - Fire detecting, manual alarm, and supervised patrol systems.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Fire detecting, manual alarm, and supervised patrol... AND MISCELLANEOUS VESSELS FIRE PROTECTION EQUIPMENT Fire Detecting and Extinguishing Equipment, Where Required § 95.05-1 Fire detecting, manual alarm, and supervised patrol systems. (a) Fire detecting,...

  2. 46 CFR 95.05-1 - Fire detecting, manual alarm, and supervised patrol systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Fire detecting, manual alarm, and supervised patrol... AND MISCELLANEOUS VESSELS FIRE PROTECTION EQUIPMENT Fire Detecting and Extinguishing Equipment, Where Required § 95.05-1 Fire detecting, manual alarm, and supervised patrol systems. (a) Fire detecting,...

  3. Emergency Vehicle Alarm System for Deaf Drivers by Using LEDs and Vibration Devices

    NASA Astrophysics Data System (ADS)

    Kuwahara, Noriaki; Morimoto, Kazunari; Kozuki, Kazumasa; Kawamura, Tomonori

    We are developing the emergency vehicle alarm system for deaf drivers by using LEDs and vibration devices. In order to design the alarm for deaf drivers, we have conducted basic experiment in order to evaluate perceptual characteristic on visibility of LED.

  4. MRDIS Standalone Central Alarm Station

    SciTech Connect

    2012-09-12

    The MRDIS Standalone Central Alarm Station(MRDIS-CAS} is a software system for receiving, storing, and reviewing radiation data collected by the Mobile Radiation Detection and Identification System (MRDIS}, a mobile radiation scanning system developed for use in foreign ports for the DOE Megaports Initiative. It is designed to run on one of the on board computers in the MRDIS cab. It will collect, store, and display data from the MRDIS without the need for wireless communications or centralized server technology. It is intended to be a lightweight replacement for a distributed Megaports communication system in ports where the necessary communications infrastructure does not exist for a full Megaports communications system.

  5. 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

  6. Development and Evaluation of New Coupling System for Lower Limb Prostheses with Acoustic Alarm System

    PubMed Central

    Eshraghi, Arezoo; Osman, Noor Azuan Abu; Gholizadeh, Hossein; Ahmadian, Jalil; Rahmati, Bizhan; Abas, Wan Abu Bakar Wan

    2013-01-01

    Individuals with lower limb amputation need a secure suspension system for their prosthetic devices. A new coupling system was developed that is capable of suspending the prosthesis. The system's safety is ensured through an acoustic alarm system. This article explains how the system works and provides an in vivo evaluation of the device with regard to pistoning during walking. The system was designed to be used with silicone liners and is based on the requirements of prosthetic suspension systems. Mechanical testing was performed using a universal testing machine. The pistoning during walking was measured using a motion analysis system. The new coupling device produced significantly less pistoning compared to a common suspension system (pin/lock). The safety alarm system would buzz if the suspension was going to fail. The new coupling system could securely suspend the prostheses in transtibial amputees and produced less vertical movement than the pin/lock system. PMID:23881340

  7. Applications of CRISPR-Cas systems in neuroscience

    PubMed Central

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome editing tools, and in particular those based on CRISPR-Cas systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in virtually any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research. PMID:26656253

  8. SILENE Benchmark Critical Experiments for Criticality Accident Alarm Systems

    SciTech Connect

    Miller, Thomas Martin; Reynolds, Kevin H.

    2011-01-01

    In October 2010 a series of benchmark experiments was conducted at the Commissariat a Energie Atomique et aux Energies Alternatives (CEA) Valduc SILENE [1] facility. These experiments were a joint effort between the US Department of Energy (DOE) and the French CEA. The purpose of these experiments was to create three benchmarks for the verification and validation of radiation transport codes and evaluated nuclear data used in the analysis of criticality accident alarm systems (CAASs). This presentation will discuss the geometric configuration of these experiments and the quantities that were measured and will present some preliminary comparisons between the measured data and calculations. This series consisted of three single-pulsed experiments with the SILENE reactor. During the first experiment the reactor was bare (unshielded), but during the second and third experiments it was shielded by lead and polyethylene, respectively. During each experiment several neutron activation foils and thermoluminescent dosimeters (TLDs) were placed around the reactor, and some of these detectors were themselves shielded from the reactor by high-density magnetite and barite concrete, standard concrete, and/or BoroBond. All the concrete was provided by CEA Saclay, and the BoroBond was provided by Y-12 National Security Complex. Figure 1 is a picture of the SILENE reactor cell configured for pulse 1. Also included in these experiments were measurements of the neutron and photon spectra with two BICRON BC-501A liquid scintillators. These two detectors were provided and operated by CEA Valduc. They were set up just outside the SILENE reactor cell with additional lead shielding to prevent the detectors from being saturated. The final detectors involved in the experiments were two different types of CAAS detectors. The Babcock International Group provided three CIDAS CAAS detectors, which measured photon dose and dose rate with a Geiger-Mueller tube. CIDAS detectors are currently in

  9. Alarm communication and display systems for high security department of energy facilities

    SciTech Connect

    Williams, J.S.

    1987-01-01

    An Alarm Communication and Display System collects alarm data, presents information to security operators, and enables the operators to enter commands affecting security operations; the ultimate goal of the system is to provide rapid assessment of alarms. This paper presents an overview of the architecture and operating principles used for alarm communication and display systems developed for application at several Department of Energy facilities. Although facilities have unique requirements and procedures, the architecture and operating principles of the ACDS presented in this paper have allowed site-specific implementations at several Department of Energy facilities. In addition, this technology has been transferred to other DOE facilities for adaptation to their requirements. Further efforts to enhance ACDS technology include the use of local area network technology to assist in peripheral switching, a distributed CCTV video switching system, and state-of-the-art hardware changes which improve system performance and effectiveness.

  10. Nuisance alarm suppression techniques for fibre-optic intrusion detection systems

    NASA Astrophysics Data System (ADS)

    Mahmoud, Seedahmed S.; Visagathilagar, Yuvaraja; Katsifolis, Jim

    2012-02-01

    The suppression of nuisance alarms without degrading sensitivity in fibre-optic intrusion detection systems is important for maintaining acceptable performance. Signal processing algorithms that maintain the POD and minimize nuisance alarms are crucial for achieving this. A level crossings algorithm is presented for suppressing torrential rain-induced nuisance alarms in a fibre-optic fence-based perimeter intrusion detection system. Results show that rain-induced nuisance alarms can be suppressed for rainfall rates in excess of 100 mm/hr, and intrusion events can be detected simultaneously during rain periods. The use of a level crossing based detection and novel classification algorithm is also presented demonstrating the suppression of nuisance events and discrimination of nuisance and intrusion events in a buried pipeline fibre-optic intrusion detection system. The sensor employed for both types of systems is a distributed bidirectional fibre-optic Mach Zehnder interferometer.

  11. 40 CFR 267.34 - When must personnel have access to communication equipment or an alarm system?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... communication equipment or an alarm system? 267.34 Section 267.34 Protection of Environment ENVIRONMENTAL... have access to communication equipment or an alarm system? (a) Whenever hazardous waste is being poured... to an internal alarm or emergency communication device, either directly or through visual or...

  12. Earthquake alarm system for the Maui-A offshore platform, New Zealand

    SciTech Connect

    Tyler, R.G.; Beck, J.L.

    1983-02-01

    Situated in the Tasman Sea, the Maui A offshore gas production platform has an earthquake alarm system that gives immediate warning when the seismic accelerations reach half the platform's design level. The system monitors only the response of the lower modes of the platform, as these make the major contribution to the stresses in the structure. In order to reduce the risk of false alarms, a radio link with similar detectors on shore confirms that an earthquake has occurred.

  13. SD-CAS: Spin Dynamics by Computer Algebra System.

    PubMed

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples. PMID:20843716

  14. Low Power Wireless Smoke Alarm System in Home Fires

    PubMed Central

    Luis, Juan Aponte; Galán, Juan Antonio Gómez; Espigado, Javier Alcina

    2015-01-01

    A novel sensing device for fire detection in domestic environments is presented. The fire detector uses a combination of several sensors that not only detect smoke, but discriminate between different types of smoke. This feature avoids false alarms and warns of different situations. Power consumption is optimized both in terms of hardware and software, providing a high degree of autonomy of almost five years. Data gathered from the device are transmitted through a wireless communication to a base station. The low cost and compact design provides wide application prospects. PMID:26307994

  15. Low Power Wireless Smoke Alarm System in Home Fires.

    PubMed

    Aponte Luis, Juan; Gómez Galán, Juan Antonio; Alcina Espigado, Javier

    2015-01-01

    A novel sensing device for fire detection in domestic environments is presented. The fire detector uses a combination of several sensors that not only detect smoke, but discriminate between different types of smoke. This feature avoids false alarms and warns of different situations. Power consumption is optimized both in terms of hardware and software, providing a high degree of autonomy of almost five years. Data gathered from the device are transmitted through a wireless communication to a base station. The low cost and compact design provides wide application prospects. PMID:26307994

  16. Mouse Genome Editing using CRISPR/Cas System

    PubMed Central

    Harms, Donald W; Quadros, Rolen M; Seruggia, Davide; Ohtsuka, Masato; Takahashi, Gou

    2015-01-01

    The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models. PMID:25271839

  17. A technical approach for determining the importance of information in computerized alarm systems

    SciTech Connect

    Fortney, D.S.; Lim, J.J.

    1994-06-10

    Computerized alarm and access control systems must be treated as special entities rather than as generic automated information systems. This distinction arises due to the real-time control and monitoring functions performed by these systems at classified facilities and the degree of centralization of a site`s safeguards system information in the associated databases. As an added requirement for these systems, DOE safeguards and security classification policy is to protect information whose dissemination has the potential for significantly increasing the probability of successful adversary action against the facility, or lowering adversary resources needed for a successful attack. Thus at issue is just how valuable would specific alarm system information be to an adversary with a higher order objective. We have developed and applied a technical approach for determining the importance of information contained in computerized alarm and access control systems. The methodology is based on vulnerability assessment rather than blanket classification rules. This method uses a system architecture diagram to guide the analysis and to develop adversary defeat methods for each node and link. These defeat methods are evaluated with respect to required adversary resources, technical difficulty, and detection capability. Then they are incorporated into site vulnerability assessments to determine the significance of alarm system information in the context of a facility attack. This methodology was successfully applied to the Argus alarm, access control, and assessment system developed at the Lawrence Livermore National Lab. Argus is software-driven, contains interrelated databases, shares host computers, and communicates with field processors and alarms through a common network. The evaluation results provided insights into the importance of alarm system information while the methodology itself provided a framework for addressing associated information protection issues.

  18. Targeted mutagenesis in chicken using CRISPR/Cas9 system.

    PubMed

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

  19. Targeted mutagenesis in chicken using CRISPR/Cas9 system

    PubMed Central

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

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

    PubMed Central

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

    2016-01-01

    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. PMID:26817415

  1. 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

    2016-01-01

    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. PMID:26817415

  2. Development of an on-line fuzzy expert system for integrated alarm processing in nuclear power plants

    SciTech Connect

    Choi, S.S.; Kang, K.S.; Kim, H.G.; Chang, S.H.

    1995-08-01

    An on-line fuzzy expert system, called alarm filtering and diagnostic system (AFDS), was developed to provide the operator with clean alarm pictures and system-wide failure information during abnormal states through alarm filtering and diagnosis. In addition, it carries out alarm prognosis to warn the operator of process abnormalities. Clean alarm pictures that have no information overlapping are generated from multiple activated alarms at the alarm filtering stage. The meta rules for dynamic filtering were established on the basis of the alarm relationship network. In the case of alarm diagnosis, the relations between alarms and abnormal states are represented by means of fuzzy relations, and the compositional inference rule of fuzzy logic is utilized to infer abnormal states from the fuzzy relations. The AFDS offers the operator related operating procedures as well as diagnostic results. At the stage of alarm prognosis, the future values of some important critical safety parameters are predicted by means of Levinson algorithm selected from the comparative experiments, and the global trends of these parameters are estimated using data smoothing and fuzzy membership. This information enables early failure detection and is also used to supplement diagnostic symptoms.

  3. Guide RNAs: A Glimpse at the Sequences that Drive CRISPR-Cas Systems.

    PubMed

    Briner, Alexandra E; Barrangou, Rodolphe

    2016-01-01

    CRISPR-Cas systems provide adaptive immunity in bacteria and archaea. Although there are two main classes of CRISPR-Cas systems defined by gene content, interfering RNA biogenesis, and effector proteins, Type II systems have recently been exploited on a broad scale to develop next-generation genetic engineering and genome-editing tools. Conveniently, Type II systems are streamlined and rely on a single protein, Cas9, and a guide RNA molecule, comprised of a CRISPR RNA (crRNA) and trans-acting CRISPR RNA (tracrRNA), to achieve effective and programmable nucleic acid targeting and cleavage. Currently, most commercially available Cas9-based genome-editing tools use the CRISPR-Cas system from Streptococcus pyogenes (SpyCas9), although many orthogonal Type II systems are available for diverse and multiplexable genome engineering applications. Here, we discuss the biological significance of Type II CRISPR-Cas elements, including the tracrRNA, crRNA, Cas9, and protospacer-adjacent motif (PAM), and look at the native function of these elements to understand how they can be engineered, enhanced, and optimized for genome editing applications. Additionally, we discuss the basis for orthogonal Cas9 and guide RNA systems that would allow researchers to concurrently use multiple Cas9-based systems for different purposes. Understanding the native function of endogenous Type II CRISPR-Cas systems can lead to new Cas9 tool development to expand the genetic manipulation toolbox. PMID:27371605

  4. An embedded telecommunication cable auto-locating system of guard against theft and alarm

    NASA Astrophysics Data System (ADS)

    Li, Yan; Li, Jun; Jin, Tian-Bo; Zhang, Li-yong

    2005-12-01

    This system adopted two-level structure that was composed of Cable Monitoring And Controlling Device (CMCD) and Cable Monitoring Center (CMC). CMC receives the alarm data via the MODEM and the telephone net. In this way, the functions of typing the fault point's map, alarming and the cable management can be picked up. CMCD takes the processor chip Atemega128 as the center of the system that adopted the alternating current and direct current on-line switching electricity-supply mode. Also, the system includes four groups of independent power, the relay and the optical isolation to separate the system from the monitoring cable and the telephone net. CMCD accomplishes the cable real-time monitoring, malfunctions auto-locating, telephone voice alarming, long-distance parameters modification, data up-loading, error verifying and the telecommunication room's environment monitoring and so on. The longest distance of monitoring cable is 20 km, and the precision is 1%.

  5. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    ERIC Educational Resources Information Center

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  6. Medical audible alarms: a review

    PubMed Central

    Edworthy, Judy

    2013-01-01

    Objectives This paper summarizes much of the research that is applicable to the design of auditory alarms in a medical context. It also summarizes research that demonstrates that false alarm rates are unacceptably high, meaning that the proper application of auditory alarm design principles are compromised. Target audience Designers, users, and manufacturers of medical information and monitoring systems that indicate when medical or other parameters are exceeded and that are indicated by an auditory signal or signals. Scope The emergence of alarms as a ‘hot topic’; an outline of the issues and design principles, including IEC 60601-1-8; the high incidence of false alarms and its impact on alarm design and alarm fatigue; approaches to reducing alarm fatigue; alarm philosophy explained; urgency in audible alarms; different classes of sound as alarms; heterogeneity in alarm set design; problems with IEC 60601-1-8 and ways of approaching this design problem. PMID:23100127

  7. CRISPR-Cas systems for genome editing, regulation and targeting

    PubMed Central

    Sander, Jeffry D.; Joung, J. Keith

    2014-01-01

    Targeted genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered regularly interspaced short palindromic repeat (CRISPR) technology, an important new platform for generating RNA-guided nucleases (RGNs), such as Cas9, with customizable specificities. RGN-mediated genome editing is facile, rapid and has enabled the efficient modification of endogenous genes in a wide variety of biomedically important cell types and novel organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the capabilities of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease. PMID:24584096

  8. 33 CFR 149.135 - What should be marked on the cargo transfer system alarm switch?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false What should be marked on the cargo transfer system alarm switch? 149.135 Section 149.135 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) DEEPWATER PORTS DEEPWATER PORTS: DESIGN, CONSTRUCTION, AND EQUIPMENT Pollution Prevention Equipment §...

  9. CSER 95-003: Exemption from Criticality Alarm System requirement for 232-Z building

    SciTech Connect

    Nirider, L.T.; Miller, E.M.

    1995-05-18

    This CSER establishes an exemption for 232-Z from the requirement for a Criticality Alarm System, because the formation of a critical configuration is not a credible event for any circumstance involving the cleaning out and removal of the Burning Hood and associated equipment.

  10. 46 CFR 154.1330 - Liquid level alarm system: Independent tank type C.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Liquid level alarm system: Independent tank type C. 154.1330 Section 154.1330 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Design, Construction and Equipment Instrumentation...

  11. 46 CFR 154.1325 - Liquid level alarm system: All cargo tanks.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Liquid level alarm system: All cargo tanks. 154.1325 Section 154.1325 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CERTAIN BULK DANGEROUS CARGOES SAFETY STANDARDS FOR SELF-PROPELLED VESSELS CARRYING BULK LIQUEFIED GASES Design, Construction and Equipment Instrumentation §...

  12. 33 CFR 149.414 - What are the requirements for a fire detection and alarm system?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...: (1) Either complies with 46 CFR 108.405 or (2) Is designed and installed in compliance with a national consensus standard, as that term is defined in 29 CFR 1910.2, for fire detection and fire alarm... is defined in 29 CFR 1910.7, for such systems or hardware. (b) Sleeping quarters must be fitted...

  13. 33 CFR 149.414 - What are the requirements for a fire detection and alarm system?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...: (1) Either complies with 46 CFR 108.405 or (2) Is designed and installed in compliance with a national consensus standard, as that term is defined in 29 CFR 1910.2, for fire detection and fire alarm... is defined in 29 CFR 1910.7, for such systems or hardware. (b) Sleeping quarters must be fitted...

  14. 33 CFR 149.414 - What are the requirements for a fire detection and alarm system?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...: (1) Either complies with 46 CFR 108.405 or (2) Is designed and installed in compliance with a national consensus standard, as that term is defined in 29 CFR 1910.2, for fire detection and fire alarm... is defined in 29 CFR 1910.7, for such systems or hardware. (b) Sleeping quarters must be fitted...

  15. 33 CFR 149.414 - What are the requirements for a fire detection and alarm system?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...: (1) Either complies with 46 CFR 108.405 or (2) Is designed and installed in compliance with a national consensus standard, as that term is defined in 29 CFR 1910.2, for fire detection and fire alarm... is defined in 29 CFR 1910.7, for such systems or hardware. (b) Sleeping quarters must be fitted...

  16. 33 CFR 149.414 - What are the requirements for a fire detection and alarm system?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...: (1) Either complies with 46 CFR 108.405 or (2) Is designed and installed in compliance with a national consensus standard, as that term is defined in 29 CFR 1910.2, for fire detection and fire alarm... is defined in 29 CFR 1910.7, for such systems or hardware. (b) Sleeping quarters must be fitted...

  17. Targeted mutagenesis using CRISPR/Cas system in medaka

    PubMed Central

    Ansai, Satoshi; Kinoshita, Masato

    2014-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka. PMID:24728957

  18. 46 CFR 58.25-25 - Indicating and alarm systems.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY... the pilothouse upon— (1) Failure of the electric power to the control system of any steering gear; (2... oil reservoir of a hydraulic, power-operated steering-gear system. (e) An audible and a visible...

  19. 46 CFR 28.240 - General alarm system.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Than 16 Individuals On Board, or for Fish Tender Vessels Engaged in the Aleutian Trade § 28.240 General... thereafter. (f) A public address system or other means of alerting all individuals on board may be used...

  20. 46 CFR 28.240 - General alarm system.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Than 16 Individuals On Board, or for Fish Tender Vessels Engaged in the Aleutian Trade § 28.240 General... thereafter. (f) A public address system or other means of alerting all individuals on board may be used...

  1. 46 CFR 28.240 - General alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Than 16 Individuals On Board, or for Fish Tender Vessels Engaged in the Aleutian Trade § 28.240 General... thereafter. (f) A public address system or other means of alerting all individuals on board may be used...

  2. 46 CFR 28.240 - General alarm system.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Than 16 Individuals On Board, or for Fish Tender Vessels Engaged in the Aleutian Trade § 28.240 General... thereafter. (f) A public address system or other means of alerting all individuals on board may be used...

  3. 46 CFR 28.240 - General alarm system.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Than 16 Individuals On Board, or for Fish Tender Vessels Engaged in the Aleutian Trade § 28.240 General... thereafter. (f) A public address system or other means of alerting all individuals on board may be used...

  4. 78 FR 2683 - Carriage Standards for Bridge Navigational Watch Alarm Systems (BNWAS) Aboard U.S. Flagged Vessels

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-14

    ... SECURITY Coast Guard Carriage Standards for Bridge Navigational Watch Alarm Systems (BNWAS) Aboard U.S... Guard announces the implementation date of carriage standards for Bridge Navigational Watch Alarm Systems (BNWAS), in accordance with the Articles of the International Convention for the Safety of Life...

  5. Neural Network Target Identification System for False Alarm Reduction

    NASA Technical Reports Server (NTRS)

    Ye, David; Edens, Weston; Lu, Thomas T.; Chao, Tien-Hsin

    2009-01-01

    A multi-stage automated target recognition (ATR) system has been designed to perform computer vision tasks with adequate proficiency in mimicking human vision. The system is able to detect, identify, and track targets of interest. Potential regions of interest (ROIs) are first identified by the detection stage using an Optimum Trade-off Maximum Average Correlation Height (OT-MACH) filter combined with a wavelet transform. False positives are then eliminated by the verification stage using feature extraction methods in conjunction with neural networks. Feature extraction transforms the ROIs using filtering and binning algorithms to create feature vectors. A feed forward back propagation neural network (NN) is then trained to classify each feature vector and remove false positives. This paper discusses the test of the system performance and parameter optimizations process which adapts the system to various targets and datasets. The test results show that the system was successful in substantially reducing the false positive rate when tested on a sonar image dataset.

  6. Applying an integrated neuro-expert system model in a real-time alarm processing system

    NASA Astrophysics Data System (ADS)

    Khosla, Rajiv; Dillon, Tharam S.

    1993-03-01

    In this paper we propose an integrated model which is derived from the combination of a generic neuro-expert system model, an object model, and unix operating system process (UOSP) model. This integrated model reflects the strengths of both artificial neural nets (ANNs) and expert systems (ESs). A formalism of ES object, ANN object, UOSP object, and problem domain object is used for developing a set of generic data structures and methods. These generic data structures and methods help us to build heterogeneous ES-ANN objects with uniform communication interface. The integrated model is applied in a real-time alarm processing system for a non-trivial terminal power station. It is shown how features like hierarchical/distributed ES/ANN objects, inter process communication, and fast concurrent execution help to cope with real-time system constraints like, continuity, data variability, and fast response time.

  7. MRDIS Standalone Central Alarm Station

    2012-09-12

    The MRDIS Standalone Central Alarm Station(MRDIS-CAS} is a software system for receiving, storing, and reviewing radiation data collected by the Mobile Radiation Detection and Identification System (MRDIS}, a mobile radiation scanning system developed for use in foreign ports for the DOE Megaports Initiative. It is designed to run on one of the on board computers in the MRDIS cab. It will collect, store, and display data from the MRDIS without the need for wireless communicationsmore » or centralized server technology. It is intended to be a lightweight replacement for a distributed Megaports communication system in ports where the necessary communications infrastructure does not exist for a full Megaports communications system.« less

  8. Silent emergency alarm system for schools and the like

    NASA Technical Reports Server (NTRS)

    Read, W. S.; Roberts, V. W. (Inventor)

    1973-01-01

    An emergency alert system is described. In a school each classroom (or other area) is instrumented with a hidden microphone and receiver tuned to a non-audible frequency. The receivers' outputs are connected to a central display unit in the school's administrative office. Each instructor is provided with a small concealable transmitter which, when hand activated by the instructor upon the occurrance of any emergency, generates a non-audible signal at the receiver's tuned frequency.

  9. Smart container UWB sensor system for situational awareness of intrusion alarms

    DOEpatents

    Romero, Carlos E.; Haugen, Peter C.; Zumstein, James M.; Leach, Jr., Richard R.; Vigars, Mark L.

    2013-06-11

    An in-container monitoring sensor system is based on an UWB radar intrusion detector positioned in a container and having a range gate set to the farthest wall of the container from the detector. Multipath reflections within the container make every point on or in the container appear to be at the range gate, allowing intrusion detection anywhere in the container. The system also includes other sensors to provide false alarm discrimination, and may include other sensors to monitor other parameters, e.g. radiation. The sensor system also includes a control subsystem for controlling system operation. Communications and information extraction capability may also be included. A method of detecting intrusion into a container uses UWB radar, and may also include false alarm discrimination. A secure container has an UWB based monitoring system

  10. Multiple-Parameter, Low-False-Alarm Fire-Detection Systems

    NASA Technical Reports Server (NTRS)

    Hunter, Gary W.; Greensburg, Paul; McKnight, Robert; Xu, Jennifer C.; Liu, C. C.; Dutta, Prabir; Makel, Darby; Blake, D.; Sue-Antillio, Jill

    2007-01-01

    Fire-detection systems incorporating multiple sensors that measure multiple parameters are being developed for use in storage depots, cargo bays of ships and aircraft, and other locations not amenable to frequent, direct visual inspection. These systems are intended to improve upon conventional smoke detectors, now used in such locations, that reliably detect fires but also frequently generate false alarms: for example, conventional smoke detectors based on the blockage of light by smoke particles are also affected by dust particles and water droplets and, thus, are often susceptible to false alarms. In contrast, by utilizing multiple parameters associated with fires, i.e. not only obscuration by smoke particles but also concentrations of multiple chemical species that are commonly generated in combustion, false alarms can be significantly decreased while still detecting fires as reliably as older smoke-detector systems do. The present development includes fabrication of sensors that have, variously, micrometer- or nanometer-sized features so that such multiple sensors can be integrated into arrays that have sizes, weights, and power demands smaller than those of older macroscopic sensors. The sensors include resistors, electrochemical cells, and Schottky diodes that exhibit different sensitivities to the various airborne chemicals of interest. In a system of this type, the sensor readings are digitized and processed by advanced signal-processing hardware and software to extract such chemical indications of fires as abnormally high concentrations of CO and CO2, possibly in combination with H2 and/or hydrocarbons. The system also includes a microelectromechanical systems (MEMS)-based particle detector and classifier device to increase the reliability of measurements of chemical species and particulates. In parallel research, software for modeling the evolution of a fire within an aircraft cargo bay has been developed. The model implemented in the software can

  11. The Performance of Earthworm Based Earthquake Alarm Reporting System in Taiwan

    NASA Astrophysics Data System (ADS)

    Chen, Ta-Yi; Hsiao, Nai-Chi; Wu, Yih-Min

    2016-04-01

    The Central Weather Bureau of Taiwan has operated an earthquake early warning (EEW) system and issued warnings to schools and government agencies since 2014. Because the real-time seismic data streams are integrated by the Earthworm software, some EEW modules were created under the Earthworm platform. The system is named Earthworm Based Earthquake Alarm Reporting (eBEAR) system, which is currently operating. The eBEAR system consists of new Earthworm modules for managing P-wave phase picking, trigger associations, hypocenter locations, magnitude estimations, and alert filtering prior to broadcasting. Here, we outline the methodology and performance of the eBEAR system. The online performance of the eBEAR system indicated that the average reporting times afforded by the system are approximately 15 and 26 s for inland and offshore earthquakes, respectively. Comparing to the earthquake catalog, the difference of the epicenters are less than 10 km for inland earthquakes; the difference of the magnitude are about 0.3. No false alarms generated by the system, but there were three false alarms issued by human. Due to the wrong operations, the EEW information created by off-line test were sent. However, we have learned from it and improved the standard operation procedure in the EEW system.

  12. The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system

    PubMed Central

    Semenova, Ekaterina; Kuznedelov, Konstantin; Datsenko, Kirill A.; Boudry, Pierre M.; Savitskaya, Ekaterina E.; Medvedeva, Sofia; Beloglazova, Natalia; Logacheva, Maria; Yakunin, Alexander F.; Severinov, Konstantin

    2015-01-01

    CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e. The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription. PMID:26013814

  13. Early warning, warning or alarm systems for natural hazards? A generic classification.

    NASA Astrophysics Data System (ADS)

    Sättele, Martina; Bründl, Michael; Straub, Daniel

    2013-04-01

    Early warning, warning and alarm systems have gained popularity in recent years as cost-efficient measures for dangerous natural hazard processes such as floods, storms, rock and snow avalanches, debris flows, rock and ice falls, landslides, flash floods, glacier lake outburst floods, forest fires and even earthquakes. These systems can generate information before an event causes loss of property and life. In this way, they mainly mitigate the overall risk by reducing the presence probability of endangered objects. These systems are typically prototypes tailored to specific project needs. Despite their importance there is no recognised system classification. This contribution classifies warning and alarm systems into three classes: i) threshold systems, ii) expert systems and iii) model-based expert systems. The result is a generic classification, which takes the characteristics of the natural hazard process itself and the related monitoring possibilities into account. The choice of the monitoring parameters directly determines the system's lead time. The classification of 52 active systems moreover revealed typical system characteristics for each system class. i) Threshold systems monitor dynamic process parameters of ongoing events (e.g. water level of a debris flow) and incorporate minor lead times. They have a local geographical coverage and a predefined threshold determines if an alarm is automatically activated to warn endangered objects, authorities and system operators. ii) Expert systems monitor direct changes in the variable disposition (e.g crack opening before a rock avalanche) or trigger events (e.g. heavy rain) at a local scale before the main event starts and thus offer extended lead times. The final alarm decision incorporates human, model and organisational related factors. iii) Model-based expert systems monitor indirect changes in the variable disposition (e.g. snow temperature, height or solar radiation that influence the occurrence probability

  14. Functional relationship-based alarm processing

    DOEpatents

    Corsberg, D.R.

    1987-04-13

    A functional relationship-based alarm processing system and method analyzes each alarm as it is activated and determines its relative importance with other currently activated alarms and signals in accordance with the relationships that the newly activated alarm has with other currently activated alarms. Once the initial level of importance of the alarm has been determined, that alarm is again evaluated if another related alarm is activated. Thus, each alarm's importance is continuously updated as the state of the process changes during a scenario. Four hierarchical relationships are defined by this alarm filtering methodology: (1) level precursor (usually occurs when there are two alarm settings on the same parameter); (2) direct precursor (based on causal factors between two alarms); (3) required action (system response or action expected within a specified time following activation of an alarm or combination of alarms and process signals); and (4) blocking condition (alarms that are normally expected and are not considered important). 11 figs.

  15. Design of DroDeASys (Drowsy Detection and Alarming System)

    NASA Astrophysics Data System (ADS)

    Juvale, Hrishikesh B.; Mahajan, Anant S.; Bhagwat, Ashwin A.; Badiger, Vishal T.; Bhutkar, Ganesh D.; Dhabe, Priyadarshan S.; Dhore, Manikrao L.

    The paper discusses the Drowsy Detection & Alarming System that has been developed, using a non-intrusive approach. The system is basically developed to detect drivers dozing at the wheel at night time driving. The system uses a small infra-red night vision camera that points directly towards the driver`s face and monitors the driver`s eyes in order to detect fatigue. In such a case when fatigue is detected, a warning signal is issued to alert the driver. This paper discusses the algorithms that have been used to detect drowsiness. The decision whether the driver is dozing or not is taken depending on whether the eyes are open for a specific number of frames. If the eyes are found to be closed for a certain number of consecutive frames then the driver is alerted with an alarm.

  16. Visual display and alarm system for wind tunnel static and dynamic loads

    NASA Technical Reports Server (NTRS)

    Hanly, Richard D.; Fogarty, James T.

    1987-01-01

    A wind tunnel balance monitor and alarm system developed at NASA Ames Research Center will produce several beneficial results. The costs of wind tunnel delays because of inadvertent balance damage and the costs of balance repair or replacement can be greatly reduced or eliminated with better real-time information on the balance static and dynamic loading. The wind tunnel itself will have enhanced utility with the elimination of overly cautious limits on test conditions. The microprocessor-based system features automatic scaling and 16 multicolored LED bargraphs to indicate both static and dynamic components of the signals from eight individual channels. Five individually programmable alarm levels are available with relay closures for internal or external visual and audible warning devices and other functions such as automatic activation of external recording devices, model positioning mechanism, or tunnel shutdown.

  17. Visual display and alarm system for wind tunnel static and dynamic loads

    NASA Technical Reports Server (NTRS)

    Hanly, Richard D.; Fogarty, James T.

    1987-01-01

    A wind tunnel balance monitor and alarm system developed at NASA Ames Research Center will produce several beneficial results. The costs of wind tunnel delays because of inadvertent balance damage and the costs of balance repair or replacement can be greatly reduced or eliminated with better real-time information on the balance static and dynamic loading. The wind tunnel itself will have enhanced utility with the elimination of overly cautious limits on test conditions. The microprocessor-based system features automatic scaling and 16 multicolored LED bargraphs to indicate both static and dynamic components of the signals from eight individual channels. Five individually programmable alarm levels are available with relay closures for internal or external visual and audible warning devices and other functions such as automatic activation of external recording devices, model positioning mechanisms, or tunnel shutdown.

  18. 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

  19. Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR-Cas I-F systems.

    PubMed

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

    2016-01-01

    Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat-intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR-Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer. PMID:27573106

  20. Development of a criticality alarm system neutron detector: Final project report

    SciTech Connect

    O'Dell, A.A.

    1989-05-01

    The primary objective of this project was to develop a prototype neutron detector for use in criticality alarm systems (CASs) at US Department of Energy (DOE) and contractor facilities wherever significant amounts of fissile material are processed or stored. Constraints placed on the design of the detector were that the overall size of the detector was to be as small as practical, the input voltage requirements were to be no more than 24 V, and that the gamma sensitivity would be as low as possible. Also, the detector should give dosimetric neutron response, and should have sufficient temporal capabilities to measure the entire range from fast (>1 ms) to slow (seconds to minutes) excursions, and sufficient dynamic range to measure from background to over 100 times background levels to insure proper activation of the Immediate Evacuation Alarm (IEA). Finally, the detector should insure rapid (<1 s) activation of the IEA in the event of a criticality excursion. 24 figs., 11 tabs.

  1. Video methods for evaluating physiologic monitor alarms and alarm responses.

    PubMed

    Bonafide, Christopher P; Zander, Miriam; Graham, Christian Sarkis; Weirich Paine, Christine M; Rock, Whitney; Rich, Andrew; Roberts, Kathryn E; Fortino, Margaret; Nadkarni, Vinay M; Lin, Richard; Keren, Ron

    2014-01-01

    False physiologic monitor alarms are extremely common in the hospital environment. High false alarm rates have the potential to lead to alarm fatigue, leading nurses to delay their responses to alarms, ignore alarms, or disable them entirely. Recent evidence from the U.S. Food and Drug Administration (FDA) and The Joint Commission has demonstrated a link between alarm fatigue and patient deaths. Yet, very little scientific effort has focused on the rigorous quantitative measurement of alarms and responses in the hospital setting. We developed a system using multiple temporarily mounted, minimally obtrusive video cameras in hospitalized patients' rooms to characterize physiologic monitor alarms and nurse responses as a proxy for alarm fatigue. This allowed us to efficiently categorize each alarm's cause, technical validity, actionable characteristics, and determine the nurse's response time. We describe and illustrate the methods we used to acquire the video, synchronize and process the video, manage the large digital files, integrate the video with data from the physiologic monitor alarm network, archive the video to secure servers, and perform expert review and annotation using alarm "bookmarks." We discuss the technical and logistical challenges we encountered, including the root causes of hardware failures as well as issues with consent, confidentiality, protection of the video from litigation, and Hawthorne-like effects. The description of this video method may be useful to multidisciplinary teams interested in evaluating physiologic monitor alarms and alarm responses to better characterize alarm fatigue and other patient safety issues in clinical settings. PMID:24847936

  2. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system

    PubMed Central

    Gleditzsch, Daniel; Müller-Esparza, Hanna; Pausch, Patrick; Sharma, Kundan; Dwarakanath, Srivatsa; Urlaub, Henning; Bange, Gert; Randau, Lennart

    2016-01-01

    Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli. Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity. PMID:27216815

  3. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system.

    PubMed

    Gleditzsch, Daniel; Müller-Esparza, Hanna; Pausch, Patrick; Sharma, Kundan; Dwarakanath, Srivatsa; Urlaub, Henning; Bange, Gert; Randau, Lennart

    2016-07-01

    Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity. PMID:27216815

  4. Development of an intein-mediated split–Cas9 system for gene therapy

    PubMed Central

    Truong, Dong-Jiunn Jeffery; Kühner, Karin; Kühn, Ralf; Werfel, Stanislas; Engelhardt, Stefan; Wurst, Wolfgang; Ortiz, Oskar

    2015-01-01

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV. PMID:26082496

  5. Development of an intein-mediated split-Cas9 system for gene therapy.

    PubMed

    Truong, Dong-Jiunn Jeffery; Kühner, Karin; Kühn, Ralf; Werfel, Stanislas; Engelhardt, Stefan; Wurst, Wolfgang; Ortiz, Oskar

    2015-07-27

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split-Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split-Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV. PMID:26082496

  6. Occurrence and Diversity of CRISPR-Cas Systems in the Genus Bifidobacterium

    PubMed Central

    Briner, Alexandra E.; Lugli, Gabriele Andrea; Milani, Christian; Duranti, Sabrina; Turroni, Francesca; Gueimonde, Miguel; Margolles, Abelardo; van Sinderen, Douwe; Ventura, Marco; Barrangou, Rodolphe

    2015-01-01

    CRISPR-Cas systems constitute adaptive immune systems for antiviral defense in bacteria. We investigated the occurrence and diversity of CRISPR-Cas systems in 48 Bifidobacterium genomes to gain insights into the diversity and co-evolution of CRISPR-Cas systems within the genus and investigate CRISPR spacer content. We identified the elements necessary for the successful targeting and inference of foreign DNA in select Type II CRISPR-Cas systems, including the tracrRNA and target PAM sequence. Bifidobacterium species have a very high frequency of CRISPR-Cas occurrence (77%, 37 of 48). We found that many Bifidobacterium species have unusually large and diverse CRISPR-Cas systems that contain spacer sequences showing homology to foreign genetic elements like prophages. A large number of CRISPR spacers in bifidobacteria show perfect homology to prophage sequences harbored in the chromosomes of other species of Bifidobacterium, including some spacers that self-target the chromosome. A correlation was observed between strains that lacked CRISPR-Cas systems and the number of times prophages in that chromosome were targeted by other CRISPR spacers. The presence of prophage-targeting CRISPR spacers and prophage content may shed light on evolutionary processes and strain divergence. Finally, elements of Type II CRISPR-Cas systems, including the tracrRNA and crRNAs, set the stage for the development of genome editing and genetic engineering tools. PMID:26230606

  7. Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

    PubMed Central

    van Belkum, Alex; Soriaga, Leah B.; LaFave, Matthew C.; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E. Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C.; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S.; Richardson, Toby H.; Peterson, Todd C.; Hubby, Bolyn

    2015-01-01

    ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. PMID:26604259

  8. 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. PMID:23439366

  9. CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.

    PubMed

    Jiang, Wenyan; Marraffini, Luciano A

    2015-01-01

    Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields. PMID:26209264

  10. Behavioral characterization of the alarm reaction and anxiolytic-like effect of acute treatment with fluoxetine in piauçu fish.

    PubMed

    Barbosa Júnior, Augusto; Alves, Fabiana Luca; Pereira, Aparecida de Sousa Fim; Ide, Liliam Midori; Hoffmann, Anette

    2012-02-01

    In Ostariophysan fish, the detection of the alarm substance liberated into the water as a consequence of an attack by a predator elicits an alarm reaction or anti-predatory behavior. In this study, experiments were performed to: (i) describe and quantitatively characterize the behavioral and ventilatory responses in piauçu fish (Leporinus macrocephalus), individually and as part of a school, to conspecific alarm substance (CAS) and; (ii) test the effect of acute fluoxetine treatment on alarm reaction. Histological analysis revealed the presence of club cells in the intermediate and superficial layers of the epidermis. The predominant behavioral response to CAS was freezing for fish held individually, characterized by the cessation of the swimming activity as the animal settles to a bottom corner of the aquarium. Fish exposed to CAS showed decrease in the mean ventilatory frequency (approximately 13%) relative to control. In schools, CAS elicited a biphasic response that was characterized by erratic movements followed by increased school cohesion and immobility, reflected as an increased school cohesion (65.5% vs. -5.8% for controls) and in the number of animals near the bottom of the aquarium (42.0% vs. 6.5% for controls). Animals treated with single i.p. injections of fluoxetine (10 μg/g b.w.) did not exhibit alarm behavior following CAS stimulation. These results show that an alarm pheromone system is present in piauçu fish, evidenced by the presence of epidermal club cells and an alarm reaction induced by CAS and consequently of a chemosensory system to transmit the appropriate information to neural structures responsible for initiating anti-predator behavioral responses. In addition, fluoxetine treatment caused an anxiolytic-like effect following CAS exposure. Thus, the alarm reaction in piauçu can be a useful model for neuroethological and pharmacological studies of anxiety-related states. PMID:22037203

  11. CRISPR-Cas systems: new players in gene regulation and bacterial physiology

    PubMed Central

    Sampson, Timothy R.; Weiss, David S.

    2014-01-01

    CRISPR-Cas systems are bacterial defenses against foreign nucleic acids derived from bacteriophages, plasmids or other sources. These systems are targeted in an RNA-dependent, sequence-specific manner, and are also adaptive, providing protection against previously encountered foreign elements. In addition to their canonical function in defense against foreign nucleic acid, their roles in various aspects of bacterial physiology are now being uncovered. We recently revealed a role for a Cas9-based Type II CRISPR-Cas system in the control of endogenous gene expression, a novel form of prokaryotic gene regulation. Cas9 functions in association with two small RNAs to target and alter the stability of an endogenous transcript encoding a bacterial lipoprotein (BLP). Since BLPs are recognized by the host innate immune protein Toll-like Receptor 2 (TLR2), CRISPR-Cas-mediated repression of BLP expression facilitates evasion of TLR2 by the intracellular bacterial pathogen Francisella novicida, and is essential for its virulence. Here we describe the Cas9 regulatory system in detail, as well as data on its role in controlling virulence traits of Neisseria meningitidis and Campylobacter jejuni. We also discuss potential roles of CRISPR-Cas systems in the response to envelope stress and other aspects of bacterial physiology. Since ~45% of bacteria and ~83% of Archaea encode these machineries, the newly appreciated regulatory functions of CRISPR-Cas systems are likely to play broad roles in controlling the pathogenesis and physiology of diverse prokaryotes. PMID:24772391

  12. From Calculus to Dynamical Systems through DGS and CAS

    ERIC Educational Resources Information Center

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  13. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems.

    PubMed

    Mohanraju, Prarthana; Makarova, Kira S; Zetsche, Bernd; Zhang, Feng; Koonin, Eugene V; van der Oost, John

    2016-08-01

    Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications. PMID:27493190

  14. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

    PubMed

    Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

    2015-09-01

    Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria. PMID:26297561

  15. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  16. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  17. Degeneration of a CRISPR/Cas system and its regulatory target during the evolution of a pathogen

    PubMed Central

    Sampson, Timothy R; Weiss, David S

    2013-01-01

    CRISPR/Cas systems are bacterial RNA-guided endonuclease machineries that target foreign nucleic acids. Recently, we demonstrated that the Cas protein Cas9 controls gene expression and virulence in Francisella novicida by altering the stability of the mRNA for an immunostimulatory bacterial lipoprotein (BLP). Genomic analyses, however, revealed that Francisella species with increased virulence harbor degenerated CRISPR/Cas systems. We hypothesize that CRISPR/Cas degeneration removed a barrier against genome alterations, which resulted in enhanced virulence. Importantly, the BLP locus was also lost; likely a necessary adaptation in the absence of Cas9-mediated repression. CRISPR/Cas systems likely play regulatory roles in numerous bacteria, and these data suggest additional genomic changes may be required to maintain fitness after CRISPR/Cas loss in such bacteria, having important evolutionary implications. PMID:24100224

  18. Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

    PubMed

    Redding, Sy; Sternberg, Samuel H; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K; Wiedenheft, Blake; Doudna, Jennifer A; Greene, Eric C

    2015-11-01

    CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA. PMID:26522594

  19. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion

    PubMed Central

    Sampson, Timothy R.; Napier, Brooke A.; Schroeder, Max R.; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K.; Llewellyn, Anna C.; Jones, Crystal L.; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P.; Weiss, David S.

    2014-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals. PMID:25024199

  20. Monitor alarm fatigue: an integrative review.

    PubMed

    Cvach, Maria

    2012-01-01

    Alarm fatigue is a national problem and the number one medical device technology hazard in 2012. The problem of alarm desensitization is multifaceted and related to a high false alarm rate, poor positive predictive value, lack of alarm standardization, and the number of alarming medical devices in hospitals today. This integrative review synthesizes research and non-research findings published between 1/1/2000 and 10/1/2011 using The Johns Hopkins Nursing Evidence-Based Practice model. Seventy-two articles were included. Research evidence was organized into five main themes: excessive alarms and effects on staff; nurse's response to alarms; alarm sounds and audibility; technology to reduce false alarms; and alarm notification systems. Non-research evidence was divided into two main themes: strategies to reduce alarm desensitization, and alarm priority and notification systems. Evidence-based practice recommendations and gaps in research are summarized. PMID:22839984

  1. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level.

    PubMed

    Lee, Seung Hwan; Bae, Sangsu

    2016-04-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207]. PMID:26923305

  2. Structural and dynamic views of the CRISPR-Cas system at the single-molecule level

    PubMed Central

    Lee, Seung Hwan; Bae, Sangsu

    2016-01-01

    The CRISPR-Cas system has emerged as a fascinating and important genome editing tool. It is now widely used in biology, biotechnology, and biomedical research in both academic and industrial settings. To improve the specificity and efficiency of Cas nucleases and to extend the applications of these systems for other areas of research, an understanding of their precise working mechanisms is crucial. In this review, we summarize current studies on the molecular structures and dynamic functions of type I and type II Cas nucleases, with a focus on target DNA searching and cleavage processes as revealed by single-molecule observations. [BMB Reports 2016; 49(4): 201-207] PMID:26923305

  3. An attributable cost model for a telecare system using advanced community alarms.

    PubMed

    Brownsell, S J; Bradley, D A; Bragg, R; Catling, P; Carlier, J

    2001-01-01

    We have developed an attributable cost model for a city-based telecare scheme involving 11,618 community alarm users. The equipment was assumed to cost 500 Pounds-1000 Pounds per installation, compared with 175 Pounds for the current system. Because of the significant additional capital cost of the proposed system, it would be necessary to borrow to finance it. For example, if the home equipment cost 500 Pounds per unit, an additional 2.2 million Pounds would be required. Nonetheless, it would be possible to achieve a return on the investment after 10 years. The principal savings would arise from reduced hospital bed costs and reduced residential care. The model suggests that the financial benefits of the proposed system would occur in the ratio of 4% to the local authority housing department, 43% to the National Health Service and 53% to the residential care provider. PMID:11331043

  4. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    PubMed

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA. PMID:21343909

  5. Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in Subtype I-C/Dvulg CRISPR-Cas system

    PubMed Central

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong

    2012-01-01

    SUMMARY 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 B. 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, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-subunit interference complex similar to E. 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. PMID:22841292

  6. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    SciTech Connect

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong

    2012-10-10

    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, 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.

  7. In Vivo Protein Interactions and Complex Formation in the Pectobacterium atrosepticum Subtype I-F CRISPR/Cas System

    PubMed Central

    Richter, Corinna; Gristwood, Tamzin; Clulow, James S.; Fineran, Peter C.

    2012-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism. PMID:23226499

  8. [Application Progress of CRISPR/Cas9 System for Gene Editing in Tumor Research].

    PubMed

    Liu, Chao; Li, Zhiwei; Zhang, Yanqiao

    2015-09-20

    TCRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated nuclease 9) gene editing system is a new type of gene editing technology developed based on the immune mechanism of archaea resisting the invasion of exogenous nucleic acid. Compared with traditional gene editing system, CRISPR/Cas9 system is more efficient, easier operating, and less cytotoxic. Currently, CRISPR/Cas9 gene editing technology has been applied to many aspects of cancer research, including research on cancer genes, constructing animal tumor models, screening tumor resistance-associated and phenotypic-related genes and cancer gene therapy. In this review, the application of the CRISPR/Cas9 system in tumor research were introduced. PMID:26383982

  9. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome.

    PubMed

    Müller, Maximilian; Lee, Ciaran M; Gasiunas, Giedrius; Davis, Timothy H; Cradick, Thomas J; Siksnys, Virginijus; Bao, Gang; Cathomen, Toni; Mussolino, Claudio

    2016-03-01

    RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications. PMID:26658966

  10. Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles.

    PubMed

    Chellapandi, P; Ranjani, J

    2015-09-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology. PMID:26279704

  11. Three-Alarm System: Revisited to treat Thumb-sucking Habit

    PubMed Central

    Shetty, Manoj; Shetty, N Shridhar; Deoghare, Anushka

    2015-01-01

    ABSTRACT Thumb and digit-sucking habits or non-nutritive sucking are considered to be the most prevalent among oral habits. Most children stop thumb sucking on their own. If the habit continues beyond 3 to 4 years of age, it not only affects the dental occlusion, but the shape of the thumb/digit may be altered as well. This article presents the management of thumb sucking by modified RURS, elbow guard incorporated with revised ‘three-alarm’ system. How to cite this article: Shetty RM, Shetty M, Shetty NS, Deoghare A. Three-Alarm System: Revisited to treat Thumb-sucking Habit. Int J Clin Pediatr Dent 2015;8(1):82-86. PMID:26124588

  12. Co-Alignment System (CAS) study. Report on task 1-3. [Solar Extreme Ultraviolet Telescope and Spectrometer pointing system

    NASA Technical Reports Server (NTRS)

    Anderson, N. T.

    1980-01-01

    The design of a suitable coalignment system (CAS) for the Solar Extreme Ultraviolet Telescope and Spectrometer (SEUTS) is presented. The CAS provides offset adjustment capabilities to SEUTS which will be mounted on a single large pointing system with other devices. The suitability of existing designs is determined and modifications are suggested.

  13. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.

    PubMed

    Pawluk, April; Staals, Raymond H J; Taylor, Corinda; Watson, Bridget N J; Saha, Senjuti; Fineran, Peter C; Maxwell, Karen L; Davidson, Alan R

    2016-01-01

    CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems. PMID:27573108

  14. Establishing a CRISPR-Cas-like immune system conferring DNA virus resistance in plants.

    PubMed

    Ji, Xiang; Zhang, Huawei; Zhang, Yi; Wang, Yanpeng; Gao, Caixia

    2015-01-01

    CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR-associated proteins) is an adaptive immune system in many archaea and bacteria that cleaves foreign DNA on the basis of sequence complementarity. Here, using the geminivirus, beet severe curly top virus (BSCTV), transient assays performed in Nicotiana benthamiana demonstrate that the sgRNA-Cas9 constructs inhibit virus accumulation and introduce mutations at the target sequences. Further, transgenic Arabidopsis and N. benthamiana plants overexpressing sgRNA-Cas9 are highly resistant to virus infection. PMID:27251395

  15. Design and validation of an intelligent patient monitoring and alarm system based on a fuzzy logic process model.

    PubMed

    Becker, K; Thull, B; Käsmacher-Leidinger, H; Stemmer, J; Rau, G; Kalff, G; Zimmermann, H J

    1997-09-01

    The process of patient care performed by an anaesthesiologist during high invasive surgery requires fundamental knowledge of the physiologic processes and a long standing experience in patient management to cope with the inter-individual variability of the patients. Biomedical engineering research improves the patient monitoring task by providing technical devices to measure a large number of a patient's vital parameters. These measurements improve the safety of the patient during the surgical procedure, because pathological states can be recognised earlier, but may also lead to an increased cognitive load of the physician. In order to reduce cognitive strain and to support intra-operative monitoring for the anaesthesiologist an intelligent patient monitoring and alarm system has been proposed and implemented which evaluates a patient's haemodynamic state on the basis of a current vital parameter constellation with a knowledge-based approach. In this paper general design aspects and evaluation of the intelligent patient monitoring and alarm system in the operating theatre are described. The validation of the inference engine of the intelligent patient monitoring and alarm system was performed in two steps. Firstly, the knowledge base was validated with real patient data which was acquired online in the operating theatre. Secondly, a research prototype of the whole system was implemented in the operating theatre. In the first step, the anaesthetists were asked to enter a state variable evaluation before a drug application or any other intervention on the patient into a recording system. These state variable evaluations were compared to those generated by the intelligent alarm system on the same vital parameter constellations. Altogether 641 state variable evaluations were entered by six different physicians. In total, the sensitivity of alarm recognition is 99.3%, the specificity is 66% and the predictability is 45%. The second step was performed using a research

  16. In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

    PubMed Central

    Liu, Yunkun; Tao, Weixin; Wen, Shishi; Li, Zhengyuan; Yang, Anna; Deng, Zixin

    2015-01-01

    ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. PMID:26556277

  17. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    PubMed

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. PMID:26979473

  18. Evidence of a suffocation alarm system within the periaqueductal gray matter of the rat.

    PubMed

    Schimitel, F G; de Almeida, G M; Pitol, D N; Armini, R S; Tufik, S; Schenberg, L C

    2012-01-01

    Dyspnea, hunger for air, and urge to flee are the cardinal symptoms of panic attacks. Patients also show baseline respiratory abnormalities and a higher rate of comorbid and antecedent respiratory diseases. Panic attacks are also precipitated by infusion of sodium lactate and inhalation of 5% CO₂ in predisposed patients but not in healthy volunteers or patients without panic disorder. Accordingly, Klein [Klein (1993) Arch Gen Psychiatry 50:306-317] suggested that clinical panic is the misfiring of an as-yet-unidentified suffocation alarm system. In rats, selective anoxia of chemoreceptor cells by potassium cyanide (KCN) and electrical and chemical stimulations of periaqueductal gray matter (PAG) produce defensive behaviors, which resemble panic attacks. Thus, here we examined the effects of single or combined administrations of CO₂ (8% and 13%) and KCN (10-80 μg, i.v.) on spontaneous and PAG-evoked behaviors of rats either intact or bearing electrolytic lesions of PAG. Exposure to CO₂ alone reduced grooming while increased exophthalmus, suggesting an arousal response to non-visual cues of environment. Unexpectedly, however, CO₂ attenuated PAG-evoked immobility, trotting, and galloping while facilitated defecation and micturition. Conversely, KCN produced all defensive behaviors of the rat and facilitated PAG-evoked trotting, galloping, and defecation. There were also facilitatory trends in PAG-evoked exophthalmus, immobility, and jumping. Moreover, whereas the KCN-evoked defensive behaviors were attenuated or even suppressed by discrete lesions of PAG, they were markedly facilitated by CO₂. Authors suggest that the PAG harbors an anoxia-sensitive suffocation alarm system which activation precipitates panic attacks and potentiates the subject responses to hypercapnia. PMID:22062132

  19. 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

  20. Efficient gene knockout in goats using CRISPR/Cas9 system.

    PubMed

    Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A; Chen, Chuangfu

    2014-01-01

    The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications. PMID:25188313

  1. Genome editing in Ustilago maydis using the CRISPR-Cas system.

    PubMed

    Schuster, Mariana; Schweizer, Gabriel; Reissmann, Stefanie; Kahmann, Regine

    2016-04-01

    This communication describes the establishment of the type II bacterial CRISPR-Cas9 system to efficiently disrupt target genes in the fungal maize pathogen Ustilago maydis. A single step transformation of a self-replicating plasmid constitutively expressing the U. maydis codon-optimized cas9 gene and a suitable sgRNA under control of the U. maydis U6 snRNA promoter was sufficient to induce genome editing. On average 70% of the progeny of a single transformant were disrupted within the respective b gene. Without selection the self-replicating plasmid was lost rapidly allowing transient expression of the CRISPR-Cas9 system to minimize potential long-term negative effects of Cas9. This technology will be an important advance for the simultaneous disruption of functionally redundant genes and gene families to investigate their contribution to virulence of U. maydis. PMID:26365384

  2. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9.

    PubMed

    Ceasar, S Antony; Rajan, Vinothkumar; Prykhozhij, Sergey V; Berman, Jason N; Ignacimuthu, S

    2016-09-01

    The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology. PMID:27350235

  3. Functional relationship-based alarm processing

    DOEpatents

    Corsberg, Daniel R.

    1988-01-01

    A functional relationship-based alarm processing system and method analyzes each alarm as it is activated and determines its relative importance with other currently activated alarms and signals in accordance with the relationships that the newly activated alarm has with other currently activated alarms. Once the initial level of importance of the alarm has been determined, that alarm is again evaluated if another related alarm is activated. Thus, each alarm's importance is continuously oupdated as the state of the process changes during a scenario. Four hierarchical relationships are defined by this alarm filtering methodology: (1) level precursor (usually occurs when there are two alarm settings on the same parameter); (2) direct precursor (based on caussal factors between two alarms); (3) required action (system response or action) expected within a specified time following activation of an alarm or combination of alarms and process signals); and (4) blocking condition (alarms that are normally expected and are not considered important). The alarm processing system and method is sensitive to the dynamic nature of the process being monitored and is capable of changing the relative importance of each alarm as necessary.

  4. Incidence of Type II CRISPR1-Cas Systems in Enterococcus Is Species-Dependent

    PubMed Central

    Lyons, Casandra; Raustad, Nicole; Bustos, Mario A.; Shiaris, Michael

    2015-01-01

    CRISPR-Cas systems, which obstruct both viral infection and incorporation of mobile genetic elements by horizontal transfer, are a specific immune response common to prokaryotes. Antiviral protection by CRISPR-Cas comes at a cost, as horizontally-acquired genes may increase fitness and provide rapid adaptation to habitat change. To date, investigations into the prevalence of CRISPR have primarily focused on pathogenic and clinical bacteria, while less is known about CRISPR dynamics in commensal and environmental species. We designed PCR primers and coupled these with DNA sequencing of products to detect and characterize the presence of cas1, a universal CRISPR-associated gene and proxy for the Type II CRISPR1-Cas system, in environmental and non-clinical Enterococcus isolates. CRISPR1-cas1 was detected in approximately 33% of the 275 strains examined, and differences in CRISPR1 carriage between species was significant. Incidence of cas1 in E. hirae was 73%, nearly three times that of E. faecalis (23.6%) and 10 times more frequent than in E. durans (7.1%). Also, this is the first report of CRISPR1 presence in E. durans, as well as in the plant-associated species E. casseliflavus and E. sulfureus. Significant differences in CRISPR1-cas1 incidence among Enterococcus species support the hypothesis that there is a tradeoff between protection and adaptability. The differences in the habitats of enterococcal species may exert varying selective pressure that results in a species-dependent distribution of CRISPR-Cas systems. PMID:26600384

  5. CRISPR-Cas9 systems: versatile cancer modelling platforms and promising therapeutic strategies.

    PubMed

    Wen, Wan-Shun; Yuan, Zhi-Min; Ma, Shi-Jie; Xu, Jiang; Yuan, Dong-Tang

    2016-03-15

    The RNA-guided nuclease CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) and its variants such as nickase Cas9, dead Cas9, guide RNA scaffolds and RNA-targeting Cas9 are convenient and versatile platforms for site-specific genome editing and epigenome modulation. They are easy-to-use, simple-to-design and capable of targeting multiple loci simultaneously. Given that cancer develops from cumulative genetic and epigenetic alterations, CRISPR-Cas9 and its variants (hereafter referred to as CRISPR-Cas9 systems) hold extensive application potentials in cancer modeling and therapy. To date, they have already been applied to model oncogenic mutations in cell lines (e.g., Choi and Meyerson, Nat Commun 2014;5:3728) and in adult animals (e.g., Xue et al., Nature 2014;514:380-4), as well as to combat cancer by disabling oncogenic viruses (e.g., Hu et al., Biomed Res Int 2014;2014:612823) or by manipulating cancer genome (e.g., Liu et al., Nat Commun 2014;5:5393). Given the importance of epigenome and transcriptome in tumourigenesis, manipulation of cancer epigenome and transcriptome for cancer modeling and therapy is a promising area in the future. Whereas (epi)genetic modifications of cancer microenvironment with CRISPR-Cas9 systems for therapeutic purposes represent another promising area in cancer research. Herein, we introduce the functions and mechanisms of CRISPR-Cas9 systems in genome editing and epigenome modulation, retrospect their applications in cancer modelling and therapy, discuss limitations and possible solutions and propose future directions, in hope of providing concise and enlightening information for readers interested in this area. PMID:26044706

  6. Exploiting CRISPR-Cas immune systems for genome editing in bacteria.

    PubMed

    Barrangou, Rodolphe; van Pijkeren, Jan-Peter

    2016-02-01

    The CRISPR-Cas immune system is a DNA-encoded, RNA-mediated, DNA-targeting defense mechanism, which provides sequence-specific targeting of DNA. This molecular machinery can be engineered into the sgRNA:Cas9 technology, for programmable cleavage of DNA. Following the genesis of double-stranded DNA breaks, the DNA repair machinery generates mutations at the cleavage site using various pathways. This technology has revolutionized eukaryotic genome editing, and we are at the cusp of full exploitation in bacteria. Here, we discuss the potential of CRISPR-based technologies for use in bacteria, and highlight the application of single stranded DNA recombineering combined with CRISPR-Cas selection to edit the genome of a probiotic organism. We envision that CRISPR-Cas technologies will play a key role in the development of next-generation industrial bacteria. PMID:26629846

  7. The postman always rings twice: two cases of shotgun deaths associated with an unconventional home security alarm system.

    PubMed

    Asirdizer, Mahmut; Turkmen, Nursel; Akan, Okan; Yavuz, Mehmet Sunay

    2014-06-01

    Injury and death cases caused by booby traps are not common in forensic medicine practice. Besides, installation of booby traps including firearms is generally for suicidal and rarely for homicidal purposes. Although few patents were described about home security alarm system that were created by firearms in the United States, 1 sample of injury with a similar unconventional mechanism of home safety system was reported by Asirdizer and Yavuz in 2009. In the published case report, the story of an electrical technician who was invited to a summer house by the homeowner to check the home security alarm system was reported. In the so-called report, he was stated to be injured by the shotgun attached to the unconventional home security alarm system while checking the system. As a result, the homeowner was convicted of a possible intent to cause a life-threatening injury to the technician.The so-called homeowner and his wife died by the same shotgun attached to the same unconventional home security alarm system 4 years on from the first event. In the present case report, we have aimed to present the findings of the crime scene and the autopsies of these unusual 2 deaths and to discuss individual and legal factors in paving the way for the deaths of 2 people. PMID:24781392

  8. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9.

    PubMed

    Terao, Miho; Tamano, Moe; Hara, Satoshi; Kato, Tomoko; Kinoshita, Masato; Takada, Shuji

    2016-07-29

    The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice. PMID:26972821

  9. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9

    PubMed Central

    Terao, Miho; Tamano, Moe; Hara, Satoshi; Kato, Tomoko; Kinoshita, Masato; Takada, Shuji

    2016-01-01

    The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice. PMID:26972821

  10. Multiplex gene editing of the Yarrowia lipolytica genome using the CRISPR-Cas9 system.

    PubMed

    Gao, Shuliang; Tong, Yangyang; Wen, Zhiqiang; Zhu, Li; Ge, Mei; Chen, Daijie; Jiang, Yu; Yang, Sheng

    2016-08-01

    Yarrowia lipolytica is categorized as a generally recognized as safe (GRAS) organism and is a heavily documented, unconventional yeast that has been widely incorporated into multiple industrial fields to produce valuable biochemicals. This study describes the construction of a CRISPR-Cas9 system for genome editing in Y. lipolytica using a single plasmid (pCAS1yl or pCAS2yl) to transport Cas9 and relevant guide RNA expression cassettes, with or without donor DNA, to target genes. Two Cas9 target genes, TRP1 and PEX10, were repaired by non-homologous end-joining (NHEJ) or homologous recombination, with maximal efficiencies in Y. lipolytica of 85.6 % for the wild-type strain and 94.1 % for the ku70/ku80 double-deficient strain, within 4 days. Simultaneous double and triple multigene editing was achieved with pCAS1yl by NHEJ, with efficiencies of 36.7 or 19.3 %, respectively, and the pCASyl system was successfully expanded to different Y. lipolytica breeding strains. This timesaving method will enable and improve synthetic biology, metabolic engineering and functional genomic studies of Y. lipolytica. PMID:27349768

  11. Large fragment deletion using a CRISPR/Cas9 system in Saccharomyces cerevisiae.

    PubMed

    Hao, Huanhuan; Wang, Xiaofei; Jia, Haiyan; Yu, Miao; Zhang, Xiaoyu; Tang, Hui; Zhang, Liping

    2016-09-15

    Large chromosomal modifications have been performed in natural and laboratory evolution studies and hold tremendous potential for use in foundational research, medicine, and biotechnology applications. Recently, the type II bacterial Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR-associated (CRISPR/Cas9) system has emerged as a powerful tool for genome editing in various organisms. In this study, we applied the CRISPR/Cas9 system to preform large fragment deletions in Saccharomyces cerevisiae and compared the performance activity to that of a traditional method that uses the Latour system. Here we report in S. Cerevisiae the CRIPR/Cas9 system has been used to delete fragments exceeding 30 kb. The use of the CRISPR/Cas9 system for generating chromosomal segment excision showed some potential advantages over the Latour system. All the results indicated that CRISPR/Cas9 system was a rapid, efficient, low-cost, and versatile method for genome editing and that it can be applied in further studies in the fields of biology, agriculture, and medicine. PMID:27402178

  12. 10 CFR 34.75 - Records of alarm system and entrance control checks at permanent radiographic installations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... permanent radiographic installations. 34.75 Section 34.75 Energy NUCLEAR REGULATORY COMMISSION LICENSES FOR INDUSTRIAL RADIOGRAPHY AND RADIATION SAFETY REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Recordkeeping Requirements § 34.75 Records of alarm system and entrance control checks at permanent...

  13. 10 CFR 34.75 - Records of alarm system and entrance control checks at permanent radiographic installations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... permanent radiographic installations. 34.75 Section 34.75 Energy NUCLEAR REGULATORY COMMISSION LICENSES FOR INDUSTRIAL RADIOGRAPHY AND RADIATION SAFETY REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Recordkeeping Requirements § 34.75 Records of alarm system and entrance control checks at permanent...

  14. 10 CFR 34.75 - Records of alarm system and entrance control checks at permanent radiographic installations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... permanent radiographic installations. 34.75 Section 34.75 Energy NUCLEAR REGULATORY COMMISSION LICENSES FOR INDUSTRIAL RADIOGRAPHY AND RADIATION SAFETY REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Recordkeeping Requirements § 34.75 Records of alarm system and entrance control checks at permanent...

  15. 10 CFR 34.75 - Records of alarm system and entrance control checks at permanent radiographic installations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... permanent radiographic installations. 34.75 Section 34.75 Energy NUCLEAR REGULATORY COMMISSION LICENSES FOR INDUSTRIAL RADIOGRAPHY AND RADIATION SAFETY REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS Recordkeeping Requirements § 34.75 Records of alarm system and entrance control checks at permanent...

  16. 10 CFR 34.75 - Records of alarm system and entrance control checks at permanent radiographic installations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Records of alarm system and entrance control checks at permanent radiographic installations. 34.75 Section 34.75 Energy NUCLEAR REGULATORY COMMISSION LICENSES FOR INDUSTRIAL RADIOGRAPHY AND RADIATION SAFETY REQUIREMENTS FOR INDUSTRIAL RADIOGRAPHIC OPERATIONS...

  17. 40 CFR 264.34 - Access to communications or alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... operation must have immediate access to an internal alarm or emergency communication device, either directly... (immediately available at the scene of operation) or a hand-held two-way radio, capable of summoning...

  18. 40 CFR 265.34 - Access to communications or alarm system.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... involved in the operation must have immediate access to an internal alarm or emergency communication device... scene of operation) or a hand-held two-way radio, capable of summoning external emergency...

  19. Removal of criticality accident alarm systems at the Y-12 Plant waste management facilities

    SciTech Connect

    Marble, R.C.; Taylor, R.G.

    1998-09-01

    This paper discusses why criticality accident alarm systems (CAASs) were installed in certain waste management buildings at the Y-12 Plant, why the plant now wants to remove them, and what steps were taken to allow the US Department of Energy (DOE) to authorize the removal of the systems. To begin with, the systems in question were installed in the mid- to late-1980s. Some of the facilities were new, and there was no operating experience with the processes. A CAAS, although expensive, is an absolute necessity where criticality accidents are credible. But, they are a superfluous and unnecessary expense in those facilities where it has been determined that a criticality accident is incredible (defined as having a probability of <1 {times} 10{sup {minus}6}/yr). The PRAs have been performed to evaluate six Y-12 Plant waste management facilities, five storage facilities, and a nondestructive analysis facility, with an additional study now being performed on the West End Treatment Facility. The results to date have shown that the probability of various criticality accident scenarios at these facilities is <1 {times} 10{sup {minus}6}/yr and that the CAASs are not needed in these facilities.

  20. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system

    PubMed Central

    Liu, Rui; Chen, Ling; Jiang, Yanping; Zhou, Zhihua; Zou, Gen

    2015-01-01

    Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

  1. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  2. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

    PubMed Central

    Xie, Kabin; Minkenberg, Bastian

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  3. The CRISPR-Cas system - from bacterial immunity to genome engineering.

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-01-01

    Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients. PMID:27594566

  4. Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems.

    PubMed

    Heussler, Gary E; O'Toole, George A

    2016-05-15

    Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPR-Cas self-targeting is vital to fully understanding the spectrum of function of these systems. PMID:26929301

  5. Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

    PubMed

    Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

    2016-04-01

    CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. PMID:27041224

  6. The CRISPR/Cas9 system for gene editing and its potential application in pain research

    PubMed Central

    Sun, Linlin; Lutz, Brianna Marie; Tao, Yuan-Xiang

    2016-01-01

    The CRISPR/Cas9 system is a research hotspot in genome editing and regulation. Currently, it is used in genomic silencing and knock-in experiments as well as transcriptional activation and repression. This versatile system consists of two components: a guide RNA (gRNA) and a Cas9 nuclease. Recognition of a genomic DNA target is mediated through base pairing with a 20-base gRNA. The latter further recruits the Cas9 endonuclease protein to the target site and creates double-stranded breaks in the target DNA. Compared with traditional genome editing directed by DNA-binding protein domains, this short RNA-directed Cas9 endonuclease system is simple and easily programmable. Although this system may have off-target effects and in vivo delivery and immune challenges, researchers have employed this system in vivo to establish disease models, study specific gene functions under certain disease conditions, and correct genomic information for disease treatment. In regards to pain research, the CRISPR/Cas9 system may act as a novel tool in gene correction therapy for pain-associated hereditary diseases and may be a new approach for RNA-guided transcriptional activation or repression of pain-related genes. In addition, this system is also applied to loss-of-function mutations in pain-related genes and knockin of reporter genes or loxP tags at pain-related genomic loci. The CRISPR/Cas9 system will likely be carried out widely in both bench work and clinical settings in the pain field. PMID:27500183

  7. A light-inducible CRISPR/Cas9 system for control of endogenous gene activation

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2015-01-01

    Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes. PMID:25664691

  8. Conservatism in SRS Criticality Alarm System 12 Rad Zone Calculations - How Much is Enough?

    SciTech Connect

    Yates, K.R.

    2002-06-13

    Savannah River Site (SRS) uses two methods (i.e., Approximate Method and MCNP) of calculating the 12-rad zone. The reasons for the two-tier approach are described in Ref. 1 and 2. Lately, there have been occasions in which the use of either the Approximate Method (AM) or MCNP3 calculations indicated potential facility impacts. For example, one or both methods may indicate that a 12-rad zone extends outside of relatively thick shielding, or extends to the roof of a facility, or extends through shielding to part of a stairwell. In such cases, a criticality alarm system may have to be installed to protect workers in a small, localized area from a potential dose that is not substantially greater than 12 rad in air. But, is the potential dose really greater than 12 rad in air? A subcommittee was appointed to look into the two 12-rad zone calculation methods for the purpose of identifying items contributing to over-conservatism and under-conservatism, and to recommend a path forward.

  9. Transformation of Ground Vibration Signal for Debris-Flow Monitoring and Detection in Alarm Systems

    PubMed Central

    Abancó, Clàudia; Hürlimann, Marcel; Fritschi, Bruno; Graf, Christoph; Moya, José

    2012-01-01

    Debris flows are fast mass movements formed by a mix of water and solid materials, which occur in steep torrents, and are a source of high risks for human settlements. Geophones are widely used to detect the ground vibration induced by passing debris flows. However, the recording of geophone signals usually requires storing a huge amount of data, which leads to problems in storage capacity and power consumption. This paper presents a method to transform and simplify the signals measured by geophones. The key input parameter is the ground velocity threshold, which removes the seismic noise that is not related to debris flows. A signal conditioner was developed to implement the transformation and the ground velocity threshold was set by electrical resistors. The signal conditioner was installed at various European monitoring sites to test the method. Results show that data amount and power consumption can be greatly reduced without losing much information on the main features of the debris flows. However, the outcome stresses the importance of choosing a ground vibration threshold, which must be accurately calibrated. The transformation is also suitable to detect other rapid mass movements and to distinguish among different processes, which points to a possible implementation in alarm systems. PMID:22666064

  10. Transformation of ground vibration signal for debris-flow monitoring and detection in alarm systems.

    PubMed

    Abancó, Clàudia; Hürlimann, Marcel; Fritschi, Bruno; Graf, Christoph; Moya, José

    2012-01-01

    Debris flows are fast mass movements formed by a mix of water and solid materials, which occur in steep torrents, and are a source of high risks for human settlements. Geophones are widely used to detect the ground vibration induced by passing debris flows. However, the recording of geophone signals usually requires storing a huge amount of data, which leads to problems in storage capacity and power consumption. This paper presents a method to transform and simplify the signals measured by geophones. The key input parameter is the ground velocity threshold, which removes the seismic noise that is not related to debris flows. A signal conditioner was developed to implement the transformation and the ground velocity threshold was set by electrical resistors. The signal conditioner was installed at various European monitoring sites to test the method. Results show that data amount and power consumption can be greatly reduced without losing much information on the main features of the debris flows. However, the outcome stresses the importance of choosing a ground vibration threshold, which must be accurately calibrated. The transformation is also suitable to detect other rapid mass movements and to distinguish among different processes, which points to a possible implementation in alarm systems. PMID:22666064

  11. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems.

    PubMed

    Burstein, David; Sun, Christine L; Brown, Christopher T; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J; Thomas, Brian C; Banfield, Jillian F

    2016-01-01

    Current understanding of microorganism-virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides. PMID:26837824

  12. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    PubMed Central

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-01-01

    Current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides. PMID:26837824

  13. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    DOE PAGESBeta

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-02-03

    Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. Wemore » correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.« less

  14. 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…

  15. Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis

    PubMed Central

    Elmore, Joshua R.; Yokooji, Yuusuke; Sato, Takaaki; Olson, Sara; Glover, III, Claiborne V.C.; Graveley, Brenton R.; Atomi, Haruyuki; Terns, Rebecca M.; Terns, Michael P.

    2013-01-01

    CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry. PMID:23535213

  16. Tandem repeat knockout utilizing the CRISPR/Cas9 system in human cells.

    PubMed

    Lv, Qingyan; Lai, Liangxue; Yuan, Lin; Song, Yuning; Sui, Tingting; Li, Zhanjun

    2016-05-15

    Tandem repeats have been shown to cause human genetic diseases and contribute significantly to genome variation and instability. Although multi-sgRNAs mediated CRISPR/Cas9 system have used to generate regional deletions previously, in this study we explored a method of generating regional deletions of tandem repeats by taking advantage of the off-target effects of CRISPR/Cas9 in 293FT cells. Our results revealed that generation of large-fragment deletions of tandem repeats located in the MAGEL2 and XIST gene was possible. In summary, we have demonstrated that large-fragment deletions of tandem repeats can be achieved using a sgRNA-directed CRISPR/Cas9 system, facilitating the functional study of tandem repeats in future studies. PMID:26873114

  17. Talking Fire Alarms Calm Kids.

    ERIC Educational Resources Information Center

    Executive Educator, 1984

    1984-01-01

    The new microprocessor-based fire alarm systems can help to control smoke movement throughout school buildings by opening vents and doors, identify the burning section, activate voice alarms, provide firefighters with telephone systems during the fire, and release fire-preventing gas. (KS)

  18. Heritable custom genomic modifications in Caenorhabditis elegans via a CRISPR-Cas9 system.

    PubMed

    Tzur, Yonatan B; Friedland, Ari E; Nadarajan, Saravanapriah; Church, George M; Calarco, John A; Colaiácovo, Monica P

    2013-11-01

    We adapted the CRISPR-Cas9 system for template-mediated repair of targeted double-strand breaks via homologous recombination in Caenorhabditis elegans, enabling customized and efficient genome editing. This system can be used to create specific insertions, deletions, and base pair changes in the germline of C. elegans. PMID:23979579

  19. A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

    PubMed

    Zhang, Haiwei; Zhang, Xixi; Fan, Cunxian; Xie, Qun; Xu, Chengxian; Zhao, Qun; Liu, Yongbo; Wu, Xiaoxia; Zhang, Haibing

    2016-03-18

    CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs. PMID:26879140

  20. The monitoring and alarm system based on distributed temperature fiber sensing

    NASA Astrophysics Data System (ADS)

    Zhao, Hong-qiang; Zhao, Yu-liang; Zhang, Yu-ye; Wang, Shu-juan

    2014-09-01

    Air material depot is a warehouse which store consumed all the parts and equipment vault of the plane. In order to ensure the various aviation equipment integrity of the backup piece rate, the inside temperature of depot must be controlled within a certain range. Therefore, the depot must be equipped a self-contained temperature real-time monitoring system. This paper presents a distributed temperature sensing alarm system to apply to real-time measure spatial distribution of temperature field. In order to eliminate influence to the scattering strength from the light source instability and the fiber bending splice loss and to improve temperature measurement accuracy, the system design used dual-channel dual- wavelength comparison method which make Anti-Stokes as signal channel and Stokes as a reference channel to collect signals of two channel respectively and detect the ratio of the two channels' signals. The light of LD directional coupling to the sensing optical fiber in the temperature field to test, domain reflect light from the sensing optical fiber directional coupling to receive channel again, Rayleigh domain reflect light is filtered after optical filter, the Anti-Stokes and Stokes are both taken out, converted and magnified, the two signals is digitalized by A/D Converter, and written to the storage machine , which linear cumulative to the content of the storage unit, The distributed measurement of the temperature field to test is finished. The collected 2900 measuring points real-time on 2km of optical fiber. The spatial resolution of the system was 0.7m, measurement range was -20-370°C, and measurement error was ± 2 °C. All index of the system achieved the desired objective. To get an accurate temperature field spatial distribution and the information of temporal variation, the system enabled real-time temperature of aviation depot monitoring and early warning . As a new sensing technology, the distributed fiber optic sensor has the functions of self

  1. RNA-guided genome editing in plants using a CRISPR-Cas system.

    PubMed

    Xie, Kabin; Yang, Yinong

    2013-11-01

    Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants. PMID:23956122

  2. Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems.

    PubMed

    Shmakov, Sergey; Abudayyeh, Omar O; Makarova, Kira S; Wolf, Yuri I; Gootenberg, Jonathan S; Semenova, Ekaterina; Minakhin, Leonid; Joung, Julia; Konermann, Silvana; Severinov, Konstantin; Zhang, Feng; Koonin, Eugene V

    2015-11-01

    Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements. PMID:26593719

  3. Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research.

    PubMed

    Liu, Degao; Hu, Rongbin; Palla, Kaitlin J; Tuskan, Gerald A; Yang, Xiaohan

    2016-04-01

    Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. This article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation of gene expression, and identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort. PMID:26896588

  4. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    PubMed

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This 'suicide' CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  5. A ‘suicide’ CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans

    PubMed Central

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  6. Applications of the CRISPR-Cas9 system in cancer biology.

    PubMed

    Sánchez-Rivera, Francisco J; Jacks, Tyler

    2015-07-01

    The prokaryotic type II CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large range of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here, we review current approaches for functional studies of cancer genes that are based on CRISPR-Cas, with emphasis on their applicability for the development of next-generation models of human cancer. PMID:26040603

  7. Applications of the CRISPR-Cas9 system in cancer biology

    PubMed Central

    Sánchez-Rivera, Francisco J.; Jacks, Tyler

    2015-01-01

    Preface The prokaryotic type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large variety of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here we review current approaches based on CRISPR-Cas9 for functional studies of cancer genes, with emphasis on its applicability for the development of the next-generation models of human cancer. PMID:26040603

  8. Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

    PubMed

    Sun, Xianjun; Hu, Zheng; Chen, Rui; Jiang, Qiyang; Song, Guohua; Zhang, Hui; Xi, Yajun

    2015-01-01

    Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules. PMID:26022141

  9. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    PubMed

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system. PMID:26950513

  10. Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

    PubMed

    Kennedy, Edward M; Kornepati, Anand V R; Mefferd, Adam L; Marshall, Joy B; Tsai, Kevin; Bogerd, Hal P; Cullen, Bryan R

    2015-12-01

    RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus. PMID:26291065

  11. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system

    PubMed Central

    Wang, Xianlong; Cao, Chunwei; Huang, Jiaojiao; Yao, Jing; Hai, Tang; Zheng, Qiantao; Wang, Xiao; Zhang, Hongyong; Qin, Guosong; Cheng, Jinbo; Wang, Yanfang; Yuan, Zengqiang; Zhou, Qi; Wang, Hongmei; Zhao, Jianguo

    2016-01-01

    Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson’s disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value. PMID:26857844

  12. The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

    PubMed Central

    Staals, Raymond H. J.; Endtz, Hubert P.; van Baarlen, Peter; van der Oost, John

    2014-01-01

    SUMMARY Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular. PMID:24600041

  13. The Fine Tuning of Pain Thresholds: A Sophisticated Double Alarm System

    PubMed Central

    Plaghki, Léon; Decruynaere, Céline; Van Dooren, Paul; Le Bars, Daniel

    2010-01-01

    illustrates the role of nociception as a “double level” and “double release” alarm system based on level detectors. By contrast, warmth detection was found to be based on difference detectors. It is hypothesized that pain results from a CNS build-up process resulting from population coding and strongly influenced by the background temperatures surrounding at large the stimulation site. We propose an alternative solution to the conventional methods that only measure a single “threshold of pain”, without knowing which of the two systems is involved. PMID:20428245

  14. Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPR-Cas system.

    PubMed

    Lück, Christian; Brzuszkiewicz, Elzbieta; Rydzewski, Kerstin; Koshkolda, Tetyana; Sarnow, Katharina; Essig, Andreas; Heuner, Klaus

    2015-12-01

    In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains. PMID:26294350

  15. Engineering Translational Activators with CRISPR-Cas System.

    PubMed

    Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

    2016-01-15

    RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility. PMID:26414660

  16. Xcel Energy implements an alarm management strategy

    SciTech Connect

    Bass, J.; Abreu, G.

    2007-11-15

    Not so long ago, Xcel Energy's Pawnee Station, a 505 MW coal-fired generating station in Brush, Colorado, USA was commonly generating 300 to 400 alarms per 8-hour shift. The article describes how the alarm system was revised and improved by tackling alarm dead-bands, and rationalising alarms for routine events. Operators are trained to understand the functions of alarm management components, their use and response, and obtain feedback. Today the power station reports about one alarm per hour. 3 photos.

  17. Foreign DNA acquisition by the I-F CRISPR–Cas system requires all components of the interference machinery

    PubMed Central

    Vorontsova, Daria; Datsenko, Kirill A.; Medvedeva, Sofia; Bondy-Denomy, Joseph; Savitskaya, Ekaterina E.; Pougach, Ksenia; Logacheva, Maria; Wiedenheft, Blake; Davidson, Alan R.; Severinov, Konstantin; Semenova, Ekaterina

    2015-01-01

    CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems. PMID:26586803

  18. Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia

    PubMed Central

    Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

    2016-01-01

    Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%–87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy—introducing Cas9 and sgRNA expression cassettes sequentially into petunia—can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation. PMID:26837606

  19. Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

    PubMed Central

    Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

    2015-01-01

    ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

  20. Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.

    PubMed

    Tao, Li; Yang, Mingyao; Wang, Xiaodong; Zhang, Zhenni; Wu, Zhonghong; Tian, Jianhui; An, Lei; Wang, Shumin

    2016-08-01

    The parthenotes represent ideal models mimicking the embryonic development and characterizing the function of maternal genomes as well as an alternative source of pluripotent cell lines. Besides, parthenogenetically activated (PA) embryos serve as a rapid assay system to maximize the efficiency of generating genetically modified pig CRISPR/Cas9 system, an efficient and multiplex gene editing tool, has been utilized to modify the genome of porcine parthenotes. However, lower biallelic mutation rate and high mosaicism frequency were observed. Here, we aimed to enhance the biallelic mutation rate with reduced mosaicism by optimization of the concentration and injection time of the Cas9/sgRNA mixture in porcine parthenotes. The results showed that the efficient biallelic mutation (93%) and low mosaicism (33%) could be achieved in porcine parthenotes by cytoplasmic injection of Cas9 mRNA/sgRNA (125/12.5 ng/μl) after 8 h of parthenogenetical activation. Thus, our study provides an effective strategy for increasing the biallelic mutation rate and population homogeneity of genetically modified parthenotes, which will strengthen the role of parthenotes in uncovering early embryonic development and assessing the mutation efficiency due to the simplicity and adaptability of CRISPR/Cas9. PMID:27221047

  1. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system

    PubMed Central

    Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J.; Hornicek, Francis J; Duan, Zhenfeng

    2014-01-01

    Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. PMID:25348612

  2. Ultrasonic Technology in Duress Alarms.

    ERIC Educational Resources Information Center

    Lee, Martha A.

    2000-01-01

    Provides the pros and cons of the most commonly used technologies in personal duress alarm systems in the school environment. Discussed are radio frequency devices, infrared systems, and ultrasonic technology. (GR)

  3. An Active Type I-E CRISPR-Cas System Identified in Streptomyces avermitilis

    PubMed Central

    Qiu, Yi; Wang, Shiwei; Chen, Zhi; Guo, Yajie; Song, Yuan

    2016-01-01

    CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA. The naive features of spacer acquisition in the type I-E system of S. avermitilis were investigated and a completely conserved PAM 5’-AAG-3’ was identified. Spacer acquisition displayed differential strand bias upstream and downstream of the priming spacer, and irregular integrations of new spacers were observed. In addition, introduction of this system into host conferred phage resistance to some extent. This study will give new insights into adaptation mechanism of the type I-E systems in vivo, and meanwhile provide theoretical foundation for applying this system on the genetic modification of S. avermitilis. PMID:26901661

  4. Dynamic alarm response procedures

    SciTech Connect

    Martin, J.; Gordon, P.; Fitch, K.

    2006-07-01

    The Dynamic Alarm Response Procedure (DARP) system provides a robust, Web-based alternative to existing hard-copy alarm response procedures. This paperless system improves performance by eliminating time wasted looking up paper procedures by number, looking up plant process values and equipment and component status at graphical display or panels, and maintenance of the procedures. Because it is a Web-based system, it is platform independent. DARP's can be served from any Web server that supports CGI scripting, such as Apache{sup R}, IIS{sup R}, TclHTTPD, and others. DARP pages can be viewed in any Web browser that supports Javascript and Scalable Vector Graphics (SVG), such as Netscape{sup R}, Microsoft Internet Explorer{sup R}, Mozilla Firefox{sup R}, Opera{sup R}, and others. (authors)

  5. Condition Assessment Survey (CAS) Program. Deficiency standards and inspections methods manual: Volume 11, 0.11 Specialty systems

    SciTech Connect

    Not Available

    1993-05-01

    General information is presented for asset determinant factor/CAS repair codes/CAS cost factors; guide sheet tool & material listing; testing methods; inspection frequency; standard system design life tables; system work breakdown structure; and general system/material data. Deficiency standards and inspection methods are presented for canopies; loading dock systems; tanks; domes (bulk storage, metal framing); louvers & vents; access floors; integrated ceilings; and mezzanine structures.

  6. Fire alarm system/fire suppression system for mobile tactical shelters

    NASA Astrophysics Data System (ADS)

    Walker, F. K.; Lecours, C. A.; Radcliff, O.

    1985-08-01

    The objective of this project was to develop a fire detection/suppression capability for DoD standard family mobile tactical shelters. The systems developed and tested provide complete protection during all employment conditions; in garrison use, storage, transportation, and deployed field conditions. The reports outlines the requirement and the test and evaluation program. Two manufacturers of detection systems and two manufacturers of suppression systems were identified and qualified to meet the fire protection requirements for mobile tactical shelters.

  7. 24 CFR 3285.703 - Smoke alarms.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 5 2014-04-01 2014-04-01 false Smoke alarms. 3285.703 Section 3285... DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Electrical Systems and Equipment § 3285.703 Smoke alarms. Smoke alarms must be functionally tested in accordance with applicable requirements of the...

  8. 24 CFR 3285.703 - Smoke alarms.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 5 2010-04-01 2010-04-01 false Smoke alarms. 3285.703 Section 3285... DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Electrical Systems and Equipment § 3285.703 Smoke alarms. Smoke alarms must be functionally tested in accordance with applicable requirements of the...

  9. 24 CFR 3285.703 - Smoke alarms.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 5 2013-04-01 2013-04-01 false Smoke alarms. 3285.703 Section 3285... DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Electrical Systems and Equipment § 3285.703 Smoke alarms. Smoke alarms must be functionally tested in accordance with applicable requirements of the...

  10. 24 CFR 3285.703 - Smoke alarms.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 5 2011-04-01 2011-04-01 false Smoke alarms. 3285.703 Section 3285... DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Electrical Systems and Equipment § 3285.703 Smoke alarms. Smoke alarms must be functionally tested in accordance with applicable requirements of the...

  11. 24 CFR 3285.703 - Smoke alarms.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 5 2012-04-01 2012-04-01 false Smoke alarms. 3285.703 Section 3285... DEVELOPMENT MODEL MANUFACTURED HOME INSTALLATION STANDARDS Electrical Systems and Equipment § 3285.703 Smoke alarms. Smoke alarms must be functionally tested in accordance with applicable requirements of the...

  12. 30 CFR 77.311 - Alarm devices.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Alarm devices. 77.311 Section 77.311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY....311 Alarm devices. Thermal dryer systems shall be equipped with both audible and visual alarm...

  13. 30 CFR 77.311 - Alarm devices.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Alarm devices. 77.311 Section 77.311 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR COAL MINE SAFETY AND HEALTH MANDATORY....311 Alarm devices. Thermal dryer systems shall be equipped with both audible and visual alarm...

  14. Improved alarm tracking for better accountability

    SciTech Connect

    Nemesure, S.; Marr, G.; Shrey, T.; Kling, N.; Hammons, L.; Ingrassia, P.; D'Ottavio, T.

    2011-03-28

    An alarm system is a vital component of any accelerator, as it provides a warning that some element of the system is not functioning properly. The severity and age of the alarm may sometimes signify whether urgent or deferred attention is required. For example, older alarms may be given a lower priority if an assumption is made that someone else is already investigating it, whereas those of higher severity or alarms that are more current may indicate the need for an immediate response. The alarm history also provides valuable information regarding the functionality of the overall system, thus careful tracking of these data is likely to improve response time, remove uncertainty about the current status and assist in the ability to promptly respond to the same warning/trigger in the future. Since one goal of every alarm display is to be free of alarms, a clear and concise presentation of an alarm along with useful historic annotations can help the end user address the warning more quickly, thus expediting the elimination of such alarm conditions. By defining a discrete set of very specific alarm management states and by utilizing database resources to maintain a complete and easily accessible alarm history, we anticipate facilitated work flow due to more efficient operator response and management of alarms.

  15. Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

    PubMed Central

    Min, Kyunghun; Ichikawa, Yuichi

    2016-01-01

    ABSTRACT Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. PMID:27340698

  16. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi

    PubMed Central

    Nødvig, Christina S.; Nielsen, Jakob B.; Kogle, Martin E.; Mortensen, Uffe H.

    2015-01-01

    The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting. PMID:26177455

  17. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli

    PubMed Central

    Reisch, Chris R.; Prather, Kristala L. J.

    2015-01-01

    Genome engineering methods in E. coli allow for easy to perform manipulations of the chromosome in vivo with the assistance of the λ-Red recombinase system. These methods generally rely on the insertion of an antibiotic resistance cassette followed by removal of the same cassette, resulting in a two-step procedure for genomic manipulations. Here we describe a method and plasmid system that can edit the genome of E. coli without chromosomal markers. This system, known as Scarless Cas9 Assisted Recombineering (no-SCAR), uses λ-Red to facilitate genomic integration of donor DNA and double stranded DNA cleavage by Cas9 to counterselect against wild-type cells. We show that point mutations, gene deletions, and short sequence insertions were efficiently performed in several genomic loci in a single-step with regards to the chromosome and did not leave behind scar sites. The single-guide RNA encoding plasmid can be easily cured due to its temperature sensitive origin of replication, allowing for iterative chromosomal manipulations of the same strain, as is often required in metabolic engineering. In addition, we demonstrate the ability to efficiently cure the second plasmid in the system by targeting with Cas9, leaving the cells plasmid-free. PMID:26463009

  18. A simple, flexible and high-throughput cloning system for plant genome editing via CRISPR-Cas system.

    PubMed

    Kim, Hyeran; Kim, Sang-Tae; Ryu, Jahee; Choi, Min Kyung; Kweon, Jiyeon; Kang, Beum-Chang; Ahn, Hyo-Min; Bae, Suji; Kim, Jungeun; Kim, Jin-Soo; Kim, Sang-Gyu

    2016-08-01

    CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant. PMID:26946469

  19. How tissue injury alarms the immune system and causes a systemic inflammatory response syndrome

    PubMed Central

    2012-01-01

    Systemic inflammation is very prevalent among critically ill patients, particularly those with extensive tissue injury. Although downstream mediators (cytokines) and effector cells (phagocytes) have been identified, proximal mediators originating from injured tissues remained elusive. Alarmins (“danger signals”) released by necrotic/injured cells have been identified recently and certainly play a role in triggering local and systemic inflammation in critically ill patients. The most promising alarmin candidates are of mitochondrial origin, i.e. mitochondrial DNA and the chemotactic factor fMet-Leu-Phe (fMLP). ATP also is released from necrotic tissues and stimulates the assembly of the inflammasome, leading to the production of proinflammatory cytokines, such as interleukin (IL)-1ß. The identification of novel alarmins opens new therapeutic avenues for the treatment of severe SIRS, and SIRS-dependent organ dysfunction. PMID:22788849

  20. Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System.

    PubMed

    Bai, Yichun; He, Linjie; Li, Pengcheng; Xu, Kun; Shao, Simin; Ren, Chonghua; Liu, Zhongtian; Wei, Zehui; Zhang, Zhiying

    2016-01-01

    In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the Puro(R) gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing. PMID:26869617

  1. Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

    PubMed Central

    Bai, Yichun; He, Linjie; Li, Pengcheng; Xu, Kun; Shao, Simin; Ren, Chonghua; Liu, Zhongtian; Wei, Zehui; Zhang, Zhiying

    2016-01-01

    In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing. PMID:26869617

  2. An alarm for monitoring CPAP.

    PubMed

    Carter, B; Clare, D; Hochmann, M; Osborne, A; Fraser, T

    1993-04-01

    We have built a device for use within the hospital and at home that is designed to warn of circuit disconnection when used in conjunction with continuous positive airway pressure (CPAP) therapy delivered via ventilators or CPAP generating systems. The Royal Children's Hospital CPAP alarm is a compact, battery operated alarm and monitor of circuit pressure. The device includes intrinsic safety features including a safety blow-off valve, a high pressure alarm and design features that make the device practical, safe and easy to use by both trained hospital personnel and home care attendants with limited training. PMID:8517514

  3. The molecular mechanism of CRISPR/Cas9 system and its application in gene therapy of human diseases.

    PubMed

    Liang, Qu; Huashan, Li; Yunhan, Jiang; Chunsheng, Dong

    2015-10-01

    CRISPR/Cas system is an adaptive immune system that confers resistance to exogenous virus or plasmid in bacteria and archaea. In recent years, the booming CRISPR/Cas9 genome editing technology modified from type2 CRISPR/Cas adaptive immune system has been widely applied to various research fields of life science and led to revolutionary changes. In this review, we summarize the origin and development of CRISPR/Cas9 genome editing technology as well as its applications in life science research. We focus on the latest application of this system in gene therapy of human diseases and the associated side/off-target effects, which may provide references for researchers in related areas. PMID:26496749

  4. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    PubMed Central

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

  5. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system.

    PubMed

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

  6. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

  7. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

    PubMed Central

    Nemudryi, A. A.; Valetdinova, K. R.; Medvedev, S. P.; Zakian, S. M.

    2014-01-01

    Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn’t enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared – this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools. PMID:25349712

  8. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

    PubMed Central

    Bialek, Julia K.; Dunay, Gábor A.; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C.

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination. PMID:27341108

  9. Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system.

    PubMed

    Fujii, Wataru; Kakuta, Shigeru; Yoshioka, Shin; Kyuwa, Shigeru; Sugiura, Koji; Naito, Kunihiko

    2016-08-26

    Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5'-NNAGAA sequence as a protospacer adjacent motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design. PMID:27318086

  10. The CRISPR/Cas9 system for plant genome editing and beyond.

    PubMed

    Bortesi, Luisa; Fischer, Rainer

    2015-01-01

    Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments. PMID:25536441

  11. Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

    PubMed Central

    Fuller, Kevin K.; Chen, Shan

    2015-01-01

    Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen. PMID:26318395

  12. A CRISPR-Cas9 sex-ratio distortion system for genetic control

    PubMed Central

    Galizi, Roberto; Hammond, Andrew; Kyrou, Kyros; Taxiarchi, Chrysanthi; Bernardini, Federica; O’Loughlin, Samantha M.; Papathanos, Philippos-Aris; Nolan, Tony; Windbichler, Nikolai; Crisanti, Andrea

    2016-01-01

    Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome. PMID:27484623

  13. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish.

    PubMed

    Ablain, Julien; Durand, Ellen M; Yang, Song; Zhou, Yi; Zon, Leonard I

    2015-03-23

    CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish, it allows the rapid generation of knockout lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies in zebrafish. PMID:25752963

  14. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system

    PubMed Central

    Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

    2016-01-01

    The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications. PMID:26813419

  15. A CRISPR-Cas9 sex-ratio distortion system for genetic control.

    PubMed

    Galizi, Roberto; Hammond, Andrew; Kyrou, Kyros; Taxiarchi, Chrysanthi; Bernardini, Federica; O'Loughlin, Samantha M; Papathanos, Philippos-Aris; Nolan, Tony; Windbichler, Nikolai; Crisanti, Andrea

    2016-01-01

    Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome. PMID:27484623

  16. Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system.

    PubMed

    Goto, Teppei; Hara, Hiromasa; Nakauchi, Hiromitsu; Hochi, Shinichi; Hirabayashi, Masumi

    2016-08-01

    The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants. PMID:26931321

  17. Detection and classification of alarm threshold violations in condition monitoring systems working in highly varying operational conditions

    NASA Astrophysics Data System (ADS)

    Strączkiewicz, M.; Barszcz, T.; Jabłoński, A.

    2015-07-01

    All commonly used condition monitoring systems (CMS) enable defining alarm thresholds that enhance efficient surveillance and maintenance of dynamic state of machinery. The thresholds are imposed on the measured values such as vibration-based indicators, temperature, pressure, etc. For complex machinery such as wind turbine (WT) the total number of thresholds might be counted in hundreds multiplied by the number of operational states. All the parameters vary not only due to possible machinery malfunctions, but also due to changes in operating conditions and these changes are typically much stronger than the former ones. Very often, such a behavior may lead to hundreds of false alarms. Therefore, authors propose a novel approach based on parameterized description of the threshold violation. For this purpose the novelty and severity factors are introduced. The first parameter refers to the time of violation occurrence while the second one describes the impact of the indicator-increase to the entire machine. Such approach increases reliability of the CMS by providing the operator with the most useful information of the system events. The idea of the procedure is presented on a simulated data similar to those from a wind turbine.

  18. Intelligent alarming

    NASA Technical Reports Server (NTRS)

    Braden, W. B.

    1992-01-01

    This talk discusses the importance of providing a process operator with concise information about a process fault including a root cause diagnosis of the problem, a suggested best action for correcting the fault, and prioritization of the problem set. A decision tree approach is used to illustrate one type of approach for determining the root cause of a problem. Fault detection in several different types of scenarios is addressed, including pump malfunctions and pipeline leaks. The talk stresses the need for a good data rectification strategy and good process models along with a method for presenting the findings to the process operator in a focused and understandable way. A real time expert system is discussed as an effective tool to help provide operators with this type of information. The use of expert systems in the analysis of actual versus predicted results from neural networks and other types of process models is discussed.

  19. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system.

    PubMed

    Wu, Mingming; Wei, Caihong; Lian, Zhengxing; Liu, Ruizao; Zhu, Caiye; Wang, Huihua; Cao, Jiaxue; Shen, Yuelei; Zhao, Fuping; Zhang, Li; Mu, Zhu; Wang, Yayu; Wang, Xiaogang; Du, Lixin; Wang, Chuduan

    2016-01-01

    Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep. PMID:27063570

  20. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.

    PubMed

    Nishitani, Chikako; Hirai, Narumi; Komori, Sadao; Wada, Masato; Okada, Kazuma; Osakabe, Keishi; Yamamoto, Toshiya; Osakabe, Yuriko

    2016-01-01

    Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome. PMID:27530958

  1. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    PubMed Central

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  2. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    PubMed

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  3. Rosa26-targeted sheep gene knock-in via CRISPR-Cas9 system

    PubMed Central

    Wu, Mingming; Wei, Caihong; Lian, Zhengxing; Liu, Ruizao; Zhu, Caiye; Wang, Huihua; Cao, Jiaxue; Shen, Yuelei; Zhao, Fuping; Zhang, Li; Mu, Zhu; Wang, Yayu; Wang, Xiaogang; Du, Lixin; Wang, Chuduan

    2016-01-01

    Recent advances in our ability to design DNA binding factors with specificity for desired sequences have resulted in a revolution in genetic engineering, enabling directed changes to the genome to be made relatively easily. Technologies that facilitate specific and precise genome editing, such as knock-in, are critical for determining the functions of genes and for understanding fundamental biological processes. The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in mammals. Rosa26 gene can encode a non-essential nuclear RNA in almost all organizations, and become a hot point of exogenous gene insertion. Here, we describe efficient, precise CRISPR/Cas9-mediated Integration using a donor vector with tGFP sequence targeted in the sheep genomic Rosa26 locus. We succeeded in integrating with high efficiency an exogenous tGFP (turboGFP) gene into targeted genes in frame. Due to its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in will become a standard method for the generation transgenic sheep. PMID:27063570

  4. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system

    PubMed Central

    Nishitani, Chikako; Hirai, Narumi; Komori, Sadao; Wada, Masato; Okada, Kazuma; Osakabe, Keishi; Yamamoto, Toshiya; Osakabe, Yuriko

    2016-01-01

    Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome. PMID:27530958

  5. Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.

    PubMed

    Zu, Yao; Zhang, Xushuai; Ren, Jianfeng; Dong, Xuehong; Zhu, Zhe; Jia, Liang; Zhang, Qinghua; Li, Weiming

    2016-01-01

    Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches. PMID:27005311

  6. Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Kankan; Ouyang, Hongsheng; Xie, Zicong; Yao, Chaogang; Guo, Nannan; Li, Mengjing; Jiao, Huping; Pang, Daxin

    2015-01-01

    Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety. PMID:26564781

  7. Biallelic editing of a lamprey genome using the CRISPR/Cas9 system

    PubMed Central

    Zu, Yao; Zhang, Xushuai; Ren, Jianfeng; Dong, Xuehong; Zhu, Zhe; Jia, Liang; Zhang, Qinghua; Li, Weiming

    2016-01-01

    Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches. PMID:27005311

  8. Evidence of a suffocation alarm system sensitive to clinically-effective treatments with the panicolytics clonazepam and fluoxetine.

    PubMed

    Schimitel, Fagna Giacomin; Müller, Cláudia Janaina Torres; Tufik, Sérgio; Schenberg, Luiz Carlos

    2014-12-01

    Dyspnea, 'hunger for air', and the urge to flee are the cardinal symptoms of respiratory-type panic attacks. Patients also show baseline respiratory abnormalities and a higher rate of comorbid and antecedent respiratory diseases. Panic attacks are also precipitated by both the infusion of 0.5 M sodium lactate and the inhalation of 5-7% carbon dioxide (CO2) in predisposed patients, but not in healthy volunteers nor patients without panic disorder. Further studies show that patients with panic are also hyper-responsive to hypoxia. These and other observations led Klein (1993) to suggest that clinical panic is the misfiring of a suffocation alarm system. In rats, cytotoxic hypoxia of chemoreceptor cells by intravenous injection of potassium cyanide (KCN) produces short-lasting flight behaviors reminiscent of panic attacks. KCN-induced flight behaviors are blocked both by denervation of chemoreceptor cells and lesion of dorsal periaqueductal gray matter, a likely substrate of panic. Herein, we show that KCN-evoked flight behaviors are also attenuated by both acute and chronic treatment with clonazepam (0.01-0.3 mg/kg, intraperitoneally (i.p.)) and fluoxetine (1-4 mg/kg/day, i.p. for 21 days), respectively. Attenuation of KCN-evoked panic-like behaviors by clinically-effective treatment with panicolytics adds fresh evidence to the false suffocation alarm theory of panic disorder. PMID:25277323

  9. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

    PubMed

    Arazoe, Takayuki; Miyoshi, Kennosuke; Yamato, Tohru; Ogawa, Tetsuo; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru

    2015-12-01

    CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi. PMID:26039904

  10. Statistical Considerations in Designing Tests of Mine Detection Systems: II - Measures Related to the False Alarm Rate

    SciTech Connect

    Simonson, K.M.

    1998-08-01

    The rate at which a mine detection system falsely identifies man-made or natural clutter objects as mines is referred to as the system's false alarm rate (FAR). Generally expressed as a rate per unit area or time, the FAR is one of the primary metrics used to gauge system performance. In this report, an overview is given of statistical methods appropriate for the analysis of data relating to FAR. Techniques are presented for determining a suitable size for the clutter collection area, for summarizing the performance of a single sensor, and for comparing different sensors. For readers requiring more thorough coverage of the topics discussed, references to the statistical literature are provided. A companion report addresses statistical issues related to the estimation of mine detection probabilities.

  11. Multidisciplinary Aerospace Systems Optimization: Computational AeroSciences (CAS) Project

    NASA Technical Reports Server (NTRS)

    Kodiyalam, S.; Sobieski, Jaroslaw S. (Technical Monitor)

    2001-01-01

    The report describes a method for performing optimization of a system whose analysis is so expensive that it is impractical to let the optimization code invoke it directly because excessive computational cost and elapsed time might result. In such situation it is imperative to have user control the number of times the analysis is invoked. The reported method achieves that by two techniques in the Design of Experiment category: a uniform dispersal of the trial design points over a n-dimensional hypersphere and a response surface fitting, and the technique of krigging. Analyses of all the trial designs whose number may be set by the user are performed before activation of the optimization code and the results are stored as a data base. That code is then executed and referred to the above data base. Two applications, one of the airborne laser system, and one of an aircraft optimization illustrate the method application.

  12. Tuning CAS Application using AIMS: An Automated Instrumentation and Monitoring System

    NASA Technical Reports Server (NTRS)

    Mehra, P.; Sarukkai, S.; Schmidt, M.; Schulbach, C.; VanVoorst, B.; Yan, Jerry; Woodrow, Thomas (Technical Monitor)

    1994-01-01

    To bring together NASA's scientists and engineers and their counterparts in industry, other government agencies, and academia working in the Computational AeroSciences (CAS) field. This workshop is part of the technology transfer plan of the High Performance Computing and Communications Program (HPCCP). Specific objectives of this Workshop are to: (1) communicate the goals and objectives of HPCCP in the area of CAS; (2) promote and disseminate CAS technology within the appropriate technical communities, including NASA, industry, academia, and other government labs; (3) help promote synergy among CAS scientists; and (4) permit feedback from peer researchers in issues pacing the CAS field in general and the HPCCP CAS program in particular.

  13. CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods.

    PubMed

    Jo, Young-Il; Suresh, Bharathi; Kim, Hyongbum; Ramakrishna, Suresh

    2015-12-01

    While human gene therapy has gained significant attention for its therapeutic promise, CRISPR/Cas9 technology has made a breakthrough as an efficient genome editing tool by emulating prokaryotic immune defense mechanisms. Although many studies have found that CRISPR/Cas9 technology is more efficient, specific and manipulable than previous generations of gene editing tools, it can be further improved by elevating its overall efficiency in a higher frequency of genome modifications and reducing its off-target effects. Here, we review the development of CRISPR/Cas9 technology, focusing on enhancement of its sequence specificity, reduction of off-target effects and delivery systems. Moreover, we describe recent successful applications of CRISPR/Cas9 technology in laboratory and clinical studies. PMID:26434948

  14. Evaluating alternative responses to safeguards alarms

    SciTech Connect

    Al-Ayat, R.A.; Judd, B.R.; McCord, R.K.

    1982-04-15

    This paper describes a quantitative approach to help evaluate and respond to safeguards alarms. These alarms may be generated internally by a facility's safeguards systems or externally by individuals claiming to have stolen special nuclear material (SNM). This approach can be used to identify the most likely cause of an alarm - theft, hoax, or error - and to evaluate alternative responses to alarms. Possible responses include conducting investigations, initiating measures to recover stolen SNM, and replying to external threats. Based on the results of each alarm investigation step, the evaluation revises the likelihoods of possible causes of an alarm, and uses this information to determine the optimal sequence of further responses. The choice of an optimal sequence of responses takes into consideration the costs and benefits of successful thefts or hoaxes. These results provide an analytical basis for setting priorities and developing contingency plans for responding to safeguards alarms.

  15. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system.

    PubMed

    Anton, Tobias; Bultmann, Sebastian; Leonhardt, Heinrich; Markaki, Yolanda

    2014-01-01

    Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture. PMID:24637835

  16. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

    PubMed Central

    Burmistrz, Michał; Dudek, Bartosz; Staniec, Dominika; Rodriguez Martinez, Jose Ignacio; Bochtler, Matthias; Potempa, Jan

    2015-01-01

    ABSTRACT The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3′ end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3′ terminus by the appropriate PAM element. IMPORTANCE The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial

  17. Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.

    PubMed

    Lin, Che-Yi; Su, Yi-Hsien

    2016-01-15

    Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60-80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67-100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos. PMID:26632489

  18. Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference

    PubMed Central

    Patterson, Adrian G.; Chang, James T.; Taylor, Corinda; Fineran, Peter C.

    2015-01-01

    The CRISPR-Cas prokaryotic ‘adaptive immune systems’ represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2–3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference. PMID:26007654

  19. 21 CFR 870.2640 - Portable leakage current alarm.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Portable leakage current alarm. 870.2640 Section... leakage current alarm. (a) Identification. A portable leakage current alarm is a device used to measure the electrical leakage current between any two points of an electrical system and to sound an alarm...

  20. 21 CFR 870.2640 - Portable leakage current alarm.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Portable leakage current alarm. 870.2640 Section... leakage current alarm. (a) Identification. A portable leakage current alarm is a device used to measure the electrical leakage current between any two points of an electrical system and to sound an alarm...

  1. 21 CFR 870.2640 - Portable leakage current alarm.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Portable leakage current alarm. 870.2640 Section... leakage current alarm. (a) Identification. A portable leakage current alarm is a device used to measure the electrical leakage current between any two points of an electrical system and to sound an alarm...

  2. 21 CFR 870.2640 - Portable leakage current alarm.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Portable leakage current alarm. 870.2640 Section... leakage current alarm. (a) Identification. A portable leakage current alarm is a device used to measure the electrical leakage current between any two points of an electrical system and to sound an alarm...

  3. 21 CFR 870.2640 - Portable leakage current alarm.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Portable leakage current alarm. 870.2640 Section... leakage current alarm. (a) Identification. A portable leakage current alarm is a device used to measure the electrical leakage current between any two points of an electrical system and to sound an alarm...

  4. 46 CFR 169.732 - Carbon dioxide alarm.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Carbon dioxide alarm. 169.732 Section 169.732 Shipping... Control, Miscellaneous Systems, and Equipment Markings § 169.732 Carbon dioxide alarm. Each carbon dioxide alarm must be conspicuously identified: “WHEN ALARM SOUNDS—VACATE AT ONCE. CARBON DIOXIDE BEING RELEASED.”...

  5. 46 CFR 169.732 - Carbon dioxide alarm.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Carbon dioxide alarm. 169.732 Section 169.732 Shipping... Control, Miscellaneous Systems, and Equipment Markings § 169.732 Carbon dioxide alarm. Each carbon dioxide alarm must be conspicuously identified: “WHEN ALARM SOUNDS—VACATE AT ONCE. CARBON DIOXIDE BEING RELEASED.”...

  6. Hypo- and Hyperglycemic Alarms

    PubMed Central

    Howsmon, Daniel; Bequette, B. Wayne

    2015-01-01

    Soon after the discovery that insulin regulates blood glucose by Banting and Best in 1922, the symptoms and risks associated with hypoglycemia became widely recognized. This article reviews devices to warn individuals of impending hypo- and hyperglycemia; biosignals used by these devices include electroencephalography, electrocardiography, skin galvanic resistance, diabetes alert dogs, and continuous glucose monitors (CGMs). While systems based on other technology are increasing in performance and decreasing in size, CGM technology remains the best method for both reactive and predictive alarming of hypo- or hyperglycemia. PMID:25931581

  7. The subtype I-F CRISPR-Cas system influences pathogenicity island retention in Pectobacterium atrosepticum via crRNA generation and Csy complex formation.

    PubMed

    Richter, Corinna; Fineran, Peter C

    2013-12-01

    CRISPR (clustered regularly interspaced short palindromic repeats) arrays and Cas (CRISPR-associated) proteins confer acquired resistance against mobile genetic elements in a wide range of bacteria and archaea. The phytopathogen Pectobacterium atrosepticum SCRI1043 encodes a single subtype I-F CRISPR system, which is composed of three CRISPR arrays and the cas operon encoding Cas1, Cas3 (a Cas2-Cas3 fusion), Csy1, Csy2, Csy3 and Cas6f (Csy4). The CRISPR arrays are transcribed into pre-crRNA (CRISPR RNA) and then processed by Cas6f to generate crRNAs. Furthermore, the formation of Cas protein complexes has been implicated in both the interference and acquisition stages of defence. In the present paper, we discuss the development of tightly controlled 'programmable' CRISPR arrays as tools to investigate CRISPR-Cas function and the effects of chromosomal targeting. Finally, we address how chromosomal targeting by CRISPR-Cas can cause large-scale genome deletions, which can ultimately influence bacterial evolution and pathogenicity. PMID:24256239

  8. Creating Genome Modifications in C. elegans Using the CRISPR/Cas9 System.

    PubMed

    Calarco, John A; Friedland, Ari E

    2015-01-01

    The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome. PMID:26423968

  9. Technical Basis for the Use of Alarming Personal Criticality Detectors to Augment Permanent Nuclear Incident Monitor (NIM) Systems in Areas Not Normally Occupied

    SciTech Connect

    Yates, K.R.

    2003-05-26

    The technical basis for the use of alarming personal criticality detectors (APCDs) to augment permanent Nuclear Incident Monitor (NIM) Systems in areas not normally occupied is evaluated. All applicable DOE O 420.1A and ANSI/ANS-8.3-1997 criticality alarm system requirements and recommendations are evaluated for applicability to APCDs. Based on this evaluation, design criteria and administrative requirements are presented for APCDs. Siemens EPD/Mk-2 and EPD-N devices are shown to meet the design criteria. A definition of not normally occupied is also presented.

  10. Efficiently Editing the Vaccinia Virus Genome by Using the CRISPR-Cas9 System

    PubMed Central

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa

    2015-01-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  11. Efficiently editing the vaccinia virus genome by using the CRISPR-Cas9 system.

    PubMed

    Yuan, Ming; Zhang, Wensheng; Wang, Jun; Al Yaghchi, Chadwan; Ahmed, Jahangir; Chard, Louisa; Lemoine, Nick R; Wang, Yaohe

    2015-05-01

    Vaccinia virus (VACV) continues to be used in immunotherapy for the prevention of infectious diseases and treatment of cancer since its use for the eradication of smallpox. However, the current method of editing the VACV genome is not efficient. Here, we demonstrate that the CRISPR-Cas9 system can be used to edit the VACV genome rapidly and efficiently. Additionally, a set of 8,964 computationally designed unique guide RNAs (gRNAs) targeting all VACV genes will be valuable for the study of VACV gene functions. PMID:25741005

  12. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Zhang, Qunyu; Zhu, Qinlong; Liu, Wei; Chen, Yan; Qiu, Rong; Wang, Bin; Yang, Zhongfang; Li, Heying; Lin, Yuru; Xie, Yongyao; Shen, Rongxin; Chen, Shuifu; Wang, Zhi; Chen, Yuanling; Guo, Jingxin; Chen, Letian; Zhao, Xiucai; Dong, Zhicheng; Liu, Yao-Guang

    2015-08-01

    CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement. PMID:25917172

  13. Speech Alarms Pilot Study

    NASA Technical Reports Server (NTRS)

    Sandor, A.; Moses, H. R.

    2016-01-01

    Currently on the International Space Station (ISS) and other space vehicles Caution & Warning (C&W) alerts are represented with various auditory tones that correspond to the type of event. This system relies on the crew's ability to remember what each tone represents in a high stress, high workload environment when responding to the alert. Furthermore, crew receive a year or more in advance of the mission that makes remembering the semantic meaning of the alerts more difficult. The current system works for missions conducted close to Earth where ground operators can assist as needed. On long duration missions, however, they will need to work off-nominal events autonomously. There is evidence that speech alarms may be easier and faster to recognize, especially during an off-nominal event. The Information Presentation Directed Research Project (FY07-FY09) funded by the Human Research Program included several studies investigating C&W alerts. The studies evaluated tone alerts currently in use with NASA flight deck displays along with candidate speech alerts. A follow-on study used four types of speech alerts to investigate how quickly various types of auditory alerts with and without a speech component - either at the beginning or at the end of the tone - can be identified. Even though crew were familiar with the tone alert from training or direct mission experience, alerts starting with a speech component were identified faster than alerts starting with a tone. The current study replicated the results from the previous study in a more rigorous experimental design to determine if the candidate speech alarms are ready for transition to operations or if more research is needed. Four types of alarms (caution, warning, fire, and depressurization) were presented to participants in both tone and speech formats in laboratory settings and later in the Human Exploration Research Analog (HERA). In the laboratory study, the alerts were presented by software and participants were

  14. Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1.

    PubMed

    Jakutyte-Giraitiene, Lina; Gasiunas, Giedrius

    2016-08-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes. PMID:27255973

  15. A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons.

    PubMed

    Williams, Michael R; Fricano-Kugler, Catherine J; Getz, Stephanie A; Skelton, Patrick D; Lee, Jeonghoon; Rizzuto, Christian P; Geller, Joseph S; Li, Meijie; Luikart, Bryan W

    2016-01-01

    Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals. PMID:27161796

  16. A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons

    PubMed Central

    Williams, Michael R.; Fricano-Kugler, Catherine J.; Getz, Stephanie A.; Skelton, Patrick D.; Lee, Jeonghoon; Rizzuto, Christian P.; Geller, Joseph S.; Li, Meijie; Luikart, Bryan W.

    2016-01-01

    Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals. PMID:27161796

  17. 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.

  18. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

    PubMed

    Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

    2016-05-15

    Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells. PMID:26525194

  19. XANES and Raman spectrometry on glasses and crystals in the CAS system.

    NASA Astrophysics Data System (ADS)

    Neuville, N.; Cormier, C.; Flank, F.; Massiot, M.

    2003-04-01

    XANES and Raman spectrometry on glasses and crystals in the CAS system. DANIEL R. NEUVILLE1, LAURENT CORMIER2 ANNE-MARIE FLANK3 and DOMINIQUE MASSIOT4 1Laboratoire de physico-chimie des fluides géologiques, IPGP-CNRS-UMR7047, 4 place Jussieu, 75252 Paris 2Laboratoire de Minéralogie et de Cristallographie, Universités PARIS 6 et 7, IPGP, UMR CNRS 7590, 4 place Jussieu, 75252 Paris 3Laboratoire pour l’Utilisation du Rayonnement Electromagnétique, Bat. 209D, B.P. 34, 91898, Orsay Cedex France 4UPR 4212, CNRS-CRMHT1d, avenue de la recherche scientifique F-45071 Orléans Cedex 2. Calcium aluminate and aluminosilicate glasses are attractive materials for a wide range of technical applications due to their highly refractory nature, their excellent optical and mechanical properties. The CaO-Al2O3-SiO2 system (CAS) is remarkable since glasses with very few SiO2 content can be synthesized, contrary to alkali or Mg aluminosilicate glasses. We have synthesized more than 40 different glasses in the CAS system using quenching method and 15 glasses using laser heating. These glasses were studied using a Raman spectrometer T64000 from Jobin-Yvon-Dilor company, X-ray absorption spectroscopy at Si, Al, Ca K edges the SA32 and D44 beamlines at LURE and NMR-700MHz. Cormier et al (2000) have shown using X-ray and neutron diffraction that aluminium is in 4-fold coordination in this ternary system. In this present study, we present Raman and XANES obtained at room temperature for these glasses. On the join SiO2-CaAl2O4 glass, we observed a decrease in Raman frequency with increasing CaAl2O4 content for all the bands. In particular, we observed a big decrease in frequency for the T4 band near 1150 cm-1 assigned to T-O0 in T4 units. This decrease suggests that aluminium substitutes principally for Si4+ in the fully polymerized structural units (TO2) according with Neuville and Mysen (1996). On the join SiO2-Ca3Al2O7 (R=CaO/AL2O3=3), we observed a decrease for all bands with

  20. 46 CFR 95.05-1 - Fire detecting, manual alarm, and supervised patrol systems.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... provided a smoke detecting or other suitable type fire detecting system. (c) Enclosed spaces which are “specially suitable for vehicles” shall be fitted with an approved fire or smoke detecting system....

  1. [CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants].

    PubMed

    Xingliang, Ma; Yaoguang, Liu

    2016-02-01

    Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression constructs, the transformation method for obtaining targeted mutations, the efficiency and features of the resulting mutations and the methods for detecting or genotyping of the mutation sites. We also discuss the existing problems and perspectives of CRISPR/Cas9-based genomic editing in plants. PMID:26907775

  2. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

    PubMed Central

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

  3. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

    PubMed

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-07-15

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

  4. Fanconi Anemia Gene Editing by the CRISPR/Cas9 System

    PubMed Central

    Osborn, Mark J.; Gabriel, Richard; Webber, Beau R.; DeFeo, Anthony P.; McElroy, Amber N.; Jarjour, Jordan; Starker, Colby G.; Wagner, John E.; Joung, J. Keith; Voytas, Daniel F.; von Kalle, Christof; Schmidt, Manfred; Blazar, Bruce R.

    2015-01-01

    Abstract Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder. PMID:25545896

  5. Genetic screens in human cells using the CRISPR/Cas9 system

    PubMed Central

    Wang, Tim; Wei, Jenny J.; Sabatini, David M.; Lander, Eric S.

    2014-01-01

    The bacterial CRISPR/Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Finally, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells. PMID:24336569

  6. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

    PubMed

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2013-01-01

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants. PMID:24112467

  7. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

    PubMed Central

    2013-01-01

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants. PMID:24112467

  8. 46 CFR 95.15-30 - Alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Alarms. 95.15-30 Section 95.15-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) CARGO AND MISCELLANEOUS VESSELS FIRE PROTECTION EQUIPMENT Carbon Dioxide Extinguishing Systems, Details § 95.15-30 Alarms. (a) Spaces which are protected by a carbon dioxide extinguishing system and...

  9. Black Juveniles in the Juvenile Justice System: A Cause for Alarm.

    ERIC Educational Resources Information Center

    LeFlore, Larry

    This report examines the representation of black youth in the juvenile justice system, describes changes in juvenile justice philosophy, and discusses policy implications. Black youth are overrepresented at all stages of the juvenile justice system compared to white youth. Positivist theories explain this overrepresentation as the result of…

  10. 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

  11. Implementation guidance for industrial-level security systems using radio frequency alarm links

    SciTech Connect

    Swank, R.G.

    1996-07-12

    Spread spectrum (SS) RF transmission technologies have properties that make the transmitted signal difficult to intercept, interpret, and jam. The digital code used in the modulation process results in a signal that has high reception reliability and supports multiple use of frequency bands and selective addressing. These attributes and the relatively low installation cost of RF systems make SSRF technologies candidate for communications links in security systems used for industrial sites, remote locations, and where trenching or other disturbances of soil or structures may not be desirable or may be costly. This guide provides a description of such a system and presents implementation methods that may be of engineering benefit.

  12. Improved detection and false alarm rejection using FLGPR and color imagery in a forward-looking system

    NASA Astrophysics Data System (ADS)

    Havens, Timothy C.; Spain, Christopher J.; Ho, K. C.; Keller, James M.; Ton, Tuan T.; Wong, David C.; Soumekh, Mehrdad

    2010-04-01

    Forward-looking ground-penetrating radar (FLGPR) has received a significant amount of attention for use in explosivehazards detection. A drawback to FLGPR is that it results in an excessive number of false detections. This paper presents our analysis of the explosive-hazards detection system tested by the U.S. Army Night Vision and Electronic Sensors Directorate (NVESD). The NVESD system combines an FLGPR with a visible-spectrum color camera. We present a target detection algorithm that uses a locally-adaptive detection scheme with spectrum-based features. The remaining FLGPR detections are then projected into the camera imagery and image-based features are collected. A one-class classifier is then used to reduce the number of false detections. We show that our proposed FLGPR target detection algorithm, coupled with our camera-based false alarm (FA) reduction method, is effective at reducing the number of FAs in test data collected at a US Army test facility.

  13. 33 CFR 149.130 - What are the requirements for the cargo transfer system alarm?

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... EQUIPMENT Pollution Prevention Equipment § 149.130 What are the requirements for the cargo transfer system... in areas of high ambient noise levels where hearing protection is required under § 150.615 of...

  14. 33 CFR 149.130 - What are the requirements for the cargo transfer system alarm?

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... EQUIPMENT Pollution Prevention Equipment § 149.130 What are the requirements for the cargo transfer system... in areas of high ambient noise levels where hearing protection is required under § 150.615 of...

  15. 33 CFR 149.130 - What are the requirements for the cargo transfer system alarm?

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... EQUIPMENT Pollution Prevention Equipment § 149.130 What are the requirements for the cargo transfer system... in areas of high ambient noise levels where hearing protection is required under § 150.615 of...

  16. 33 CFR 149.130 - What are the requirements for the cargo transfer system alarm?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... EQUIPMENT Pollution Prevention Equipment § 149.130 What are the requirements for the cargo transfer system... in areas of high ambient noise levels where hearing protection is required under § 150.615 of...

  17. 33 CFR 149.130 - What are the requirements for the cargo transfer system alarm?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... EQUIPMENT Pollution Prevention Equipment § 149.130 What are the requirements for the cargo transfer system... in areas of high ambient noise levels where hearing protection is required under § 150.615 of...

  18. High-Efficiency Multiplex Genome Editing of Streptomyces Species Using an Engineered CRISPR/Cas System

    PubMed Central

    2015-01-01

    Actinobacteria, particularly those of genus Streptomyces, remain invaluable hosts for the discovery and engineering of natural products and their cognate biosynthetic pathways. However, genetic manipulation of these bacteria is often labor and time intensive. Here, we present an engineered CRISPR/Cas system for rapid multiplex genome editing of Streptomyces strains, demonstrating targeted chromosomal deletions in three different Streptomyces species and of various sizes (ranging from 20 bp to 30 kb) with efficiency ranging from 70 to 100%. The designed pCRISPomyces plasmids are amenable to assembly of spacers and editing templates via Golden Gate assembly and isothermal assembly (or traditional digestion/ligation), respectively, allowing rapid plasmid construction to target any genomic locus of interest. As such, the pCRISPomyces system represents a powerful new tool for genome editing in Streptomyces. PMID:25458909

  19. Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

    PubMed

    Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

    2016-01-01

    Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations. PMID:27557690

  20. False alarms and missed events: the impact and origins of perceived inaccuracy in tornado warning systems.

    PubMed

    Ripberger, Joseph T; Silva, Carol L; Jenkins-Smith, Hank C; Carlson, Deven E; James, Mark; Herron, Kerry G

    2015-01-01

    Theory and conventional wisdom suggest that errors undermine the credibility of tornado warning systems and thus decrease the probability that individuals will comply (i.e., engage in protective action) when future warnings are issued. Unfortunately, empirical research on the influence of warning system accuracy on public responses to tornado warnings is incomplete and inconclusive. This study adds to existing research by analyzing two sets of relationships. First, we assess the relationship between perceptions of accuracy, credibility, and warning response. Using data collected via a large regional survey, we find that trust in the National Weather Service (NWS; the agency responsible for issuing tornado warnings) increases the likelihood that an individual will opt for protective action when responding to a hypothetical warning. More importantly, we find that subjective perceptions of warning system accuracy are, as theory suggests, systematically related to trust in the NWS and (by extension) stated responses to future warnings. The second half of the study matches survey data against NWS warning and event archives to investigate a critical follow-up question--Why do some people perceive that their warning system is accurate, whereas others perceive that their system is error prone? We find that subjective perceptions are--in part-a function of objective experience, knowledge, and demographic characteristics. When considered in tandem, these findings support the proposition that errors influence perceptions about the accuracy of warning systems, which in turn impact the credibility that people assign to information provided by systems and, ultimately, public decisions about how to respond when warnings are issued. PMID:25082540

  1. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

    PubMed

    Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

    2014-01-01

    Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice. PMID:25027812

  2. W-026, acceptance test report fire alarm system (submittal number 1571.1)

    SciTech Connect

    Watson, T.L., Westinghouse Hanford

    1996-12-16

    This Acceptance Test Report was written by 3-D Protection Systems, Inc., and PCL Construction. WRAP I Facility Engineering, Solid Waste Fire Safety, Kaiser Acceptance Inspector and Hanford Fire Department personnel witnessed this test. All exceptions were resolved. The resolutions are attached. Contractor`s Material and Test Certificates are attached. Results from Solid Waste Industrial Hygiene sound level surveys are also included.

  3. 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

  4. 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

    Pearson, Bruce M; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H M

    2015-09-01

    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

  5. 46 CFR 193.15-30 - Alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Alarms. 193.15-30 Section 193.15-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS FIRE PROTECTION EQUIPMENT Carbon Dioxide and Clean Agent Extinguishing Systems, Details § 193.15-30 Alarms. (a) Space normally accessible to persons on board while...

  6. 46 CFR 95.15-30 - Alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Dioxide Extinguishing Systems, Details § 95.15-30 Alarms. (a) Spaces which are protected by a carbon... audible alarm in such spaces which will be automatically sounded when the carbon dioxide is admitted to... sound during the 20 second delay period prior to the discharge of carbon dioxide into the space, and...

  7. 46 CFR 130.470 - Fire alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Fire alarms. 130.470 Section 130.470 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.470 Fire alarms. (a)...

  8. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  9. 46 CFR 130.470 - Fire alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Fire alarms. 130.470 Section 130.470 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.470 Fire alarms. (a)...

  10. 46 CFR 130.470 - Fire alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Fire alarms. 130.470 Section 130.470 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.470 Fire alarms. (a)...

  11. 46 CFR 130.470 - Fire alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Fire alarms. 130.470 Section 130.470 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.470 Fire alarms. (a)...

  12. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  13. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  14. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  15. 46 CFR 130.470 - Fire alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Fire alarms. 130.470 Section 130.470 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.470 Fire alarms. (a)...

  16. 46 CFR 193.15-30 - Alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Alarms. 193.15-30 Section 193.15-30 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OCEANOGRAPHIC RESEARCH VESSELS FIRE PROTECTION EQUIPMENT Carbon Dioxide and Clean Agent Extinguishing Systems, Details § 193.15-30 Alarms. (a) Space normally accessible to persons on board while...

  17. 46 CFR 130.450 - Machinery alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Machinery alarms. 130.450 Section 130.450 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) OFFSHORE SUPPLY VESSELS VESSEL CONTROL, AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.450 Machinery alarms....

  18. Gene inactivation using the CRISPR/Cas9 system in the nematode Pristionchus pacificus.

    PubMed

    Witte, Hanh; Moreno, Eduardo; Rödelsperger, Christian; Kim, Jungeun; Kim, Jin-Soo; Streit, Adrian; Sommer, Ralf J

    2015-01-01

    The diplogastrid nematode Pristionchus pacificus is a nematode model system for comparative studies to Caenorhabditis elegans and integrative evolutionary biology aiming for interdisciplinary approaches of evo-devo, population genetics, and ecology. For this, fieldwork can be combined with laboratory studies, and P. pacificus has a well-developed methodological toolkit of forward genetics, whole genome sequencing, DNA-mediated transformation, and various -omics platforms. Here, we establish CRISPR/Cas9-based gene inactivation and describe various boundary conditions of this methodology for P. pacificus. Specifically, we demonstrate that most mutations arise within the first 9 hours after injections. We systematically tested the efficiency of sgRNAs targeting different exons in Ppa-dpy-1 and characterized the molecular nature of the induced mutations. Finally, we provide a protocol that might also be useful for researchers working with other non-Caenorhabditis nematodes. PMID:25548084

  19. Research of pulse signal processing based on sleep-monitoring alarm system

    NASA Astrophysics Data System (ADS)

    Zhang, Kaisheng; Zeng, Yuan

    2009-07-01

    From pulse diagnosis of Chinese herbalist doctor to the research of cardiovascular system by modem iatrology,they all have showed and proved that human pulse has a good affinity with diseases,especially cardiovascular diseases. Human pulse contains much physical information, and it will be propitious to know the human healthy state early so as to get therapy and recovery early when pulse signal is often detected and analyzed. study how to use the embedded microcontroller to transmit physiological signal from human to personal computer by infrared communication, and the normal sphygmic parameter in one's sleeping is compared with the one measured in order to judge whether one's sleeping condition is normal, finally ascertain the best control plan.

  20. Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

    PubMed

    Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

    2016-01-01

    Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability. PMID:24615461

  1. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice

    PubMed Central

    2014-01-01

    Background Microinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3 days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos. Results T7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25–100% of the embryos were knockout embryos and 7–57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay. Conclusions The single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos. PMID:25042988

  2. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    PubMed

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-01-01

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN. PMID:25236476

  3. Search for contact systems among EB-type binaries. IV - V375 Cas, UW Ori, DO Cas, RU ERI

    NASA Astrophysics Data System (ADS)

    Barone, F.; di Fiore, L.; Milano, L.; Pirozzi, L.; Russo, G.

    1992-12-01

    We present the analysis of the data of four EB-type eclipsing binaries, continuing our search for contact or almost contact systems. The Price algorithm has been used in conjunction to the Wilson-Devinney model to try to obtain, where possible, unambiguous solutions for all the systems.

  4. 8. INTERIOR, FIRE ALARM CONTROL ROOM (NORTH OF MAIN GARAGE), ...

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

    8. INTERIOR, FIRE ALARM CONTROL ROOM (NORTH OF MAIN GARAGE), FROM ENTRYWAY, LOOKING NORTH, SHOWING ADDITIONAL 'GAMEWELL' FIRE ALARM SYSTEMS. - Oakland Naval Supply Center, Firehouse, East of Fourth Street, between A & B Streets, Oakland, Alameda County, CA

  5. Decision-making and response strategies in interaction with alarms: the impact of alarm reliability, availability of alarm validity information and workload.

    PubMed

    Manzey, Dietrich; Gérard, Nina; Wiczorek, Rebecca

    2014-01-01

    Responding to alarm systems which usually commit a number of false alarms and/or misses involves decision-making under uncertainty. Four laboratory experiments including a total of 256 participants were conducted to gain comprehensive insight into humans' dealing with this uncertainty. Specifically, it was investigated how responses to alarms/non-alarms are affected by the predictive validities of these events, and to what extent response strategies depend on whether or not the validity of alarms/non-alarms can be cross-checked against other data. Among others, the results suggest that, without cross-check possibility (experiment 1), low levels of predictive validity of alarms ( ≤ 0.5) led most participants to use one of two different strategies which both involved non-responding to a significant number of alarms (cry-wolf effect). Yet, providing access to alarm validity information reduced this effect dramatically (experiment 2). This latter result emerged independent of the effort needed for cross-checkings of alarms (experiment 3), but was affected by the workload imposed by concurrent tasks (experiment 4). Theoretical and practical consequences of these results for decision-making and response selection in interaction with alarm systems, as well as the design of effective alarm systems, are discussed. PMID:25224606

  6. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.

    PubMed

    Whitworth, Kristin M; Lee, Kiho; Benne, Joshua A; Beaton, Benjamin P; Spate, Lee D; Murphy, Stephanie L; Samuel, Melissa S; Mao, Jiude; O'Gorman, Chad; Walters, Eric M; Murphy, Clifton N; Driver, John; Mileham, Alan; McLaren, David; Wells, Kevin D; Prather, Randall S

    2014-09-01

    Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications. PMID:25100712

  7. CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

    PubMed

    Yan, Meng; Zhou, Shi-Rong; Xue, Hong-Wei

    2015-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system. PMID:25319067

  8. Smart smoke alarm

    DOEpatents

    Warmack, Robert J. Bruce; Wolf, Dennis A; Frank, Steven Shane

    2015-04-28

    Methods and apparatus for smoke detection are disclosed. In one embodiment, a smoke detector uses linear discriminant analysis (LDA) to determine whether observed conditions indicate that an alarm is warranted.

  9. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.

    PubMed

    Hisano, Yu; Sakuma, Tetsushi; Nakade, Shota; Ohga, Rie; Ota, Satoshi; Okamoto, Hitoshi; Yamamoto, Takashi; Kawahara, Atsuo

    2015-01-01

    The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish. PMID:25740433

  10. Speech Alarms Pilot Study

    NASA Technical Reports Server (NTRS)

    Sandor, Aniko; Moses, Haifa

    2016-01-01

    Speech alarms have been used extensively in aviation and included in International Building Codes (IBC) and National Fire Protection Association's (NFPA) Life Safety Code. However, they have not been implemented on space vehicles. Previous studies conducted at NASA JSC showed that speech alarms lead to faster identification and higher accuracy. This research evaluated updated speech and tone alerts in a laboratory environment and in the Human Exploration Research Analog (HERA) in a realistic setup.

  11. Lymphoid-rich effusions. Diagnosis by morphometry using the CAS 200 System.

    PubMed

    Walts, A E; Svidler, R; Tolmachoff, T; Marchevsky, A M

    1994-04-01

    Lymphoid-rich effusions frequently present diagnostic problems in clinical cytology. In the authors' previous studies, most lymphoid-rich effusions had been correctly classified as benign lymphocytosis or malignant lymphoma by an experimental computerized interactive morphometry system, in which randomly selected lymphoid nuclear profile images were measured in Papanicolaou fixed and stained cytospin smears. The present study used the CAS 200 System and criteria from the previously described rule-based expert system to classify similar preparations of 134 lymphoid rich pleural, peritoneal, and pericardial effusions (90 benign lymphocytoses, 36 malignant lymphomas, and 8 chronic lymphocytic leukemias). A total of 98.9% of the benign lymphocytoses and 88.9% of the malignant lymphomas were correctly classified (predictive values of correct diagnoses 95.7% and 97.3%, respectively). Chronic lymphocytic leukemias could not be distinguished from benign lymphocytoses by nuclear profile areas. Optical density histograms of benign, lymphomatous, and chronic lymphocytic leukemias effusions are described. Advantages and limitations of image analysis and immunocytochemistry are discussed. PMID:8160646

  12. A CRISPR/Cas9 system adapted for gene editing in marine algae

    PubMed Central

    Nymark, Marianne; Sharma, Amit Kumar; Sparstad, Torfinn; Bones, Atle M.; Winge, Per

    2016-01-01

    Here we report that the CRISPR/Cas9 technology can be used to efficiently generate stable targeted gene mutations in microalgae, using the marine diatom Phaeodactylum tricornutum as a model species. Our vector design opens for rapid and easy adaption of the construct to the target chosen. To screen for CRISPR/Cas9 mutants we employed high resolution melting based PCR assays, mutants were confirmed by sequencing and further validated by functional analyses. PMID:27108533

  13. A CRISPR/Cas9 system adapted for gene editing in marine algae.

    PubMed

    Nymark, Marianne; Sharma, Amit Kumar; Sparstad, Torfinn; Bones, Atle M; Winge, Per

    2016-01-01

    Here we report that the CRISPR/Cas9 technology can be used to efficiently generate stable targeted gene mutations in microalgae, using the marine diatom Phaeodactylum tricornutum as a model species. Our vector design opens for rapid and easy adaption of the construct to the target chosen. To screen for CRISPR/Cas9 mutants we employed high resolution melting based PCR assays, mutants were confirmed by sequencing and further validated by functional analyses. PMID:27108533

  14. Complete genome sequence of Pedobacter cryoconitis PAMC 27485, a CRISPR-Cas system-containing psychrophile isolated from Antarctica.

    PubMed

    Lee, Jaejin; Jung, You-Jung; Lee, Hong Kum; Hong, Soon Gyu; Kim, Ok-Sun

    2016-05-20

    Pedobacter cryoconitis PAMC 27485, an aerobic, Gram-negative, facultatively psychrophilic bacterium, was isolated from Antarctic soil. Here we report the complete genome of P. cryoconitis PAMC 27485, which contains a type II CRISPR-Cas system and genes encoding useful enzymes (e.g. proteases). The genome sequence of P. cryoconitis PAMC 27485 could provide insights into its adaptive immune system against foreign genetic elements and biotechnological potential. PMID:27015980

  15. Predominance of Single Prophage Carrying a CRISPR/cas System in "Candidatus Liberibacter asiaticus" Strains in Southern China.

    PubMed

    Zheng, Zheng; Bao, Minli; Wu, Fengnian; Chen, Jianchi; Deng, Xiaoling

    2016-01-01

    "Candidatus Liberibacter asiaticus" (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the "Ca. Liberibacter" genera. PMID:26741827

  16. Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

    PubMed Central

    Zheng, Zheng; Bao, Minli; Wu, Fengnian; Chen, Jianchi; Deng, Xiaoling

    2016-01-01

    “Candidatus Liberibacter asiaticus” (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the “Ca. Liberibacter” genera. PMID:26741827

  17. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  18. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  19. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  20. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  1. 46 CFR 119.530 - Bilge high level alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Bilge high level alarms. 119.530 Section 119.530... Bilge and Ballast Systems § 119.530 Bilge high level alarms. (a) Each vessel must be provided with a visual and audible alarm at the operating station to indicate a high water level in each of the...

  2. 46 CFR 169.731 - General alarm bells.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false General alarm bells. 169.731 Section 169.731 Shipping... Control, Miscellaneous Systems, and Equipment Markings § 169.731 General alarm bells. On vessels of 100 gross tons and over each general alarm bell must be identified by red lettering at least 1/2 inch...

  3. 46 CFR 169.730 - General alarm bell switch.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false General alarm bell switch. 169.730 Section 169.730... Vessel Control, Miscellaneous Systems, and Equipment Markings § 169.730 General alarm bell switch. On vessels of 100 gross tons and over there must be a general alarm bell switch in the pilothouse,...

  4. 46 CFR 130.460 - Placement of machinery alarms.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Placement of machinery alarms. 130.460 Section 130.460..., AND MISCELLANEOUS EQUIPMENT AND SYSTEMS Automation of Unattended Machinery Spaces § 130.460 Placement of machinery alarms. (a) Visible and audible alarms must be installed at the pilothouse to...

  5. CAS. Controlled Access Security

    SciTech Connect

    Martinez, B.; Pomeroy, G.

    1989-12-01

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  6. Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B

    PubMed Central

    Maier, Lisa-Katharina; Lange, Sita J.; Stoll, Britta; Haas, Karina A.; Fischer, Susan; Fischer, Eike; Duchardt-Ferner, Elke; Wöhnert, Jens; Backofen, Rolf; Marchfelder, Anita

    2013-01-01

    To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5′ handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid. PMID:23594992

  7. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system.

    PubMed

    Song, Yuning; Yuan, Lin; Wang, Yong; Chen, Mao; Deng, Jichao; Lv, Qingyan; Sui, Tingting; Li, Zhanjun; Lai, Liangxue

    2016-08-01

    The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms. PMID:26817461

  8. Smoke alarm tests may not adequately indicate smoke alarm function.

    PubMed

    Peek-Asa, Corinne; Yang, Jingzhen; Hamann, Cara; Young, Tracy

    2011-01-01

    Smoke alarms are one of the most promoted prevention strategies to reduce residential fire deaths, and they can reduce residential fire deaths by half. Smoke alarm function can be measured by two tests: the smoke alarm button test and the chemical smoke test. Using results from a randomized trial of smoke alarms, we compared smoke alarm response to the button test and the smoke test. The smoke alarms found in the study homes at baseline were tested, as well as study alarms placed into homes as part of the randomized trial. Study alarms were tested at 12 and 42 months postinstallation. The proportion of alarms that passed the button test but not the smoke test ranged from 0.5 to 5.8% of alarms; this result was found most frequently among ionization alarms with zinc or alkaline batteries. These alarms would indicate to the owner (through the button test) that the smoke alarm was working, but the alarm would not actually respond in the case of a fire (as demonstrated by failing the smoke test). The proportion of alarms that passed the smoke test but not the button test ranged from 1.0 to 3.0%. These alarms would appear nonfunctional to the owner (because the button test failed), even though the alarm would operate in response to a fire (as demonstrated by passing the smoke test). The general public is not aware of the potential for inaccuracy in smoke alarm tests, and burn professionals can advocate for enhanced testing methods. The optimal test to determine smoke alarm function is the chemical smoke test. PMID:21747329

  9. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system

    PubMed Central

    Maddalo, Danilo; Manchado, Eusebio; Concepcion, Carla P.; Bonetti, Ciro; Vidigal, Joana A.; Han, Yoon-Chi; Ogrodowski, Paul; Crippa, Alessandra; Rekhtman, Natasha; de Stanchina, Elisa; Lowe, Scott W.; Ventura, Andrea

    2014-01-01

    Chromosomal rearrangements play a central role in the pathogenesis of human cancers and often result in the expression of therapeutically actionable gene fusions1. A recently discovered example is a fusion between the Echinoderm Microtubule-associated Protein-like 4 (EML4) and the Anaplastic Lymphoma Kinase (ALK) genes, generated by an inversion on the short arm of chromosome 2: inv(2)(p21p23). The EML4-ALK oncogene is detected in a subset of human non-small cell lung cancers (NSCLC)2 and is clinically relevant because it confers sensitivity to ALK inhibitors3. Despite their importance, modeling such genetic events in mice has proven challenging and requires complex manipulation of the germline. Here we describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. We apply it to generate a mouse model of Eml4-Alk-driven lung cancer. The resulting tumors invariably harbor the Eml4-Alkinversion, express the Eml4-Alk fusion gene, display histo-pathologic and molecular features typical of ALK+ human NSCLCs, and respond to treatment with ALK-inhibitors. The general strategy described here substantially expands our ability to model human cancers in mice and potentially in other organisms. PMID:25337876

  10. Generation of muscular dystrophy model rats with a CRISPR/Cas system.

    PubMed

    Nakamura, Katsuyuki; Fujii, Wataru; Tsuboi, Masaya; Tanihata, Jun; Teramoto, Naomi; Takeuchi, Shiho; Naito, Kunihiko; Yamanouchi, Keitaro; Nishihara, Masugi

    2014-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD. PMID:25005781

  11. Alarm points for fixed oxygen monitors

    SciTech Connect

    Miller, G.C.

    1987-05-01

    Oxygen concentration monitors were installed in a vault where numerous pipes carried inert cryogens and gases to the Mirror Fusion Test Facility (MFTF-B) experimental vessel at Lawrence Livermore National Laboratory (LLNL). The problems associated with oxygen-monitoring systems and the reasons why such monitors were installed were reviewed. As a result of this review, the MFTF-B monitors were set to sound an evacuation alarm when the oxygen concentration fell below 18%. We chose the 18% alarm criterion to minimize false alarms and to allow time for personnel to escape in an oxygen-deficient environment.

  12. Clinical Alarms in Intensive Care Units: Perceived Obstacles of Alarm Management and Alarm Fatigue in Nurses

    PubMed Central

    Cho, Ok Min; Lee, Young Whee; Cho, Insook

    2016-01-01

    Objectives The purpose of this descriptive study was to investigate the current situation of clinical alarms in intensive care unit (ICU), nurses' recognition of and fatigue in relation to clinical alarms, and obstacles in alarm management. Methods Subjects were ICU nurses and devices from 48 critically ill patient cases. Data were collected through direct observation of alarm occurrence and questionnaires that were completed by the ICU nurses. The observation time unit was one hour block. One bed out of 56 ICU beds was randomly assigned to each observation time unit. Results Overall 2,184 clinical alarms were counted for 48 hours of observation, and 45.5 clinical alarms occurred per hour per subject. Of these, 1,394 alarms (63.8%) were categorized as false alarms. The alarm fatigue score was 24.3 ± 4.0 out of 35. The highest scoring item was "always get bothered due to clinical alarms". The highest scoring item in obstacles was "frequent false alarms, which lead to reduced attention or response to alarms". Conclusions Nurses reported that they felt some fatigue due to clinical alarms, and false alarms were also obstacles to proper management. An appropriate hospital policy should be developed to reduce false alarms and nurses' alarm fatigue. PMID:26893950

  13. Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

    PubMed Central

    Li, Kai; Wang, Gang; Andersen, Troels; Zhou, Pingzhu; Pu, William T.

    2014-01-01

    Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease. PMID:25166277

  14. Multistation alarm system for eruptive activity based on the automatic classification of volcanic tremor: specifications and performance

    NASA Astrophysics Data System (ADS)

    Langer, Horst; Falsaperla, Susanna; Messina, Alfio; Spampinato, Salvatore

    2015-04-01

    With over fifty eruptive episodes (Strombolian activity, lava fountains, and lava flows) between 2006 and 2013, Mt Etna, Italy, underscored its role as the most active volcano in Europe. Seven paroxysmal lava fountains at the South East Crater occurred in 2007-2008 and 46 at the New South East Crater between 2011 and 2013. Month-lasting lava emissions affected the upper eastern flank of the volcano in 2006 and 2008-2009. On this background, effective monitoring and forecast of volcanic phenomena are a first order issue for their potential socio-economic impact in a densely populated region like the town of Catania and its surroundings. For example, explosive activity has often formed thick ash clouds with widespread tephra fall able to disrupt the air traffic, as well as to cause severe problems at infrastructures, such as highways and roads. For timely information on changes in the state of the volcano and possible onset of dangerous eruptive phenomena, the analysis of the continuous background seismic signal, the so-called volcanic tremor, turned out of paramount importance. Changes in the state of the volcano as well as in its eruptive style are usually concurrent with variations of the spectral characteristics (amplitude and frequency content) of tremor. The huge amount of digital data continuously acquired by INGV's broadband seismic stations every day makes a manual analysis difficult, and techniques of automatic classification of the tremor signal are therefore applied. The application of unsupervised classification techniques to the tremor data revealed significant changes well before the onset of the eruptive episodes. This evidence led to the development of specific software packages related to real-time processing of the tremor data. The operational characteristics of these tools - fail-safe, robustness with respect to noise and data outages, as well as computational efficiency - allowed the identification of criteria for automatic alarm flagging. The

  15. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  16. [High-throughput functional screening using CRISPR/Cas9 system].

    PubMed

    Wang, Gancheng; Ming, Ma; Ye, Yanzhen; Xi, Jianzhong

    2016-05-01

    High-throughput screening, a powerful tool for the discovery of functionally important genes responsible for certain phenotypes, is performed according to loss-of-function or gain-of-function strategies. RNAi technology or knockout approaches have been widely used in high throughput screening due to their advantages of ease use, low cost and so on. However, imcomplete knockdown activity and off-target effect hindered their utility. More recently, CRISPR/Cas9 technology is becoming a robust tool for genome editing in diverse cells or animals, since it could generate a gene mutation in a target-specific manner. In this review, we first summarize the characterization of CRISPR/Cas9 and make comparison with traditional genetic tools, then describe recent achievements of genetic screen in several model organisms using CRISPR/Cas9, finally discuss on its future challenges and opportunities. PMID:27232487

  17. Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system.

    PubMed

    Wang, Xiaolong; Yu, Honghao; Lei, Anmin; Zhou, Jiankui; Zeng, Wenxian; Zhu, Haijing; Dong, Zhiming; Niu, Yiyuan; Shi, Bingbo; Cai, Bei; Liu, Jinwang; Huang, Shuai; Yan, Hailong; Zhao, Xiaoe; Zhou, Guangxian; He, Xiaoling; Chen, Xiaoxu; Yang, Yuxin; Jiang, Yu; Shi, Lei; Tian, Xiue; Wang, Yongjun; Ma, Baohua; Huang, Xingxu; Qu, Lei; Chen, Yulin

    2015-01-01

    Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding. PMID:26354037

  18. A CRISPR-Cas9 Gene Drive System Targeting Female Reproduction in the Malaria Mosquito vector Anopheles gambiae

    PubMed Central

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony

    2016-01-01

    Gene-drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years [AU please provide a real estimate, this seems vague]. We describe CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria [AU:OK?]. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene-drive constructs designed to target and edit each gene [AU:OK?]. For each locus targeted we observed strong gene drive at the molecular level, with transmission rates to progeny of 91 to 99.6%. Population modelling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to control suppress mosquito populations to levels that do not support malaria transmission. PMID:26641531

  19. Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli.

    PubMed

    Pyne, Michael E; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2015-08-01

    To date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less. PMID:26002895

  20. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

    PubMed Central

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L.; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

  1. [A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system].

    PubMed

    Qihan, Wang; Cong, Huai; Ruilin, Sun; Hua, Zhuang; Hongyan, Chen; Jian, Fei; Daru, Lu

    2015-11-01

    Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factor Ⅸ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtained FⅨ-knockout mouse strains with phenotype of hemophilia B with the CRISPR/Cas system efficiently. We chose the 8th exon as the target locus, and co-injected codon-optimized Cas9 mRNA with sgRNA of FⅨ into C57BL/6 mice zygotes. We obtained 60 mice in total and genotyped them by high resolution melting (HRM) and sequencing. The results showed the mutation rate was 85.0% in total, and 79.5% and 95.2% in males and females, respectively. No off-targets were detected in the similar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significantly below the normal level and reduced to 6.82% of wild-type mice. The activity assay demonstrated that all the mutant mice were lack of FⅨ. In summary, we have generated hemophilia B model mice with extreme efficiency, using the RNA-guided Cas9 nuclease gene editing system. PMID:26582528

  2. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

    PubMed

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

  3. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

    PubMed

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony

    2016-01-01

    Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission. PMID:26641531

  4. Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems.

    PubMed

    Jin, Li-Fang; Li, Jin-Song

    2016-07-18

    With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

  5. Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

    PubMed Central

    JIN, Li-Fang; LI, Jin-Song

    2016-01-01

    With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

  6. Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

    PubMed Central

    Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

    2015-01-01

    CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

  7. 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…

  8. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

    PubMed

    Shao, Ming; Xu, Tian-Rui; Chen, Ce-Shi

    2016-07-18

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine. PMID:27469250

  9. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

    PubMed Central

    SHAO, Ming; XU, Tian-Rui; CHEN, Ce-Shi

    2016-01-01

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and biomedicine. PMID:27469250

  10. Applications of the Petri net to simulate, test, and validate the performance and safety of complex, heterogeneous, multi-modality patient monitoring alarm systems.

    PubMed

    Sloane, E B; Gelhot, V

    2004-01-01

    This research is motivated by the rapid pace of medical device and information system integration. Although the ability to interconnect many medical devices and information systems may help improve patient care, there is no way to detect if incompatibilities between one or more devices might cause critical events such as patient alarms to go unnoticed or cause one or more of the devices to become stuck in a disabled state. Petri net tools allow automated testing of all possible states and transitions between devices and/or systems to detect potential failure modes in advance. This paper describes an early research project to use Petri nets to simulate and validate a multi-modality central patient monitoring system. A free Petri net tool, HPSim, is used to simulate two wireless patient monitoring networks: one with 44 heart monitors and a central monitoring system and a second version that includes an additional 44 wireless pulse oximeters. In the latter Petri net simulation, a potentially dangerous heart arrhythmia and pulse oximetry alarms were detected. PMID:17271039

  11. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

    PubMed Central

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B.

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing. PMID:26053390

  12. Sensor fusion for intelligent alarm analysis

    SciTech Connect

    Nelson, C.L.; Fitzgerald, D.S.

    1995-03-01

    The purpose of an intelligent alarm analysis system is to provide complete and manageable information to a central alarm station operator by applying alarm processing and fusion techniques to sensor information. This paper discusses the sensor fusion approach taken to perform intelligent alarm analysis for the Advanced Exterior Sensor (AES). The AES is an intrusion detection and assessment system designed for wide-area coverage, quick deployment, low false/nuisance alarm operation, and immediate visual assessment. It combines three sensor technologies (visible, infrared, and millimeter wave radar) collocated on a compact and portable remote sensor module. The remote sensor module rotates at a rate of 1 revolution per second to detect and track motion and provide assessment in a continuous 360` field-of-regard. Sensor fusion techniques are used to correlate and integrate the track data from these three sensors into a single track for operator observation. Additional inputs to the fusion process include environmental data, knowledge of sensor performance under certain weather conditions, sensor priority, and recent operator feedback. A confidence value is assigned to the track as a result of the fusion process. This helps to reduce nuisance alarms and to increase operator confidence in the system while reducing the workload of the operator.

  13. 46 CFR 113.25-9 - Location of general emergency alarm signal.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Location of general emergency alarm signal. 113.25-9 Section 113.25-9 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT General Emergency Alarm Systems § 113.25-9 Location of general emergency alarm signal....

  14. Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

    PubMed Central

    Box, Allison M.; McGuffie, Matthew J.; O'Hara, Brendan J.

    2015-01-01

    ABSTRACT The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3′ TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCE Cholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor

  15. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

    PubMed

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-07-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  16. Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    PubMed Central

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-01-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  17. Examinations in the Final Year of Transition to Mathematical Methods Computer Algebra System (CAS)

    ERIC Educational Resources Information Center

    Leigh-Lancaster, David; Les, Magdalena; Evans, Michael

    2010-01-01

    2009 was the final year of parallel implementation for Mathematical Methods Units 3 and 4 and Mathematical Methods (CAS) Units 3 and 4. From 2006-2009 there was a common technology-free short answer examination that covered the same function, algebra, calculus and probability content for both studies with corresponding expectations for key…

  18. Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

    PubMed

    Qazi, Shefah; Miettinen, Heini M; Wilkinson, Royce A; McCoy, Kimberly; Douglas, Trevor; Wiedenheft, Blake

    2016-03-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9. PMID:26894836

  19. 105KE Basin Area Radiation Monitor System (ARMS) Acceptance Test Procedure

    SciTech Connect

    KINKEL, C.C.

    1999-12-14

    This procedure is intended for the Area Radiation Monitoring System, ARMS, that is replacing the existing Programmable Input-Output Processing System, PIOPS, radiation monitoring system in the 105KE basin. The new system will be referred to as the 105KE ARMS, 105KE Area Radiation Monitoring System. This ATP will ensure calibration integrity of the 105KE radiation detector loops. Also, this ATP will test and document the display, printing, alarm output, alarm acknowledgement, upscale check, and security functions. This ATP test is to be performed after completion of the 105KE ARMS installation. The alarm outputs of the 105KE ARMS will be connected to the basin detector alarms, basin annunciator system, and security Alarm Monitoring System, AMS, located in the 200 area Central Alarm Station (CAS).

  20. The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

    PubMed Central

    Diomandé, Sara Esther; Chamot, Stéphanie; Antolinos, Vera; Vasai, Florian; Guinebretière, Marie-Hélène; Bornard, Isabelle; Nguyen-the, Christophe; Broussolle, Véronique

    2014-01-01

    The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at Tmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains. PMID:24509924

  1. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System

    PubMed Central

    Butler, Nathaniel M.; Atkins, Paul A.; Voytas, Daniel F.; Douches, David S.

    2015-01-01

    Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3–60% per transformation and from 0–29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87–100%. This

  2. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System.

    PubMed

    Butler, Nathaniel M; Atkins, Paul A; Voytas, Daniel F; Douches, David S

    2015-01-01

    Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration

  3. Analyzing safeguards alarms and response decisions

    SciTech Connect

    Al-Ayat, R.A.; Judd, B.R.; McCord, R.K.

    1982-01-01

    This report describes a quantitative model designed to help the Nuclear Regulatory Commission (NRC) and its licensees evaluate and respond to alarms indicating that special nuclear material (SNM) may be missing. The model is called the Alarm/Response (A/R) Model. The report demonstrates three principal uses of the A/R Model. The first is determining the most likely cause of an alarm - theft, hoax, or error. The second is evaluating alternative responses to alarms. Possible responses include conducting investigations, initiating measures to recover stolen SNM, and replying to extortion threats from individuals claiming to possess SNM. For each possible alarm, the model identifies the best response, which can be used to develop contingency plans that the licensee and the NRC can carry out. The third use is to assist the NRC in setting performance standards, especially detection time requirements. In this application, the model helps to determine the value of more timely alarms produced by licensee safeguards systems. All three uses are demonstrated with hypothetical examples. A preliminary computer code produced sample results and determined the sensitivity of those results to subjective factors in the example.

  4. Control of ELT false alarms

    NASA Technical Reports Server (NTRS)

    Toth, S.; Gershkoff, I.

    1979-01-01

    The statistics of emergency locator transmitter (ELT) alarms are presented. The primary sources of data include ELT Incident Logs, Service Difficulty Reports, and Frequency Interference Reports. The number of reported and unreported alarms is discussed, as are seasonal variations, duration of ELT transmissions, and cost of silencing. Origin, causes, and possible strategies for reducing the impact of alarms on the aviation community are considered.

  5. δ Sct-type pulsations in eclipsing binary systems: RZ Cas

    NASA Astrophysics Data System (ADS)

    Rodríguez, E.; García, J. M.; Mkrtichian, D. E.; Costa, V.; Kim, S.-L.; López-González, M. J.; Hintz, E.; Kusakin, A. V.; Gamarova, A. Y.; Lee, J. W.; Youn, J.-H.; Janiashvili, E. B.; Garrido, R.; Moya, A.; Kang, Y. W.

    2004-02-01

    We present the results of a three-continent multisite photometric campaign carried out on the Algol-type eclipsing binary system RZ Cas, in which the primary component has recently been discovered to be a δ Sct-type pulsator. The present observations include, for the first time, complete simultaneous Strömgren uvby light curves together with a few Crawford Hβ data collected around the orbital phase of the first quadrature. The new observations confirm the pulsational behaviour of the primary component. A detailed photometric analysis, based on these observations, is presented for both binarity and pulsation. The results indicate a semidetached system where the secondary fills its Roche lobe. The appearance of the light curves reveals the presence of the mass stream from the secondary component and a hotspot where this stream impacts on the surface of the primary star. There are also some indications of chromospheric activity in the secondary. On the other hand, the pulsational behaviour out-of-primary eclipse can be well described with only one frequency at 64.1935 cd-1 similar to the main peak found by Ohshima et al. The existence of multiperiodicity is not confirmed in our data. Concerning the mode identification, our results indicate non-radial pulsation in a high radial order (n= 6), with l= 2, |m|= 1, 2 as the most suitable. However, additional effects must be taken into account in the predictions. Moreover, the pulsation amplitude in the u band is larger than in b and v, which is unusual among the δ Sct-type variables. This can be explained as due to pulsation in a high n value and close to the blue edge of the δ Sct region. On the other hand, the early data of Ohshima et al. have also been analysed and similar results are found concerning the frequency content and pulsational amplitude. Finally, a revision of all the photometric out-of-primary-eclipse data sets available in the literature is made together with some additional unpublished data leading to

  6. HOME INSECURITY: NO ALARMS, FALSE ALARMS, AND SIGINT

    SciTech Connect

    Lamb, Logan M

    2014-01-01

    The market share of home security systems has substantially increased as vendors incorporate more desirable features: intrusion detection, automation, wireless, and LCD touch panel controls. Wireless connectivity allows vendors to manufacture cheaper, more featureful products that require little to no home modification to install. Consumer win, since adding devices is easier. The result: an ostensibly more secure, convenient, and connected home for a larger number of citizens. Sadly, this hypothesis is flawed; the idea of covering a home with more security sensors does not translate into a more secure home. Additionally, the number of homes using these vulnerable systems is large, and the growth rate is increasing producing a even larger problem. In this talk, I will demonstrate a generalized approach for compromising three systems: ADT, the largest home security dealer in North America; Honeywell, one of the largest manufacturers of security devices; and Vivint, a top 5 security dealer. We will suppress alarms, create false alarms, and collect artifacts that facilitate tracking the movements of individuals in their homes.

  7. 46 CFR 113.27-1 - Engineers' assistance-needed alarm.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Engineers' assistance-needed alarm. 113.27-1 Section 113.27-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Engineers' Assistance-Needed Alarm § 113.27-1 Engineers' assistance-needed alarm. Each self-propelled...

  8. 46 CFR 113.27-1 - Engineers' assistance-needed alarm.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Engineers' assistance-needed alarm. 113.27-1 Section 113.27-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Engineers' Assistance-Needed Alarm § 113.27-1 Engineers' assistance-needed alarm. Each self-propelled...

  9. 46 CFR 113.27-1 - Engineers' assistance-needed alarm.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Engineers' assistance-needed alarm. 113.27-1 Section 113.27-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING COMMUNICATION AND ALARM SYSTEMS AND EQUIPMENT Engineers' Assistance-Needed Alarm § 113.27-1 Engineers' assistance-needed alarm. Each self-propelled...

  10. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  11. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  12. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true Types of CAS coverage. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  13. 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…

  14. 48 CFR 9903.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false Types of CAS coverage... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-2 Types of CAS coverage. (a) Full coverage. Full coverage requires that the business unit comply with all of the CAS specified in part...

  15. Heritable genome editing in C. elegans via a CRISPR-Cas9 system.

    PubMed

    Friedland, Ari E; Tzur, Yonatan B; Esvelt, Kevin M; Colaiácovo, Monica P; Church, George M; Calarco, John A

    2013-08-01

    We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants. PMID:23817069

  16. Classification of alarm processing techniques and human performance issues

    SciTech Connect

    Kim, I.S.; O'Hara, J.M.

    1993-01-01

    Human factors reviews indicate that conventional alarm systems based on the one sensor, one alarm approach, have many human engineering deficiencies, a paramount example being too many alarms during major disturbances. As an effort to resolve these deficiencies, various alarm processing systems have been developed using different techniques. To ensure their contribution to operational safety, the impacts of those systems on operating crew performance should be carefully evaluated. This paper briefly reviews some of the human factors research issues associated with alarm processing techniques and then discusses a framework with which to classify the techniques. The dimensions of this framework can be used to explore the effects of alarm processing systems on human performance.

  17. Classification of alarm processing techniques and human performance issues

    SciTech Connect

    Kim, I.S.; O`Hara, J.M.

    1993-05-01

    Human factors reviews indicate that conventional alarm systems based on the one sensor, one alarm approach, have many human engineering deficiencies, a paramount example being too many alarms during major disturbances. As an effort to resolve these deficiencies, various alarm processing systems have been developed using different techniques. To ensure their contribution to operational safety, the impacts of those systems on operating crew performance should be carefully evaluated. This paper briefly reviews some of the human factors research issues associated with alarm processing techniques and then discusses a framework with which to classify the techniques. The dimensions of this framework can be used to explore the effects of alarm processing systems on human performance.

  18. [CRISPR-Cas9, a new chance for somatic gene therapy].

    PubMed

    Jordan, Bertrand

    2015-11-01

    Targeted modification of genes ("gene editing") is made much easier by the recently developed CRISPR-Cas9 system. This has raised alarm about possible uses of this technology for germline modification of the human genome; however this technology has less controversial applications, notably for somatic gene therapy with already some striking demonstrations in animal systems. Because of its precision and relative ease of use, CRISPR can be expected to drive a revolution in gene therapy and to turn it into a more mainstream approach. PMID:26576611

  19. Expression of the genes encoding the CasK/R two-component system and the DesA desaturase during Bacillus cereus cold adaptation.

    PubMed

    Diomandé, Sara Esther; Doublet, Bénédicte; Vasaï, Florian; Guinebretière, Marie-Hélène; Broussolle, Véronique; Brillard, Julien

    2016-08-01

    Two-component systems (TCS) allow a cell to elaborate a variety of adaptive responses to environment changes. The recently discovered CasK/R TCS plays a role in the optimal unsaturation of fatty acids necessary for cold adaptation of the foodborne-pathogen Bacillus cereus Here, we showed that the promoter activity of the operon encoding this TCS was repressed during growth at low temperature in the stationary phase in the parental strain when compared to the casK/R mutant, suggesting that CasR negatively regulates the activity of its own promoter in these conditions. The promoter activity of the desA gene encoding the Δ5 fatty acid desaturase, providing unsaturated fatty acids (UFAs) required for low temperature adaptation, was repressed in the casK/R mutant grown at 12°C versus 37°C. This result suggests that CasK/R activates desA expression during B. cereus growth at low temperature, allowing an optimal unsaturation of the fatty acids. In contrast, desA expression was repressed during the lag phase at low temperature in presence of UFAs, in a CasK/R-independent manner. Our findings confirm that the involvement of this major TCS in B. cereus cold adaptation is linked to the upregulation of a fatty acid desaturase. PMID:27435329

  20. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome.

    PubMed

    Liu, Xing; Hao, Ruidong; Chen, Shuliang; Guo, Deyin; Chen, Yu

    2015-08-01

    Hepatitis B virus (HBV) remains a global health threat as chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV covalently closed circular DNA. The newly developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using the CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight guide RNAs (gRNAs) that targeted the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both in vitro and in vivo. Moreover, the HBV-specific gRNA/Cas9 system could inhibit the replication of HBV of different genotypes in cells, and the viral DNA was significantly reduced by a single gRNA/Cas9 system and cleared by a combination of different gRNA/Cas9 systems. PMID:25904148

  1. Evaluation of nine different types of enuresis alarms.

    PubMed

    Goel, K M; Thomson, R B; Gibb, E M; McAinsh, T F

    1984-08-01

    One hundred enuretic children were treated in closely supervised trial conditions with nine commonly used enuresis alarm systems available commercially in the United Kingdom. Although there was little difference between the systems in terms of their effectiveness in stopping bed wetting, parents preferred the Eastleigh and Urilarm De-Luxe models which had distinct advantages in respect of false alarms, breakdowns, and durability of pads. Enuresis alarms that perform poorly in these respects may lead to loss of enthusiasm and non-compliance. The systems vary widely in price, but a private buyer may find a cheaper alarm just as effective. PMID:6476872

  2. FAULT DIAGNOSIS WITH MULTI-STATE ALARMS IN A NUCLEAR POWER CONTROL SIMULATOR

    SciTech Connect

    Austin Ragsdale; Roger Lew; Brian P. Dyre; Ronald L. Boring

    2012-10-01

    This research addresses how alarm systems can increase operator performance within nuclear power plant operations. The experiment examined the effect of two types of alarm systems (two-state and three-state alarms) on alarm compliance and diagnosis for two types of faults differing in complexity. We hypothesized three-state alarms would improve performance in alarm recognition and fault diagnoses over that of two-state alarms. We used sensitivity and criterion based on Signal Detection Theory to measure performance. We further hypothesized that operator trust would be highest when using three-state alarms. The findings from this research showed participants performed better and had more trust in three-state alarms compared to two-state alarms. Furthermore, these findings have significant theoretical implications and practical applications as they apply to improving the efficiency and effectiveness of nuclear power plant operations.

  3. Fault Diagnosis with Multi-State Alarms in a Nuclear Power Control Simulation

    SciTech Connect

    Stuart A. Ragsdale; Roger Lew; Ronald L. Boring

    2014-09-01

    This research addresses how alarm systems can increase operator performance within nuclear power plant operations. The experiment examined the effects of two types of alarm systems (two-state and three-state alarms) on alarm compliance and diagnosis for two types of faults differing in complexity. We hypothesized the use of three-state alarms would improve performance in alarm recognition and fault diagnoses over that of two-state alarms. Sensitivity and criterion based on the Signal Detection Theory were used to measure performance. We further hypothesized that operator trust would be highest when using three-state alarms. The findings from this research showed participants performed better and had more trust in three-state alarms compared to two-state alarms. Furthermore, these findings have significant theoretical implications and practical applications as they apply to improving the efficiency and effectiveness of nuclear power plant operations.

  4. Likelihood alarm displays. [for human operator

    NASA Technical Reports Server (NTRS)

    Sorkin, Robert D.; Kantowitz, Barry H.; Kantowitz, Susan C.

    1988-01-01

    In a likelihood alarm display (LAD) information about event likelihood is computed by an automated monitoring system and encoded into an alerting signal for the human operator. Operator performance within a dual-task paradigm was evaluated with two LADs: a color-coded visual alarm and a linguistically coded synthetic speech alarm. The operator's primary task was one of tracking; the secondary task was to monitor a four-element numerical display and determine whether the data arose from a 'signal' or 'no-signal' condition. A simulated 'intelligent' monitoring system alerted the operator to the likelihood of a signal. The results indicated that (1) automated monitoring systems can improve performance on primary and secondary tasks; (2) LADs can improve the allocation of attention among tasks and provide information integrated into operator decisions; and (3) LADs do not necessarily add to the operator's attentional load.

  5. Physical model of the immune response of bacteria against bacteriophage through the adaptive CRISPR-Cas immune system

    NASA Astrophysics Data System (ADS)

    Han, Pu; Niestemski, Liang Ren; Barrick, Jeffrey E.; Deem, Michael W.

    2013-04-01

    Bacteria and archaea have evolved an adaptive, heritable immune system that recognizes and protects against viruses or plasmids. This system, known as the CRISPR-Cas system, allows the host to recognize and incorporate short foreign DNA or RNA sequences, called ‘spacers’ into its CRISPR system. Spacers in the CRISPR system provide a record of the history of bacteria and phage coevolution. We use a physical model to study the dynamics of this coevolution as it evolves stochastically over time. We focus on the impact of mutation and recombination on bacteria and phage evolution and evasion. We discuss the effect of different spacer deletion mechanisms on the coevolutionary dynamics. We make predictions about bacteria and phage population growth, spacer diversity within the CRISPR locus, and spacer protection against the phage population.

  6. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  7. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9.

    PubMed

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5'-NGG-3' protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  8. The first photometric analysis and period investigation of the W UMa type binary system V1139 Cas

    NASA Astrophysics Data System (ADS)

    Li, K.; Hu, S.-M.; Guo, D.-F.; Jiang, Y.-G.; Gao, D.-Y.; Chen, X.

    2015-01-01

    V1139 Cas, which is a very short period W UMa type binary star, was a neglected object since its discovery. BVRI light curves of this system observed using the 1 m telescope at Weihai Observatory of Shandong University are presented and are analyzed using the Wilson-Devinney code. It is discovered that V1139 Cas is a shallow contact binary system (f=3.6%) with a mass ratio of q=1.583. By using all available times of minimum light, the orbital period variation is studied for the first time. We found that the orbital period has varied by a combination of an downward parabola and a sinusoid. The downward parabola means continuous period decrease at a rate of dP/dt=3.66×10-7 d yr-1 and may be caused by angular momentum loss via stellar wind. The sinusoidal variation with a period of 12.8 yr and a semi-amplitude of 0.0064 days can most likely be interpreted as the light travel time effect due to the existence of an unseen tertiary companion.

  9. Polyglutamine Disease Modeling: Epitope Based Screen for Homologous Recombination using CRISPR/Cas9 System

    PubMed Central

    An, Mahru C.; O'Brien, Robert N.; Zhang, Ningzhe; Patra, Biranchi N.; De La Cruz, Michael; Ray, Animesh; Ellerby, Lisa M.

    2014-01-01

    We have previously reported the genetic correction of Huntington’s disease (HD) patient-derived induced pluripotent stem cells using traditional homologous recombination (HR) approaches. To extend this work, we have adopted a CRISPR-based genome editing approach to improve the efficiency of recombination in order to generate allelic isogenic HD models in human cells. Incorporation of a rapid antibody-based screening approach to measure recombination provides a powerful method to determine relative efficiency of genome editing for modeling polyglutamine diseases or understanding factors that modulate CRISPR/Cas9 HR. PMID:24761311

  10. Alarm toe switch

    DOEpatents

    Ganyard, Floyd P.

    1982-01-01

    An alarm toe switch inserted within a shoe for energizing an alarm circuit n a covert manner includes an insole mounting pad into which a miniature reed switch is fixedly molded. An elongated slot perpendicular to the reed switch is formed in the bottom surface of the mounting pad. A permanent cylindrical magnet positioned in the forward portion of the slot with a diameter greater than the pad thickness causes a bump above the pad. A foam rubber block is also positioned in the slot rearwardly of the magnet and holds the magnet in normal inoperative relation. A non-magnetic support plate covers the slot and holds the magnet and foam rubber in the slot. The plate minimizes bending and frictional forces to improve movement of the magnet for reliable switch activation. The bump occupies the knuckle space beneath the big toe. When the big toe is scrunched rearwardly the magnet is moved within the slot relative to the reed switch, thus magnetically activating the switch. When toe pressure is released the foam rubber block forces the magnet back into normal inoperative position to deactivate the reed switch. The reed switch is hermetically sealed with the magnet acting through the wall so the switch assembly S is capable of reliable operation even in wet and corrosive environments.

  11. 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. PMID:25987542

  12. Framework for analyzing safeguards alarms and response decisions

    SciTech Connect

    Al-Ayat, R.A.; Judd, B.R.; McCord, R.K.

    1982-06-11

    This paper describes a quantitative approach to help evaluate and respond to safeguards alarms. These alrms may be generated internally by a facility's safeguards systems or externally by individuals claiming to possess stolen Special Nuclear Material (SNM). This approach can be used to identify the most likely cause of an alarm - theft, hoax, or error - and to evaluate alternative responses to alarms. Possible responses include conducting investigations, initiating measures to recover stolen SNM, and replying to external threats. Based on the results of each alarm investigation step, the evaluation revises the likelihoods of possible causes of an alarm, and uses this information to determine the optimal sequence of further responses. The choice of an optimal sequence of responses takes into consideration the costs and benefits of successful thefts or hoaxes. These results provide an analytical basis for setting priorities and developing contingency plans for responding to safeguards alarms.

  13. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

    PubMed

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  14. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

    PubMed Central

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  15. 46 CFR 111.33-7 - Alarms and shutdowns.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Alarms and shutdowns. 111.33-7 Section 111.33-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Power Semiconductor Rectifier Systems § 111.33-7 Alarms and shutdowns. Each power...

  16. 46 CFR 111.33-7 - Alarms and shutdowns.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Alarms and shutdowns. 111.33-7 Section 111.33-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Power Semiconductor Rectifier Systems § 111.33-7 Alarms and shutdowns. Each power...

  17. 46 CFR 111.33-7 - Alarms and shutdowns.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Alarms and shutdowns. 111.33-7 Section 111.33-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Power Semiconductor Rectifier Systems § 111.33-7 Alarms and shutdowns. Each power...

  18. 46 CFR 111.33-7 - Alarms and shutdowns.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 4 2010-10-01 2010-10-01 false Alarms and shutdowns. 111.33-7 Section 111.33-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Power Semiconductor Rectifier Systems § 111.33-7 Alarms and shutdowns. Each power...

  19. 46 CFR 111.33-7 - Alarms and shutdowns.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Alarms and shutdowns. 111.33-7 Section 111.33-7 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Power Semiconductor Rectifier Systems § 111.33-7 Alarms and shutdowns. Each power semiconductor rectifier must have a...

  20. 46 CFR 108.445 - Alarm and means of escape.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Alarm and means of escape. 108.445 Section 108.445 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) A-MOBILE OFFSHORE DRILLING UNITS DESIGN AND EQUIPMENT Fire Extinguishing Systems Fixed Carbon Dioxide Fire Extinguishing Systems § 108.445 Alarm and means of escape. (a) Each CO2...