Human Reliability Program Workshop

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

Landers, John; Rogers, Erin; Gerke, Gretchen

A Human Reliability Program (HRP) is designed to protect national security as well as worker and public safety by continuously evaluating the reliability of those who have access to sensitive materials, facilities, and programs. Some elements of a site HRP include systematic (1) supervisory reviews, (2) medical and psychological assessments, (3) management evaluations, (4) personnel security reviews, and (4) training of HRP staff and critical positions. Over the years of implementing an HRP, the Department of Energy (DOE) has faced various challenges and overcome obstacles. During this 4-day activity, participants will examine programs that mitigate threats to nuclear security andmore » the insider threat to include HRP, Nuclear Security Culture (NSC) Enhancement, and Employee Assistance Programs. The focus will be to develop an understanding of the need for a systematic HRP and to discuss challenges and best practices associated with mitigating the insider threat.« less

10 CFR 712.12 - HRP implementation.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false HRP implementation. 712.12 Section 712.12 Energy... Program Procedures § 712.12 HRP implementation. (a) The implementation of the HRP is the responsibility of... by April 22, 2004. (b) The HRP Management Official must: (1) Prepare an initial HRP implementation...

10 CFR 712.12 - HRP implementation.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false HRP implementation. 712.12 Section 712.12 Energy... Program Procedures § 712.12 HRP implementation. (a) The implementation of the HRP is the responsibility of... by April 22, 2004. (b) The HRP Management Official must: (1) Prepare an initial HRP implementation...

Evidence Report: Risk of Decompression Sickness (DCS)

NASA Technical Reports Server (NTRS)

Conkin, Johnny; Norcross, Jason R.; Wessel, James H. III; Abercromby, Andrew F. J.; Klein, Jill S.; Dervay, Joseph P.; Gernhardt, Michael L.

2013-01-01

The Risk of Decompression Sickness (DCS) is identified by the NASA Human Research Program (HRP) as a recognized risk to human health and performance in space, as defined in the HRP Program Requirements Document (PRD). This Evidence Report provides a summary of the evidence that has been used to identify and characterize this risk. Given that tissue inert gas partial pressure is often greater than ambient pressure during phases of a mission, primarily during extravehicular activity (EVA), there is a possibility that decompression sickness may occur.

NASA 2010 Pharmacology Evidence Review

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2011-01-01

In 2008, the Institute of Medicine reviewed NASA's Human Research Program Evidence in assessing the Pharmacology risk identified in NASA's Human Research Program Requirements Document (PRD). Since this review there was a major reorganization of the Pharmacology discipline within the HRP, as well as a re-evaluation of the Pharmacology evidence. This panel is being asked to review the latest version of the Pharmacology Evidence Report. Specifically, this panel will: (1) Appraise the descriptions of the human health-related risk in the HRP PRD. (2) Assess the relevance and comprehensiveness of the evidence in identifying potential threats to long-term space missions. (3) Assess the associated gaps in knowledge and identify additional areas for research as necessary.

Human Research Program: 2012 Fiscal Year Annual Report

NASA Technical Reports Server (NTRS)

Effenhauser, Laura

2012-01-01

Crew health and performance are critical to successful human exploration beyond low Earth orbit. Risks to health and performance include physiologic effects from radiation, hypogravity, and planetary environments, as well as unique challenges in medical treatment, human factors, and support of behavioral health. The scientists and engineers of the Human Research Program (HRP) investigate and reduce the greatest risks to human health and performance, and provide essential countermeasures and technologies for human space exploration. In its seventh year of operation, the HRP continued to refine its management architecture of evidence, risks, gaps, tasks, and deliverables. Experiments continued on the International Space Station (ISS), on the ground in analog environments that have features similar to those of spaceflight, and in laboratory environments. Data from these experiments furthered the understanding of how the space environment affects the human system. These research results contributed to scientific knowledge and technology developments that address the human health and performance risks. As shown in this report, HRP has made significant progress toward developing medical care and countermeasure systems for space exploration missions which will ultimately reduce risks to crew health and performance.

10 CFR 712.19 - Removal from HRP.

Code of Federal Regulations, 2010 CFR

2010-01-01

... OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability Program... immediately remove that individual from HRP duties pending a determination of the individual's reliability. A... HRP duties pending a determination of the individual's reliability is an interim, precautionary action...

Human Research Program Human Health Countermeasures Element Sensorimotor Risk Standing Review Panel (SRP) Final Report

NASA Technical Reports Server (NTRS)

Peterson, Barry

2009-01-01

The Sensorimotor Risk Standing Review Panel (SRP) met at the NASA Johnson Space Center on October 4-6, 2009 to discuss the areas of future research targeted by the Human Health Countermeasures (HHC) Element of the Human Research Program (HRP). Using evidence-based knowledge as a background for risks, NASA had identified gaps in knowledge to address those risks. Ongoing and proposed tasks were presented to address the gaps. The charge to the Sensorimotor Risk SRP was to review the gaps, evaluate whether the tasks addressed these gaps and to make recommendations to NASA s HRP Science Management Office regarding the SRP's review. The SRP was requested to evaluate the practicality of the proposed efforts in light of the realistic demands placed on the HRP. In short, all tasks presented in the Integrated Research Plan (IRP) should address specific risks related to the challenges faced by the astronauts as a result of prolonged exposure to microgravity. All tasks proposed to fill the gaps in knowledge should provide applied, translational data necessary to address the specific risks. Several presentations were made to the SRP during the site visit and the SRP spent sufficient time to address the panel charge, either as a group or in separate sessions. The SRP made a final debriefing to the HRP Program Scientist. Taking the evidence and the risk as givens, the SRP reached the following conclusions: 1) the panel is very supportive of and endorses the present activities of the Sensorimotor Risk; and the panel is likewise supportive of the gaps and associated tasks in the IRP; 2) overall, the tasks addressed the gaps in the IRP; 3) there were some gaps and tasks that merit further enhancement and some new gaps/tasks that the SRP recommends.

Human Research Program Space Radiation Standing Review Panel (SRP)

NASA Technical Reports Server (NTRS)

Woloschak, Gayle; Steinberg-Wright, S.; Coleman, Norman; Grdina, David; Hill, Colin; Iliakis, George; Metting, Noelle; Meyers, Christina

2010-01-01

The Space Radiation Standing Review Panel (SRP) met at the NASA Johnson Space Center (JSC) on December 9-11, 2009 to discuss the areas of current and future research targeted by the Space Radiation Program Element (SRPE) of the Human Research Program (HRP). Using evidence-based knowledge as a background for identified risks to astronaut health and performance, NASA had identified gaps in knowledge to address those risks. Ongoing and proposed tasks were presented to address the gaps. The charge to the Space Radiation SRP was to review the gaps, evaluate whether the tasks addressed these gaps and to make recommendations to NASA s HRP Science Management Office regarding the SRP's review. The SRP was requested to evaluate the practicality of the proposed efforts in light of the demands placed on the HRP. Several presentations were made to the SRP during the site visit and the SRP spent sufficient time to address the SRP charge. The SRP made a final debriefing to the HRP Program Scientist, Dr. John B. Charles, on December 11, 2009. The SRP noted that current SRPE strategy is properly science-based and views this as the best assurance of the likelihood that answers to the questions posed as gaps in knowledge can be found, that the uncertainty in risk estimates can be reduced, and that a solid, cost-effective approach to risk reduction solutions is being developed. The current approach of the SRPE, based on the use of carefully focused research solicitations, requiring thorough peer-review and approaches demonstrated to be on the path to answering the NASA strategic questions, addressed to a broad extramural community of qualified scientists, optimally positioned to take advantage of serendipitous discoveries and to leverage scientific advances made elsewhere, is sound and appropriate. The SRP viewed with concern statements by HRP implying that the only science legitimately deserving support should be "applied" or, in some instances that the very term "research" might be frowned upon. We understand the desire of management to ensure that research stay focused on mission objectives, but the terms used are code words fraught with different meaning for scientists. Such expressions, taken at face value, convey a profoundly flawed view of science, can easily lead down counterproductive paths, and have the potential to irretrievably corrupt NASA requirements. The SRP understands and endorses the mandate to keep research efforts focused on the mission needs. However, thoughtful application of knowledge gained by understanding the mechanisms and pathways of biological effects cannot be replaced.

Managing Research in a Risk World

NASA Technical Reports Server (NTRS)

Anton, W.; Havenhill, M.

2014-01-01

The Office of Chief Medical Officer (OCHMO) owns all human health and performance risks managed by the Human System Risk Board (HSRB). While the HSRB manages the risks, the Human Research Program (HRP) manages the research portion of the overall risk mitigation strategy for these risks. The HSRB manages risks according to a process that identifies and analyzes risks, plans risk mitigation and tracks and reviews the implementation of these strategies according to its decisions pertaining to the OCHMO risk posture. HRP manages risk research work using an architecture that describes evidence-based risks, gaps in our knowledge about characterizing or mitigating the risk, and the tasks needed to produce deliverables to fill the gaps and reduce the risk. A planning schedule reflecting expected research milestones is developed, and as deliverables and new evidence are generated, research progress is tracked via the Path to Risk Reduction (PRR) that reflects a risk's research plan for a design reference mission. HRP's risk research process closely interfaces with the HSRB risk management process. As research progresses, new deliverables and evidence are used by the HSRB in conjunction with other operational and non-research evidence to inform decisions pertaining to the likelihood and consequence of the risk and risk posture. Those decisions in turn guide forward work for research as it contributes to overall risk mitigation strategies. As HRP tracks its research work, it aligns its priorities by assessing the effectiveness of its contributions and maintaining specific core competencies that would be invaluable for future work for exploration missions.

Human Research Program Opportunities

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.

2014-01-01

The goal of HRP is to provide human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration. The Human Research Program was designed to meet the needs of human space exploration, and understand and reduce the risk to crew health and performance in exploration missions.

Developing a Gap Taxonomy to Address Crew Health Risks in NASA's Human Research Program

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.; Edwards, J. Michelle

2009-01-01

The mission of NASA's Human Research Program (HRP) is to understand and reduce the risk to crew health and performance in exploration missions. The HRP addresses 27 specific risks by identifying and then filling gaps in understanding the risks and in the ability to disposition the risks. The primary bases for identifying gaps have been past experience and requirements definition. This approach has been very effective in identifying some important, relevant gaps, but may be inadequate for identifying gaps outside the past experience base. We are exploring the use of a gap taxonomy as a comprehensive, underlying conceptual framework that allows a more systematic identification of gaps. The taxonomy is based on these stages in medical care: prediction, prevention, detection/diagnosis, treatment, monitoring, rehabilitation, and lifetime surveillance. This gap taxonomy approach identifies new gaps in HRP health risks. Many of the new gaps suggest risk reduction approaches that are more cost effective than present approaches. A major benefit of the gap taxonomy approach is to identify new, economical approaches that reduce the likelihood and/or consequence of a risk.

10 CFR 712.18 - Transferring HRP certification.

Code of Federal Regulations, 2011 CFR

2011-01-01

... into a designated HRP position; (2) Incorporate the individual into the new site's alcohol and drug-testing program; (3) Ensure that the 12-month time period for HRP requirements that was established at the...

Concepts to Support HRP Integration Using Publications and Modeling

NASA Technical Reports Server (NTRS)

Mindock, J.; Lumpkins, S.; Shelhamer, M.

2014-01-01

Initial efforts are underway to enhance the Human Research Program (HRP)'s identification and support of potential cross-disciplinary scientific collaborations. To increase the emphasis on integration in HRP's science portfolio management, concepts are being explored through the development of a set of tools. These tools are intended to enable modeling, analysis, and visualization of the state of the human system in the spaceflight environment; HRP's current understanding of that state with an indication of uncertainties; and how that state changes due to HRP programmatic progress and design reference mission definitions. In this talk, we will discuss proof-of-concept work performed using a subset of publications captured in the HRP publications database. The publications were tagged in the database with words representing factors influencing health and performance in spaceflight, as well as with words representing the risks HRP research is reducing. Analysis was performed on the publication tag data to identify relationships between factors and between risks. Network representations were then created as one type of visualization of these relationships. This enables future analyses of the structure of the networks based on results from network theory. Such analyses can provide insights into HRP's current human system knowledge state as informed by the publication data. The network structure analyses can also elucidate potential improvements by identifying network connections to establish or strengthen for maximized information flow. The relationships identified in the publication data were subsequently used as inputs to a model captured in the Systems Modeling Language (SysML), which functions as a repository for relationship information to be gleaned from multiple sources. Example network visualization outputs from a simple SysML model were then also created to compare to the visualizations based on the publication data only. We will also discuss ideas for building upon this proof-of-concept work to further support an integrated approach to human spaceflight risk reduction.

The American Board of Radiology Holman Research Pathway: 10-year retrospective review of the program and participant performance.

PubMed

Wallner, Paul E; Ang, K Kian; Zietman, Anthony L; Harris, Jay R; Ibbott, Geoffrey S; Mahoney, Mary C; Mezwa, Duane G; Wilson, Lynn D; Becker, Gary J

2013-01-01

In 1999, the American Board of Radiology (ABR) implemented an innovative training program track in diagnostic radiology (DR) and radiation oncology (RO) designed to stimulate development of a cadre of future academic researchers and educators in the 2 disciplines. The program was designated the Holman Research Pathway (HRP). An in-depth retrospective review of initial certification examination performance, post-training career choices, and academic productivity has not been written. This report represents a 10-year retrospective review of post-training performance of a cohort of trainees who have had sufficient time to complete their training and initial certification process and to enter practice. All pertinent proceedings of the ABR and Accreditation Council for Graduate Medical Education (ACGME) Residency Review Committees for DR and RO between 1997 and May 2011 were reviewed. Thirty-four HRP candidates who fulfilled the established evaluation criteria were identified, and their ABR data files were analyzed regarding performance on the qualifying and certifying examinations. All candidates were contacted directly to obtain a current curriculum vitae. Twenty candidates in RO and 14 candidates in DR were identifiable for review. All candidates attained initial certification. At the time of analysis, 23 of 33 (66.6%) candidates were employed in full-time academic practice (1 DR candidate remained in a fellowship and was not evaluated regarding employment status). Fifteen of 20 (75%) RO candidates were in faculty positions compared with 7 of 13 (53.8%) DR trainees. Additional academic productivity metrics are reported. A high percentage of HRP trainees remained in academic practice and demonstrated significant academic productivity as measured by manuscript authorship and research support. Additional time and observation will be needed to determine whether these findings will be sustained by past, current, and future HRP trainees. Copyright © 2013 Elsevier Inc. All rights reserved.

The American Board of Radiology Holman Research Pathway: 10-Year Retrospective Review of the Program and Participant Performance

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

Wallner, Paul E., E-mail: pwallner@theabr.org; Ang, K. Kian; Zietman, Anthony L.

2013-01-01

Introduction: In 1999, the American Board of Radiology (ABR) implemented an innovative training program track in diagnostic radiology (DR) and radiation oncology (RO) designed to stimulate development of a cadre of future academic researchers and educators in the 2 disciplines. The program was designated the Holman Research Pathway (HRP). An in-depth retrospective review of initial certification examination performance, post-training career choices, and academic productivity has not been written. This report represents a 10-year retrospective review of post-training performance of a cohort of trainees who have had sufficient time to complete their training and initial certification process and to enter practice.more » Methods and Materials: All pertinent proceedings of the ABR and Accreditation Council for Graduate Medical Education (ACGME) Residency Review Committees for DR and RO between 1997 and May 2011 were reviewed. Thirty-four HRP candidates who fulfilled the established evaluation criteria were identified, and their ABR data files were analyzed regarding performance on the qualifying and certifying examinations. All candidates were contacted directly to obtain a current curriculum vitae. Results: Twenty candidates in RO and 14 candidates in DR were identifiable for review. All candidates attained initial certification. At the time of analysis, 23 of 33 (66.6%) candidates were employed in full-time academic practice (1 DR candidate remained in a fellowship and was not evaluated regarding employment status). Fifteen of 20 (75%) RO candidates were in faculty positions compared with 7 of 13 (53.8%) DR trainees. Additional academic productivity metrics are reported. Conclusions: A high percentage of HRP trainees remained in academic practice and demonstrated significant academic productivity as measured by manuscript authorship and research support. Additional time and observation will be needed to determine whether these findings will be sustained by past, current, and future HRP trainees.« less

Human Research Program Integrated Research Plan: December 20, 2007, Interim Baseline

NASA Technical Reports Server (NTRS)

2008-01-01

The Human Research Program (HRP) delivers human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration. This Integrated Research Plan (IRP) describes the program s research activities that are intended to address the needs of human space exploration and serve HRP customers. The timescale of human space exploration is envisioned to take many decades. The IRP illustrates the program s research plan through the timescale of early lunar missions of extended duration. The document serves several purposes for the Human Research Program: The IRP provides a means to assure that the most significant risks to human space explorers are being adequately mitigated and/or addressed, The IRP shows the relationship of research activities to expected outcomes and need dates, The IRP shows the interrelationships among research activities that may interact to produce products that are integrative or cross defined research disciplines, The IRP illustrates the non-deterministic nature of research and technology activities by showing expected decision points and potential follow-on activities, The IRP shows the assignments of responsibility within the program organization and, as practical, the intended solicitation approach, The IRP shows the intended use of research platforms such as the International Space Station, NASA Space Radiation Laboratory, and various space flight analogs. The IRP does not show all budgeted activities of the Human research program, as some of these are enabling functions, such as management, facilities and infrastructure

Evaluation of Maximal Oxygen Uptake (V02max) and Submaximal Estimates of VO2max Before, During and After Long Duration ISS Missions

NASA Technical Reports Server (NTRS)

Moore, Alan; Evetts, Simon; Feiveson, Alan; Lee, Stuart; McCleary, Frank; Platts, Steven

2009-01-01

NASA's Human Research Program Integrated Research Plan (HRP-47065) serves as a road-map identifying critically needed information for future space flight operations (Lunar, Martian). VO2max (often termed aerobic capacity) reflects the maximum rate at which oxygen can be taken up and utilized by the body during exercise. Lack of in-flight and immediate postflight VO2max measurements was one area identified as a concern. The risk associated with not knowing this information is: Unnecessary Operational Limitations due to Inaccurate Assessment of Cardiovascular Performance (HRP-47065).

An Evidence Base for Human Spaceflight Risks in Wikipedia

NASA Technical Reports Server (NTRS)

Kundrot, Craig; Steil, Jennifer; Lumpkins, Sarah; Pellis, Neal

2013-01-01

NASA's Human Research Program (HRP) is focused on understanding and mitigating thirty two risks to crew health and performance in exploration missions beyond low Earth orbit. The HRP has developed an evidence report for each of the risks. Most evidence reports are a brief review article describing the evidence related to a specified risk, written at a level appropriate for the scientifically educated, non-specialist reader. Each evidence report captured the current state of knowledge from both research and operations. Two limitations of the evidence reports have become apparent: 1) they are updated infrequently and 2) they do not take full advantage of the expertise available in other space agencies and in related fields of terrestrial research. Therefore, the HRP is experimenting with the use of Wikipedia articles as a repository for evidence. Wikipedia's accessibility to the international space flight community and researchers in related terrestrial fields creates the opportunity to generate a more timely and comprehensive evidence base. Initial Wikipedia articles were populated for seven risks using a subset of the information in the HRP-approved evidence reports: Fatigue and Sleep Loss, Treating An Ill or Injured Crew Member, Radiation Carcinogenesis, Visual Impairment and Intracranial Pressure, Renal Stone Formation, Team Cohesion, and Intervertebral Disc Damage. Since the initial articles were created, there have been additions to these Wikipedia articles, including content from sources outside the HRP, and editorial changes to the pages. We will report on the nature of the contributions made after the initial articles were created, the comprehensiveness of the resulting Wikipedia articles, and the effort required to maintain quality control of the content. The Wikipedia approach will also be compared to wiki efforts that exert more traditional editorial control of content prior to posting.

Evidence Report: Risk of Performance Errors Due to Training Deficiencies

NASA Technical Reports Server (NTRS)

Barshi, Immanuel

2012-01-01

The Risk of Performance Errors Due to Training Deficiencies is identified by the National Aeronautics and Space Administration (NASA) Human Research Program (HRP) as a recognized risk to human health and performance in space. The HRP Program Requirements Document (PRD) defines these risks. This Evidence Report provides a summary of the evidence that has been used to identify and characterize this risk. Given that training content, timing, intervals, and delivery methods must support crew task performance, and given that training paradigms will be different for long-duration missions with increased crew autonomy, there is a risk that operators will lack the skills or knowledge necessary to complete critical tasks, resulting in flight and ground crew errors and inefficiencies, failed mission and program objectives, and an increase in crew injuries.

Scientific Merit Review of Directed Research Tasks Within the NASA Human Research Program

NASA Technical Reports Server (NTRS)

Charles, John B.

2010-01-01

The Human Research Program is instrumental in developing and delivering research findings, health countermeasures, and human systems technologies for spacecraft. :HRP is subdivided into 6 research entities, or Elements. Each Element is charged with providing the Program with knowledge and capabilities to conduct research to address the human health and performance risks as well as advance the readiness levels of technology and countermeasures. Project: An Element may be further subdivided into Projects, which are defined as an integrated set of tasks undertaken to deliver a product or set of products

Human-Robot Interaction

NASA Technical Reports Server (NTRS)

Rochlis-Zumbado, Jennifer; Sandor, Aniko; Ezer, Neta

2012-01-01

Risk of Inadequate Design of Human and Automation/Robotic Integration (HARI) is a new Human Research Program (HRP) risk. HRI is a research area that seeks to understand the complex relationship among variables that affect the way humans and robots work together to accomplish goals. The DRP addresses three major HRI study areas that will provide appropriate information for navigation guidance to a teleoperator of a robot system, and contribute to the closure of currently identified HRP gaps: (1) Overlays -- Use of overlays for teleoperation to augment the information available on the video feed (2) Camera views -- Type and arrangement of camera views for better task performance and awareness of surroundings (3) Command modalities -- Development of gesture and voice command vocabularies

Evidence Report: Risk of Crew Adverse Health Event Due to Altered Immune Response

NASA Technical Reports Server (NTRS)

Crucian, Brian; Sams, Clarence F.

2013-01-01

The Risk of Crew Adverse Health Event Due to Altered Immune Response is identified by the National Aeronautics and Space Administration (NASA) Human Research Program (HRP) as a recognized risk to human health and performance in space. The HRP Program Requirements Document (PRD) defines these risks. This Evidence Report provides a summary of the evidence that has been used to identify and characterize this risk. It is known that human immune function is altered in- and post-flight, but it is unclear at present if such alterations lead to increased susceptibility to disease. Reactivation of latent viruses has been documented in crewmembers, although this reactivation has not been directly correlated with immune changes or with observed diseases. As described in this report, further research is required to better characterize the relationships between altered immune response and susceptibility to disease during and after spaceflight. This is particularly important for future deep-space exploration missions.

10 CFR 712.11 - General requirements for HRP certification.

Code of Federal Regulations, 2010 CFR

2010-01-01

..., “Drug-Free Federal Workplace Testing Implementation Program,” for DOE employees; (9) An initial alcohol... the HRP and complete initial instruction on the importance of security, safety, reliability, and... the Manager, the NNSA Administrator, his or her designee, or the appropriate Lead Program Secretarial...

Human Research Program: Long Duration, Exploration-Class Mission Training Design

NASA Technical Reports Server (NTRS)

Barshi, Immanuel; Dempsey, Donna L.

2016-01-01

This is a presentation to the International Training Control Board that oversees astronaut training for ISS. The presentation explains the structure of HRP, the training-related work happening under the different program elements, and discusses in detail the research plan for the Training Risk under SHFHSHFE. The group includes the crew training leads for all the space agencies involved in ISS: Japan, Europe, Russia, Canada, and the US.

10 CFR 712.11 - General requirements for HRP certification.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 4 2013-01-01 2013-01-01 false General requirements for HRP certification. 712.11 Section 712.11 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the..., “Drug-Free Federal Workplace Testing Implementation Program,” for DOE employees; (9) An initial alcohol...

10 CFR 712.11 - General requirements for HRP certification.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false General requirements for HRP certification. 712.11 Section 712.11 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the..., “Drug-Free Federal Workplace Testing Implementation Program,” for DOE employees; (9) An initial alcohol...

10 CFR 712.11 - General requirements for HRP certification.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false General requirements for HRP certification. 712.11 Section 712.11 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the..., “Drug-Free Federal Workplace Testing Implementation Program,” for DOE employees; (9) An initial alcohol...

Minimizing Human Risk: Human Performance Models in the Human Factors and Behavioral Performance Element

NASA Technical Reports Server (NTRS)

Gore, Brian F.

2017-01-01

Human space exploration has never been more exciting than it is today. Human presence to outer worlds is becoming a reality as humans are leveraging much of our prior knowledge to the new mission of going to Mars. Exploring the solar system at greater distances from Earth than ever before will possess some unique challenges, which can be overcome thanks to the advances in modeling and simulation technologies. The National Aeronautics and Space Administration (NASA) is at the forefront of exploring our solar system. NASA's Human Research Program (HRP) focuses on discovering the best methods and technologies that support safe and productive human space travel in the extreme and harsh space environment. HRP uses various methods and approaches to answer questions about the impact of long duration missions on the human in space including: gravitys impact on the human body, isolation and confinement on the human, hostile environments impact on the human, space radiation, and how the distance is likely to impact the human. Predictive models are included in the HRP research portfolio as these models provide valuable insights into human-system operations. This paper will provide an overview of NASA's HRP and will present a number of projects that have used modeling and simulation to provide insights into human-system issues (e.g. automation, habitat design, schedules) in anticipation of space exploration.

Minimizing Human Risk: Human Performance Models in the Space Human Factors and Habitability and Behavioral Health and Performance Elements

NASA Technical Reports Server (NTRS)

Gore, Brian F.

2016-01-01

Human space exploration has never been more exciting than it is today. Human presence to outer worlds is becoming a reality as humans are leveraging much of our prior knowledge to the new mission of going to Mars. Exploring the solar system at greater distances from Earth than ever before will possess some unique challenges, which can be overcome thanks to the advances in modeling and simulation technologies. The National Aeronautics and Space Administration (NASA) is at the forefront of exploring our solar system. NASA's Human Research Program (HRP) focuses on discovering the best methods and technologies that support safe and productive human space travel in the extreme and harsh space environment. HRP uses various methods and approaches to answer questions about the impact of long duration missions on the human in space including: gravity's impact on the human body, isolation and confinement on the human, hostile environments impact on the human, space radiation, and how the distance is likely to impact the human. Predictive models are included in the HRP research portfolio as these models provide valuable insights into human-system operations. This paper will provide an overview of NASA's HRP and will present a number of projects that have used modeling and simulation to provide insights into human-system issues (e.g. automation, habitat design, schedules) in anticipation of space exploration.

Capability for Integrated Systems Risk-Reduction Analysis

NASA Technical Reports Server (NTRS)

Mindock, J.; Lumpkins, S.; Shelhamer, M.

2016-01-01

NASA's Human Research Program (HRP) is working to increase the likelihoods of human health and performance success during long-duration missions, and subsequent crew long-term health. To achieve these goals, there is a need to develop an integrated understanding of how the complex human physiological-socio-technical mission system behaves in spaceflight. This understanding will allow HRP to provide cross-disciplinary spaceflight countermeasures while minimizing resources such as mass, power, and volume. This understanding will also allow development of tools to assess the state of and enhance the resilience of individual crewmembers, teams, and the integrated mission system. We will discuss a set of risk-reduction questions that has been identified to guide the systems approach necessary to meet these needs. In addition, a framework of factors influencing human health and performance in space, called the Contributing Factor Map (CFM), is being applied as the backbone for incorporating information addressing these questions from sources throughout HRP. Using the common language of the CFM, information from sources such as the Human System Risk Board summaries, Integrated Research Plan, and HRP-funded publications has been combined and visualized in ways that allow insight into cross-disciplinary interconnections in a systematic, standardized fashion. We will show examples of these visualizations. We will also discuss applications of the resulting analysis capability that can inform science portfolio decisions, such as areas in which cross-disciplinary solicitations or countermeasure development will potentially be fruitful.

HRP's Healthcare Spin-Offs Through Computational Modeling and Simulation Practice Methodologies

NASA Technical Reports Server (NTRS)

Mulugeta, Lealem; Walton, Marlei; Nelson, Emily; Peng, Grace; Morrison, Tina; Erdemir, Ahmet; Myers, Jerry

2014-01-01

Spaceflight missions expose astronauts to novel operational and environmental conditions that pose health risks that are currently not well understood, and perhaps unanticipated. Furthermore, given the limited number of humans that have flown in long duration missions and beyond low Earth-orbit, the amount of research and clinical data necessary to predict and mitigate these health and performance risks are limited. Consequently, NASA's Human Research Program (HRP) conducts research and develops advanced methods and tools to predict, assess, and mitigate potential hazards to the health of astronauts. In this light, NASA has explored the possibility of leveraging computational modeling since the 1970s as a means to elucidate the physiologic risks of spaceflight and develop countermeasures. Since that time, substantial progress has been realized in this arena through a number of HRP funded activates such as the Digital Astronaut Project (DAP) and the Integrated Medical Model (IMM). Much of this success can be attributed to HRP's endeavor to establish rigorous verification, validation, and credibility (VV&C) processes that ensure computational models and simulations (M&S) are sufficiently credible to address issues within their intended scope. This presentation summarizes HRP's activities in credibility of modeling and simulation, in particular through its outreach to the community of modeling and simulation practitioners. METHODS: The HRP requires all M&S that can have moderate to high impact on crew health or mission success must be vetted in accordance to NASA Standard for Models and Simulations, NASA-STD-7009 (7009) [5]. As this standard mostly focuses on engineering systems, the IMM and DAP have invested substantial efforts to adapt the processes established in this standard for their application to biological M&S, which is more prevalent in human health and performance (HHP) and space biomedical research and operations [6,7]. These methods have also generated substantial interest by the broader medical community though institutions like the National Institutes of Health (NIH) and the Food and Drug Administration (FDA) to develop similar standards and guidelines applicable to the larger medical operations and research community. DISCUSSION: Similar to NASA, many leading government agencies, health institutions and medical product developers around the world are recognizing the potential of computational M&S to support clinical research and decision making. In this light, substantial investments are being made in computational medicine and notable discoveries are being realized [8]. However, there is a lack of broadly applicable practice guidance for the development and implementation of M&S in clinical care and research in a manner that instills confidence among medical practitioners and biological researchers [9,10]. In this presentation, we will give an overview on how HRP is working with the NIH's Interagency Modeling and Analysis Group (IMAG), the FDA and the American Society of Mechanical Engineers (ASME) to leverage NASA's biomedical VV&C processes to establish a new regulatory standard for Verification and Validation in Computational Modeling of Medical Devices, and Guidelines for Credible Practice of Computational Modeling and Simulation in Healthcare.

Multilateral Research Opportunities in Ground Analogs

NASA Technical Reports Server (NTRS)

Corbin, Barbara J.

2015-01-01

The global economy forces many nations to consider their national investments and make difficult decisions regarding their investment in future exploration. International collaboration provides an opportunity to leverage other nations' investments to meet common goals. The Humans In Space Community shares a common goal to enable safe, reliable, and productive human space exploration within and beyond Low Earth Orbit. Meeting this goal requires efficient use of limited resources and International capabilities. The International Space Station (ISS) is our primary platform to conduct microgravity research targeted at reducing human health and performance risks for exploration missions. Access to ISS resources, however, is becoming more and more constrained and will only be available through 2020 or 2024. NASA's Human Research Program (HRP) is actively pursuing methods to effectively utilize the ISS and appropriate ground analogs to understand and mitigate human health and performance risks prior to embarking on human exploration of deep space destinations. HRP developed a plan to use ground analogs of increasing fidelity to address questions related to exploration missions and is inviting International participation in these planned campaigns. Using established working groups and multilateral panels, the HRP is working with multiple Space Agencies to invite International participation in a series of 30- day missions that HRP will conduct in the US owned and operated Human Exploration Research Analog (HERA) during 2016. In addition, the HRP is negotiating access to Antarctic stations (both US and non-US), the German :envihab and Russian NEK facilities. These facilities provide unique capabilities to address critical research questions requiring longer duration simulation or isolation. We are negotiating release of international research opportunities to ensure a multilateral approach to future analog research campaigns, hoping to begin multilateral campaigns in the latter facilities by 2017. Collaborative use of analog facilities and shared investment in the development of spaceflight countermeasures through multilateral campaigns or missions that leverage the global scientific community will focus high quality research and provide sufficient power to accelerate the development of countermeasures and drive sound recommendations for exploration missions. This panel will provide an overview of efforts to encourage and facilitate multilateral collaboration in analog missions or campaigns and describe the facilities currently under consideration to reach the common goal of enabling safe, reliable, and productive human space exploration.

GamR, the LysR-Type Galactose Metabolism Regulator, Regulates hrp Gene Expression via Transcriptional Activation of Two Key hrp Regulators, HrpG and HrpX, in Xanthomonas oryzae pv. oryzae.

PubMed

Rashid, M Mamunur; Ikawa, Yumi; Tsuge, Seiji

2016-07-01

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial leaf blight of rice. For the virulence of the bacterium, the hrp genes, encoding components of the type III secretion system, are indispensable. The expression of hrp genes is regulated by two key hrp regulators, HrpG and HrpX: HrpG regulates hrpX, and HrpX regulates other hrp genes. Several other regulators have been shown to be involved in the regulation of hrp genes. Here, we found that a LysR-type transcriptional regulator that we named GamR, encoded by XOO_2767 of X. oryzae pv. oryzae strain MAFF311018, positively regulated the transcription of both hrpG and hrpX, which are adjacent to each other but have opposite orientations, with an intergenic upstream region in common. In a gel electrophoresis mobility shift assay, GamR bound directly to the middle of the upstream region common to hrpG and hrpX The loss of either GamR or its binding sites decreased hrpG and hrpX expression. Also, GamR bound to the upstream region of either a galactose metabolism-related gene (XOO_2768) or a galactose metabolism-related operon (XOO_2768 to XOO_2771) located next to gamR itself and positively regulated the genes. The deletion of the regulator gene resulted in less bacterial growth in a synthetic medium with galactose as a sole sugar source. Interestingly, induction of the galactose metabolism-related gene was dependent on galactose, while that of the hrp regulator genes was galactose independent. Our results indicate that the LysR-type transcriptional regulator that regulates the galactose metabolism-related gene(s) also acts in positive regulation of two key hrp regulators and the following hrp genes in X. oryzae pv. oryzae. The expression of hrp genes encoding components of the type III secretion system is essential for the virulence of many plant-pathogenic bacteria, including Xanthomonas oryzae pv. oryzae. It is specifically induced during infection. Research has revealed that in this bacterium, hrp gene expression is controlled by two key hrp regulators, HrpG and HrpX, along with several other regulators in the complex regulatory network, but the details remain unclear. Here, we found that a novel LysR-type transcriptional activator, named GamR, functions as an hrp regulator by directly activating the transcription of both hrpG and hrpX Interestingly, GamR also regulates a galactose metabolism-related gene (or operon) in a galactose-dependent manner, while the regulation of hrpG and hrpX is independent of the sugar. Our finding of a novel hrp regulator that directly and simultaneously regulates two key hrp regulators provides new insights into an important and complex regulation system of X. oryzae pv. oryzae hrp genes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

International Space Station Medical Project

NASA Technical Reports Server (NTRS)

Starkey, Blythe A.

2008-01-01

The goals and objectives of the ISS Medical Project (ISSMP) are to: 1) Maximize the utilization the ISS and other spaceflight platforms to assess the effects of longduration spaceflight on human systems; 2) Devise and verify strategies to ensure optimal crew performance; 3) Enable development and validation of a suite of integrated physical (e.g., exercise), pharmacologic and/or nutritional countermeasures against deleterious effects of space flight that may impact mission success or crew health. The ISSMP provides planning, integration, and implementation services for Human Research Program research tasks and evaluation activities requiring access to space or related flight resources on the ISS, Shuttle, Soyuz, Progress, or other spaceflight vehicles and platforms. This includes pre- and postflight activities; 2) ISSMP services include operations and sustaining engineering for HRP flight hardware; experiment integration and operation, including individual research tasks and on-orbit validation of next generation on-orbit equipment; medical operations; procedures development and validation; and crew training tools and processes, as well as operation and sustaining engineering for the Telescience Support Center; and 3) The ISSMP integrates the HRP approved flight activity complement and interfaces with external implementing organizations, such as the ISS Payloads Office and International Partners, to accomplish the HRP's objectives. This effort is led by JSC with Baseline Data Collection support from KSC.

Portable Load Measurement Device for Use During ARED Exercise on ISS

NASA Technical Reports Server (NTRS)

Hanson, A.; Peters, B.; Caldwell, E.; Sinka, J.; Kreutzburg, G.; Ploutz-Snyder, L.

2014-01-01

The Advanced Resistive Exercise Device (ARED) (Fig.1) is unique countermeasure hardware available to crewmembers aboard the International Space Station (ISS) used for resistance exercise training to protect against bone and muscle loss during long duration space missions. ARED instrumentation system was designed to measure and record exercise load data, but: - Reliably accurate data has not been available due to a defective force platform. - No ARED data has been recorded since mid-2011 due to failures in the instrumentation power system. ARED load data supports on-going HRP funded research, and is available to extramural researchers through LSDA-Repository. Astronaut Strength, Conditioning, and Rehabilitation specialists (ASCRs) use ARED data to track training progress and advance exercise prescriptions. ARED load data is necessary to fulfill medical requirements. HRP directed task intends to reduce to program risk (HRP IRMA Risk 1735), and evaluate the XSENS ForceShoeTM as a means of obtaining ARED load data during exercise sessions. The XSENS ForceShoes"TM" will fly as a hardware demonstration to ISS in May 2014 (39S). Additional portable load monitoring devices (PLMDs) are under evaluation in the ExPC Lab. PLMDs are favored over platform redesign as they support future exploration needs.

Human Research Program Human Health Countermeasures Element Nutrition Risk Standing Review Panel

NASA Technical Reports Server (NTRS)

Bistrian, Bruce

2009-01-01

The Nutrition Risk Standing Review Panel (SRP) reviewed and discussed the specific gaps and tasks for the Human Health Countermeasures (HHC) Element related to nutrition identified in the Human Research Program (HRP) Integrated Research Plan. There was general consensus that the described gaps and proposed tasks were critical to future NASA mission success. The SRP acknowledged the high scientific quality of the work currently being undertaken by the Nutritional Biochemistry group under the direction of Dr. Scott Smith. In review of the entire HRP, four new gaps were identified that complement the Element's existing research activities. Given the limitations of ground-based analogs for many of the unique physiological and metabolic alterations in space, future studies are needed to quantify nutritional factors that change during actual space flight. In addition, future tasks should seek to better evaluate the time course of physiological and metabolic alterations during flight to better predict alterations during longer duration missions. Finally, given the recent data suggesting a potential role for increased inflammatory responses during space flight, the role of inflammation needs to be explored in detail, including the development of potential countermeasures and new ground based analogs, if this possibility is confirmed.

Weaving Together Space Biology and the Human Research Program: Selecting Crops and Manipulating Plant Physiology to Produce High Quality Food for ISS Astronauts

NASA Technical Reports Server (NTRS)

Massa, Gioia; Hummerick, Mary; Douglas, Grace; Wheeler, Raymond

2015-01-01

Researchers from the Human Research Program (HRP) have teamed up with plant biologists at KSC to explore the potential for plant growth and food production on the international space station (ISS) and future exploration missions. KSC Space Biology (SB) brings a history of plant and plant-microbial interaction research for station and for future bioregenerative life support systems. JSC HRP brings expertise in Advanced Food Technology (AFT), Advanced Environmental Health (AEH), and Behavioral Health and Performance (BHP). The Veggie plant growth hardware on the ISS is the platform that first drove these interactions. As we prepared for the VEG-01 validation test of Veggie, we engaged with BHP to explore questions that could be asked of the crew that would contribute both to plant and to behavioral health research. AFT, AEH and BHP stakeholders were engaged immediately after the return of the Veggie flight samples of space-grown lettuce, and this team worked with the JSC human medical offices to gain approvals for crew consumption of the lettuce on ISS. As we progressed with Veggie testing we began performing crop selection studies for Veggie that were initiated through AFT. These studies consisted of testing and down selecting leafy greens, dwarf tomatoes, and dwarf pepper crops based on characteristics of plant growth and nutritional levels evaluated at KSC, and organoleptic quality evaluated at JSCs Sensory Analysis lab. This work has led to a successful collaborative proposal to the International Life Sciences Research Announcement for a jointly funded HRP-SB investigation of the impacts of light quality and fertilizer on salad crop productivity, nutrition, and flavor in Veggie on the ISS. With this work, and potentially with other pending joint projects, we will continue the synergistic research that will advance the space biology knowledge base, help close gaps in the human research roadmap, and enable humans to venture out to Mars and beyond.

The Nitty Gritty: How We Make Analogs Work

NASA Technical Reports Server (NTRS)

Self, A. L.; Huppman, S. R.; Spence, L. A.

2017-01-01

NASA's Human Research Program (HRP) is becoming increasingly reliant on Isolated, Confined and Controlled (ICC) analogs to accomplish many of its research objectives. Compared to other research platforms, ICC analogs present a unique set of operational challenges that must be addressed in order to ensure a high fidelity research environment. In particular, the Human Exploration Research Analog (HERA) habitat, which is classified as an ICC environment, has been developed over the past three years to accommodate the operational needs of research investigations from each of the HRP Elements. During the development period, various types of requirements have contributed to the current operational model, which strives to achieve the highest possible level of mission fidelity with limited resources. This presentation will focus on the operational aspects of the HERA habitat, with emphasis on how we develop the analog research environment to meet researchers' needs. Specific discussion topics include mission scenario development, operational tasks, mission timeline integration, stressor implementation, console support, and improvements based on lessons learned. The information is intended to help investigators better understand the details behind HERA operations and the benefits to their research goals.

2013 Immune Risk Standing Review Panel Research Plan Review for: The Risk of Crew Adverse Health Event Due to Altered Immune Response

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

The 2013 Immune Risk Standing Review Panel (from here on referred to as the SRP) participated in a meeting with representatives from the Human Research Program (HRP) Human Health Countermeasures (HHC) Element and HRP management on February 3-4, 2014 in Houston, TX to review the updated Research Plan for the Risk of Crew Adverse Health Event Due to Altered Immune Response in the HRP Integrated Research Plan. The SRP is impressed with the work the immune discipline has done since the 2012 SRP review and agrees with the new wording of the Gaps, no longer questions, now statements. The SRP also likes the addition of adding targets for closing the Gaps, but it is not clear how they got to some of the interim stages (interval percentages). A major concern that the SRP has mentioned since the initial 2009 SRP meeting is that there is still not enough emphasis on the interdisciplinary aspect of the immune risk associated with other risks (i.e., nutrition, radiation, etc.). The SRP recommends that a "translational SRP" or advisory group be developed that is composed of members from all of the HRP SRPs. The SRP also thinks that the immune discipline should consider a more systems biology approach. Lastly, the SRP is concerned that the risks observed in research from low Earth orbit (LEO) missions may not accurately reflect all the risks of longer duration flight beyond LEO. Also, there does not seem to be a concern for immune responses that may occur when someone is in space longer than six months, for example, a Mars mission would take three years. The absence of disease in past and current flight scenarios does not mean the risk may not be there in future flight settings.

10 CFR 712.12 - HRP implementation.

Code of Federal Regulations, 2012 CFR

2012-01-01

... DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...) Report any observed or reported behavior or condition of another HRP-certified individual that could indicate a reliability concern, including those behaviors and conditions listed in § 712.13(c), to a...

Human Research Program Human Health Countermeasures Element Bone and Muscle Risk Standing Review Panel (SRP)

NASA Technical Reports Server (NTRS)

Glowacki, Julie; Gregor, Robert

2009-01-01

The Bone and Muscle Risk Standing Review Panel (SRP) met at the NASA Johnson Space Center (JSC) on October 4-6, 2009 to discuss the areas of current and future research targeted by the Human Health Countermeasures (HHC) Element of the Human Research Program (HRP). Using evidence-based knowledge as a background for identified risks to astronaut health and performance, NASA had identified gaps in knowledge to address those risks. Ongoing and proposed tasks were presented to address the gaps. The charge to the Bone and Muscle Risk SRP was to review the gaps, evaluate whether the tasks addressed these gaps and to make recommendations to NASA s HRP Science Management Office regarding the Panel's review. The Bone and Muscle Risk SRP consisted of scientists who are experts in muscle, bone, or both and could evaluate the existing evidence with sufficient knowledge of the potential effects of long duration exposure to microgravity. More important, although expertise in basic science is important, the SRP was requested to evaluate the practicality of the proposed efforts in light of the realistic demands placed on the HRP. In short, all tasks presented in the Integrated Research Plan (IRP) should address specific questions related to the challenges faced by the astronauts as a result of prolonged exposure to microgravity. All tasks proposed to fill the gaps in knowledge should provide applied, translational data necessary to answer the specific questions. Several presentations were made to the SRP during the site visit and the SRP spent sufficient time to address the panel charge, either as a group or in separate sessions for the Bone and Muscle Risk subgroups. The SRP made a final debriefing to the HRP Program Scientist, Dr. John B. Charles, on October 6, 2009. Taking the evidence and identified risks as givens, the SRP concluded that 1) integration of information should lead to a more comprehensive approach to identifying the gaps, 2) not all tasks addressed the gaps as stated in the IRP, 3) better access should be given to the SRP to existing data to include in its review, and 4) there were some missing gaps and tasks. As a result the SRP recommended 1) combining certain gaps, which in some cases were addressed with identical tasks, 2) additional gaps and tasks to address some of the gaps, 3) deleting some gaps and tasks to better focus the efforts of NASA s HRP in prioritizing their efforts, and 4) prioritizing gaps to address significant issues needing resolution in the short term while maintaining an awareness of long-term goals. The SRP commended the efforts by the HHC Element to integrate knowledge gaps and design tasks to address the higher order questions. This integration did not apply to the Bone and Muscle Risk alone, but the musculoskeletal system as its functions integrate with issues in

2015 Dust Risk Standing Review Panel

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2015-01-01

The 2015 Dust Risk Standing Review Panel (SRP) participated in a WebEx/teleconference with members of the Space Human Factors and Habitability (SHFH) Element, representatives from the Human Research Program (HRP), NASA Headquarters, and the NASA Research Education and Support Services on November 12, 2015. The SRP reviewed the updated Evidence Report for The Risk of Adverse Health and Performance Effects of Celestial Dust Exposure, as well as the Research Plan for this Risk.

10 CFR 712.10 - Designation of HRP positions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... duties or has responsibility for working with, protecting, or transporting nuclear explosives, nuclear... 10 Energy 4 2012-01-01 2012-01-01 false Designation of HRP positions. 712.10 Section 712.10 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...

10 CFR 712.10 - Designation of HRP positions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... duties or has responsibility for working with, protecting, or transporting nuclear explosives, nuclear... 10 Energy 4 2013-01-01 2013-01-01 false Designation of HRP positions. 712.10 Section 712.10 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...

10 CFR 712.10 - Designation of HRP positions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... duties or has responsibility for working with, protecting, or transporting nuclear explosives, nuclear... 10 Energy 4 2010-01-01 2010-01-01 false Designation of HRP positions. 712.10 Section 712.10 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...

10 CFR 712.10 - Designation of HRP positions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... duties or has responsibility for working with, protecting, or transporting nuclear explosives, nuclear... 10 Energy 4 2011-01-01 2011-01-01 false Designation of HRP positions. 712.10 Section 712.10 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...

10 CFR 712.10 - Designation of HRP positions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... duties or has responsibility for working with, protecting, or transporting nuclear explosives, nuclear... 10 Energy 4 2014-01-01 2014-01-01 false Designation of HRP positions. 712.10 Section 712.10 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability...

Analogs and the BHP Risk Reduction Strategy for Future Spaceflight Missions

NASA Technical Reports Server (NTRS)

Whitmire, Sandra; Leveton, Lauren

2011-01-01

In preparation for future exploration missions to distant destinations (e.g., Moon, Near Earth Objects (NEO), and Mars), the NASA Human Research Program s (HRP) Behavioral Health and Performance Element (BHP) conducts and supports research to address four human health risks: Risk of Behavioral Conditions; Risk of Psychiatric Conditions; Risk of Performance Decrements Due to Inadequate Cooperation, Coordination, Communication, and Psychosocial Adaptation within a Team; and Risk of Performance Errors due to Sleep Loss, Fatigue, Circadian Desynchronization, and Work Overload (HRP Science Management Plan, 2008). BHP Research, in collaboration with internal and external research investigators, as well as subject matter experts within NASA operations including flight surgeons, astronauts, and mission planners and others within the Mission Operations Directorate (MOD), identifies knowledge and technology gaps within each Risk. BHP Research subsequently manages and conducts research tasks to address and close the gaps, either through risk assessment and quantification, or the development of countermeasures and monitoring technologies. The resulting deliverables, in many instances, also support current Medical Operations and/or Mission Operations for the International Space Station (ISS).

Programmatic Considerations to Reduce the Risk of Adverse Renal Stone Events in Spaceflight

NASA Technical Reports Server (NTRS)

Antonsen, Erik; Pietrzyk, Robert

2017-01-01

Introduction: Microgravity exposure may alter the likelihood that astronauts will experience renal stones. The potential risk includes both acute and chronic health issues, with the potential for significant impact on mission objectives. Methods: To understand the role of the NASA's Human Research Program (HRP) research agenda in both preventing and addressing renal stones in spaceflight, current astronaut epidemiologic data and a summary of programmatic considerations are reviewed. Results: Although there has never been a symptomatic renal stone event in a U.S. crewmember during spaceflight, urine chemistry has been altered - likely due to induced changes in renal physiology as a result of exposure to microgravity. This may predispose astronauts to stone formation, leading the HRP to conduct and sponsor research to: 1) understand the risk of stone formation in space; 2) prevent stones from forming; and 3) address stones that may form by providing novel diagnostic and therapeutic approaches. Discussion: The development of a renal stone during spaceflight is a significant medical concern that requires the HRP to minimize this risk by providing the ability to prevent, diagnose, monitor and treat the condition during spaceflight. A discussion of the risk as NASA understands it is followed by an overview of the multiple mitigations currently under study, including novel ultrasound techniques for stone detection and manipulation, and how they may function as part of a larger exploration medical system.

10 CFR 712.38 - Maintenance of medical records.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Maintenance of medical records. 712.38 Section 712.38 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Medical Standards § 712.38 Maintenance of medical records. (a) The medical records of HRP candidates and HRP-certified individuals must be maintained in...

10 CFR 712.38 - Maintenance of medical records.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Maintenance of medical records. 712.38 Section 712.38 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Medical Standards § 712.38 Maintenance of medical records. (a) The medical records of HRP candidates and HRP-certified individuals must be maintained in...

Human Research Program (HRP) Exploration Medical Capability (ExMC) Standing Review Panel (SRP)

NASA Technical Reports Server (NTRS)

Cintron, Nitza; Dutson, Eric; Friedl, Karl; Hyman, William; Jemison, Mae; Klonoff, David

2009-01-01

The SRP believes strongly that regularly performed in-flight crew assessments are needed in order to identify a change in health status before a medical condition becomes clinically apparent. It is this early recognition in change that constitutes the foundation of the "occupational health model" expounded in the HRP Requirements Document as a key component of the HRP risk mitigation strategy that will enable its objective of "prevention and mitigation of human health and performance risks". A regular crew status examination of physiological and clinical performance is needed. This can be accomplished through instrumented monitoring of routine embedded tasks. The SRP recommends addition of a new gap to address this action under Category 3.0 Mitigate the Risk. This new gap is closely associated with Task 4.19 which addresses the lack of adequate biomedical monitoring capabilities for performing periodic clinical status evaluations and contingency medical monitoring. A corollary to these gaps is the critical emphasis on preventive medicine, not only during pre- and post-flight phases of a mission as is the current practice, but continued into the in-flight phases of exploration class missions.

10 CFR 712.11 - General requirements for HRP certification.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false General requirements for HRP certification. 712.11 Section 712.11 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the... provisions of 10 CFR part 707 for DOE contractors, and DOE Order 3792.3, “Drug-Free Federal Workplace Testing...

Risk of Adverse Health Outcomes and Decrements in Performance Due to In-flight Medical Conditions

NASA Technical Reports Server (NTRS)

Antonsen,Erik

2017-01-01

The drive to undertake long-duration space exploration missions at greater distances from Earth gives rise to many challenges concerning human performance under extreme conditions. At NASA, the Human Research Program (HRP) has been established to investigate the specific risks to astronaut health and performance presented by space exploration, in addition to developing necessary countermeasures and technology to reduce risk and facilitate safer, more productive missions in space (NASA Human Research Program 2009). The HRP is divided into five subsections, covering behavioral health, space radiation, habitability, and other areas of interest. Within this structure is the ExMC Element, whose research contributes to the overall development of new technologies to overcome the challenges of expanding human exploration and habitation of space. The risk statement provided by the HRP to the ExMC Element states: "Given that medical conditions/events will occur during human spaceflight missions, there is a possibility of adverse health outcomes and decrements in performance in mission and for long term health" (NASA Human Research Program 2016). Within this risk context, the Exploration Medical Capabilities (ExMC) Element is specifically concerned with establishing evidenced-based methods of monitoring and maintaining astronaut health. Essential to completing this task is the advancement in techniques that identify, prevent, and treat any health threats that may occur during space missions. The ultimate goal of the ExMC Element is to develop and demonstrate a pathway for medical system integration into vehicle and mission design to mitigate the risk of medical issues. Integral to this effort is inclusion of an evidence-based medical and data handling system appropriate for long-duration, exploration-class missions. This requires a clear Concept of Operations, quantitative risk metrics or other tools to address changing risk throughout a mission, and system scoping and system engineering. Because of the novel nature of the risks involved in exploration missions, new and complex ethical challenges are likely to be encountered. This document describes the relevant background and evidence that informs the development of an exploration medical system.

Integrating Spaceflight Human System Risk Research

NASA Technical Reports Server (NTRS)

Mindock, J.; Lumpkins, S.; Anton, W.; Havenhill, M.; Shelhamer, M.; Canga, M.

2016-01-01

NASA is working to increase the likelihoods of human health and performance success during exploration missions, and subsequent crew long-term health. To manage the risks in achieving these goals, a system modeled after a Continuous Risk Management framework is in place. "Human System Risks" (Risks) have been identified, and approximately 30 are being actively addressed by NASA's Human Research Program (HRP). Research plans for each of HRP's Risks have been developed and are being executed. Ties between the research efforts supporting each Risk have been identified, however, this has been in an ad hoc fashion. There is growing recognition that solutions developed to address the full set of Risks covering medical, physiological, behavioral, vehicle, and organizational aspects of the exploration missions must be integrated across Risks and disciplines. We will discuss how a framework of factors influencing human health and performance in space is being applied as the backbone for bringing together sometimes disparate information relevant to the individual Risks. The resulting interrelated information is allowing us to identify and visualize connections between Risks and research efforts in a systematic and standardized way. We will discuss the applications of the visualizations and insights to research planning, solicitation, and decision-making processes.

An Integrated Extravehicular Activity Research Plan

NASA Technical Reports Server (NTRS)

Abercromby, Andrew F. J.; Ross, Amy J.; Cupples, J. Scott

2016-01-01

Multiple organizations within NASA and outside of NASA fund and participate in research related to extravehicular activity (EVA). In October 2015, representatives of the EVA Office, the Crew and Thermal Systems Division (CTSD), and the Human Research Program (HRP) at NASA Johnson Space Center agreed on a formal framework to improve multi-year coordination and collaboration in EVA research. At the core of the framework is an Integrated EVA Research Plan and a process by which it will be annually reviewed and updated. The over-arching objective of the collaborative framework is to conduct multi-disciplinary cost-effective research that will enable humans to perform EVAs safely, effectively, comfortably, and efficiently, as needed to enable and enhance human space exploration missions. Research activities must be defined, prioritized, planned and executed to comprehensively address the right questions, avoid duplication, leverage other complementary activities where possible, and ultimately provide actionable evidence-based results in time to inform subsequent tests, developments and/or research activities. Representation of all appropriate stakeholders in the definition, prioritization, planning and execution of research activities is essential to accomplishing the over-arching objective. A formal review of the Integrated EVA Research Plan will be conducted annually. External peer review of all HRP EVA research activities including compilation and review of published literature in the EVA Evidence Book is already performed annually. Coordination with stakeholders outside of the EVA Office, CTSD, and HRP is already in effect on a study-by-study basis; closer coordination on multi-year planning with other EVA stakeholders including academia is being actively pursued. Details of the current Integrated EVA Research Plan are presented including description of ongoing and planned research activities in the areas of: Benchmarking; Anthropometry and Suit Fit; Sensors; Human-Suit Modeling; Suit Trauma Monitoring and Countermeasures; EVA Workload and Duration Effects; Decompression Sickness Risk Mitigation; Deconditioned EVA Performance; and Exploration EVA Concept of Operations.

An improved method for undertaking limiting dilution assays for in vitro cloning of Plasmodium falciparum parasites.

PubMed

Butterworth, Alice S; Robertson, Alan J; Ho, Mei-Fong; Gatton, Michelle L; McCarthy, James S; Trenholme, Katharine R

2011-04-18

Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11 ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue. © 2011 Butterworth et al; licensee BioMed Central Ltd.

Harnessing the Risk-Related Data Supply Chain: An Information Architecture Approach to Enriching Human System Research and Operations Knowledge

NASA Technical Reports Server (NTRS)

Buquo, Lynn; Johnson-Throop, Kathy

2010-01-01

NASA's Human Research Program (HRP) and Space Life Sciences Directorate (SLSD), not unlike many NASA organizations today, struggle with the inherent inefficiencies caused by dependencies on heterogeneous data systems and silos of data and information spread across decentralized discipline domains. The capture of operational and research-based data/information (both in-flight and ground-based) in disparate IT systems impedes the extent to which that data/information can be efficiently and securely shared, analyzed, and enriched into knowledge that directly and more rapidly supports HRP's research-focused human system risk mitigation efforts and SLSD s operationally oriented risk management efforts. As a result, an integrated effort is underway to more fully understand and document how specific sets of risk-related data/information are generated and used and in what IT systems that data/information currently resides. By mapping the risk-related data flow from raw data to useable information and knowledge (think of it as the data supply chain), HRP and SLSD are building an information architecture plan to leverage their existing, shared IT infrastructure. In addition, it is important to create a centralized structured tool to represent risks including attributes such as likelihood, consequence, contributing factors, and the evidence supporting the information in all these fields. Representing the risks in this way enables reasoning about the risks, e.g. revisiting a risk assessment when a mitigation strategy is unavailable, updating a risk assessment when new information becomes available, etc. Such a system also provides a concise way to communicate the risks both within the organization as well as with collaborators. Understanding and, hence, harnessing the human system risk-related data supply chain enhances both organizations' abilities to securely collect, integrate, and share data assets that improve human system research and operations.

Habitability and Human Factors Contributions to Human Space Flight

NASA Technical Reports Server (NTRS)

Sumaya, Jennifer Boyer

2011-01-01

This slide presentation reviews the work of the Habitability and Human Factors Branch in support of human space flight in two main areas: Applied support to major space programs, and Space research. The field of Human Factors applies knowledge of human characteristics for the design of safer, more effective, and more efficient systems. This work is in several areas of the human space program: (1) Human-System Integration (HSI), (2) Orion Crew Exploration Vehicle, (3) Extravehicular Activity (EVA), (4) Lunar Surface Systems, (5) International Space Station (ISS), and (6) Human Research Program (HRP). After detailing the work done in these areas, the facilities that are available for human factors work are shown.

Integration and Cooperation in the Next Golden Age of Human Space Flight Data Repositories: Tools for Retrospective Analysis and Future Planning

NASA Technical Reports Server (NTRS)

Thomas, D.; Fitts, M.; Wear, M.; VanBaalen, M.

2011-01-01

As NASA transitions from the Space Shuttle era into the next phase of space exploration, the need to ensure the capture, analysis, and application of its research and medical data is of greater urgency than at any other previous time. In this era of limited resources and challenging schedules, the Human Research Program (HRP) based at NASA s Johnson Space Center (JSC) recognizes the need to extract the greatest possible amount of information from the data already captured, as well as focus current and future research funding on addressing the HRP goal to provide human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration. To this end, the Science Management Office and the Medical Informatics and Health Care Systems Branch within the HRP and the Space Medicine Division have been working to make both research data and clinical data more accessible to the user community. The Life Sciences Data Archive (LSDA), the research repository housing data and information regarding the physiologic effects of microgravity, and the Lifetime Surveillance of Astronaut Health Repository (LSAH-R), the clinical repository housing astronaut data, have joined forces to achieve this goal. The task of both repositories is to acquire, preserve, and distribute data and information both within the NASA community and to the science community at large. This is accomplished via the LSDA s public website (http://lsda.jsc.nasa.gov), which allows access to experiment descriptions including hardware, datasets, key personnel, mission descriptions and a mechanism for researchers to request additional data, research and clinical, that is not accessible from the public website. This will result in making the work of NASA and its partners available to the wider sciences community, both domestic and international. The desired outcome is the use of these data for knowledge discovery, retrospective analysis, and planning of future research studies.

NASA Life Sciences Data Repositories: Tools for Retrospective Analysis and Future Planning

NASA Technical Reports Server (NTRS)

Thomas, D.; Wear, M.; VanBaalen, M.; Lee, L.; Fitts, M.

2011-01-01

As NASA transitions from the Space Shuttle era into the next phase of space exploration, the need to ensure the capture, analysis, and application of its research and medical data is of greater urgency than at any other previous time. In this era of limited resources and challenging schedules, the Human Research Program (HRP) based at NASA s Johnson Space Center (JSC) recognizes the need to extract the greatest possible amount of information from the data already captured, as well as focus current and future research funding on addressing the HRP goal to provide human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration. To this end, the Science Management Office and the Medical Informatics and Health Care Systems Branch within the HRP and the Space Medicine Division have been working to make both research data and clinical data more accessible to the user community. The Life Sciences Data Archive (LSDA), the research repository housing data and information regarding the physiologic effects of microgravity, and the Lifetime Surveillance of Astronaut Health (LSAH-R), the clinical repository housing astronaut data, have joined forces to achieve this goal. The task of both repositories is to acquire, preserve, and distribute data and information both within the NASA community and to the science community at large. This is accomplished via the LSDA s public website (http://lsda.jsc.nasa.gov), which allows access to experiment descriptions including hardware, datasets, key personnel, mission descriptions and a mechanism for researchers to request additional data, research and clinical, that is not accessible from the public website. This will result in making the work of NASA and its partners available to the wider sciences community, both domestic and international. The desired outcome is the use of these data for knowledge discovery, retrospective analysis, and planning of future research studies.

2014 SRP Integration Transcript

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

HRP's mission is to reduce the risks to human health and performance during long-duration spaceflight. The HRP Integrated Research Plan (IRP) contains the research plans for the 32 risks that require research to characterize and mitigate. From its inception the "integrate" aspect of the IRP has denoted the integrated nature of risks to human health and performance. Even though each risk in the IRP has its own research plan and is tracked separately, the interrelated nature of health and performance requires that they be addressed in an integrative or holistic fashion so that the connectedness of physiological systems within the human body and the integrated response to spaceflight can be addressed. Common characteristics of the spaceflight environment include altered gravity, atmospheres, and light/dark cycles; space radiation; isolation; noise; and periods of high or low workload. Long-term exposure to this unique environment produces a suite of physiological effects such as stress; vision, neurocognitive, and anthropometric changes; circadian misalignment; fluid shifts; cardiovascular deconditioning; immune dysregulation; and altered nutritional requirements. Expanding cross-disciplinary integrative approaches that synthesize concepts or data from two or more disciplines would improve the identification and characterization risk factors, and enable the development of countermeasures relevant to multiple risks. Cross-disciplinary approaches might also help to illuminate problem areas that may arise when a countermeasure adversely impacts risks other than those which it was developed to mitigate, or to identify groupings of physiological changes that are likely to occur that may impact the overall risk posture. In 2014 HRP embarked on a pilot study that combined four SRPs (and 12 HRP risks) - Behavioral Health, Sensorimotor, Cardiovascular, and Bone/Muscle - specifically to discuss cross-disciplinary integration. The points outlined below were suggested to seed the discussion, within the bounding constraint that research plans must be feasible and relevant to the HRP mission. While these were suggested starting points, the overall guiding principle was to allow free discussion from panel members on any aspect of integrated research that they felt was important, Existing cross-disciplinary integration as documented in the IRP (HRR), Existing or needed integration already identified by HRP, but not yet well defined within the IRP, Areas of integration that are missing.

Advancing Translational Space Research Through Biospecimen Sharing: Amplified Impact of Studies Utilizing Analogue Space Platforms

NASA Technical Reports Server (NTRS)

Staten, B.; Moyer, E.; Vizir, V.; Gompf, H.; Hoban-Higgins, T.; Lewis, L.; Ronca, A.; Fuller, C. A.

2016-01-01

Biospecimen Sharing Programs (BSPs) have been organized by NASA Ames Research Center since the 1960s with the goal of maximizing utilization and scientific return from rare, complex and costly spaceflight experiments. BSPs involve acquiring otherwise unused biological specimens from primary space research experiments for distribution to secondary experiments. Here we describe a collaboration leveraging Ames expertise in biospecimen sharing to magnify the scientific impact of research informing astronaut health funded by the NASA Human Research Program (HRP) Human Health Countermeasures (HHC) Element. The concept expands biospecimen sharing to one-off ground-based studies utilizing analogue space platforms (e.g., Hindlimb Unloading (HLU), Artificial Gravity) for rodent experiments, thereby significantly broadening the range of research opportunities with translational relevance for protecting human health in space and on Earth.

Integrating Spaceflight Human System Risk Research

NASA Technical Reports Server (NTRS)

Mindock, Jennifer; Lumpkins, Sarah; Anton, Wilma; Havenhill, Maria; Shelhamer, Mark; Canga, Michael

2016-01-01

NASA is working to increase the likelihood of human health and performance success during exploration missions as well as to maintain the subsequent long-term health of the crew. To manage the risks in achieving these goals, a system modelled after a Continuous Risk Management framework is in place. "Human System Risks" (Risks) have been identified, and approximately 30 are being actively addressed by NASA's Human Research Program (HRP). Research plans for each of HRP's Risks have been developed and are being executed. Inter-disciplinary ties between the research efforts supporting each Risk have been identified; however, efforts to identify and benefit from these connections have been mostly ad hoc. There is growing recognition that solutions developed to address the full set of Risks covering medical, physiological, behavioural, vehicle, and organizational aspects of exploration missions must be integrated across Risks and disciplines. This paper discusses how a framework of factors influencing human health and performance in space is being applied as the backbone for bringing together sometimes disparate information relevant to the individual Risks. The resulting interrelated information enables identification and visualization of connections between Risks and research efforts in a systematic and standardized manner. This paper also discusses the applications of the visualizations and insights into research planning, solicitation, and decision-making processes.

Integrating spaceflight human system risk research

NASA Astrophysics Data System (ADS)

Mindock, Jennifer; Lumpkins, Sarah; Anton, Wilma; Havenhill, Maria; Shelhamer, Mark; Canga, Michael

2017-10-01

NASA is working to increase the likelihood of exploration mission success and to maintain crew health, both during exploration missions and long term after return to Earth. To manage the risks in achieving these goals, a system modelled after a Continuous Risk Management framework is in place. ;Human System Risks; (Risks) have been identified, and 32 are currently being actively addressed by NASA's Human Research Program (HRP). Research plans for each of HRP's Risks have been developed and are being executed. Inter-disciplinary ties between the research efforts supporting each Risk have been identified; however, efforts to identify and benefit from these connections have been mostly ad hoc. There is growing recognition that solutions developed to address the full set of Risks covering medical, physiological, behavioural, vehicle, and organizational aspects of exploration missions must be integrated across Risks and disciplines. This paper discusses how a framework of factors influencing human health and performance in space is being applied as the backbone for bringing together sometimes disparate information relevant to the individual Risks. The resulting interrelated information enables identification and visualization of connections between Risks and research efforts in a systematic and standardized manner. This paper also discusses the applications of the visualizations and insights into research planning, solicitation, and decision-making processes.

Effect of pulsed light on activity and structural changes of horseradish peroxidase

USDA-ARS?s Scientific Manuscript database

The objective of this research was to investigate the effects of pulsed light (PL) on the activity and structure of horseradish peroxidase (HRP) in buffer solution. Enzyme residual activities were measured after PL. Surface topography, secondary, and tertiary structures of HRP were determined using ...

An Evidence Based Approach to Designing Medical Support for Long Duration, Interplanetary Missions

NASA Technical Reports Server (NTRS)

Watkins, S. D.; McGrath, T. L.; Bauman, D. K.; Wu, J. H.; Barsten, K. N.; Barr, Y. R.; Kerstman, E. L.

2011-01-01

The Exploration Medical Capability (ExMC) element is one of six elements under NASA's Human Research Program (HRP). The goal of the ExMC element is to address the risk of the "inability to adequately recognize or treat an ill or injured crewmember." This poster highlights the evidence-based approach that the ExMC element has taken to address this goal, and the ExMC element's current areas of interest.

In-Flight Laboratory Analysis

NASA Technical Reports Server (NTRS)

Baumann, David; Perusek, Gail; Nelson, Emily; Krihak, Michael; Brown, Dan

2012-01-01

One-year study objectives align with HRP requirements. HRP requirements include measurement panels for research and medical operations - These measurement panels are distinctly different. Instrument requirements are defined - Power, volume and mass not quite a critical limitation as for medical operations (deep space exploration missions). One-year evaluation goals will lead HHC towards in-flight laboratory analysis capability.

HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

PubMed Central

Kim, Jihyun F.; Beer, Steven V.

1998-01-01

Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream of hrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwinia species, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts. PMID:9748455

Harnessing the Risk-Related Data Supply Chain: An Information Architecture Approach to Enriching Human System Research and Operations Knowledge

NASA Technical Reports Server (NTRS)

Buquo, Lynn E.; Johnson-Throop, Kathy A.

2011-01-01

An Information Architecture facilitates the understanding and, hence, harnessing of the human system risk-related data supply chain which enhances the ability to securely collect, integrate, and share data assets that improve human system research and operations. By mapping the risk-related data flow from raw data to useable information and knowledge (think of it as a data supply chain), the Human Research Program (HRP) and Space Life Science Directorate (SLSD) are building an information architecture plan to leverage their existing, and often shared, IT infrastructure.

Clinical Outcome Metrics for Optimization of Robust Training

NASA Technical Reports Server (NTRS)

Ebert, D.; Byrne, V. E.; McGuire, K. M.; Hurst, V. W., IV; Kerstman, E. L.; Cole, R. W.; Sargsyan, A. E.; Garcia, K. M,; Foy, M. H.; Dulchavsky, S. A.;

Quantification of Histidine-Rich Protein 3 of Plasmodium falciparum.

PubMed

Palani, Balraj

2018-04-01

Malaria is a life-threatening infectious disease and continues to be a major public health crisis in many parts of the tropical world. Plasmodium falciparum is responsible for the majority of mortality and morbidity associated with malaria. During the intraerythrocytic cycle, P. falciparum releases three proteins with high histidine content as follows: histidine-rich protein 1 (HRP1), histidine-rich protein 2 (HRP2), and histidine-rich protein 3 (HRP3). Currently, most of the diagnostic tests of P. falciparum infection target HRP2, and a number of monoclonal antibodies (mAbs) against HRP2 have been developed for use in HRP2 detection and quantification. When parasites have HRP2 deletions, the detection of HRP3 could augment the sensitivity of the detection system. The combination of both HRP2 and HRP3 mAbs in the detection system will enhance the test sensitivity. In the HRP quantitative enzyme-linked immunosorbent assay (ELISA), both HRP2 and HRP3 contribute to the result, but the relative contribution of HRP2 and HRP3 was unable to investigate, because of the nonavailability of HRP3 specific antibody ELISA. Hence an ELISA test system based on HRP3 is also essential for detection and quantification. There is not much documented in the literature on HRP3 antigen and HRP3 specific mAbs and polyclonal antibodies (pAbs). In the present study, recombinant HRP3 was expressed in Escherichia coli and purified with Ni-NTA agarose column. The purified rHRP3 was used for the generation and characterization of monoclonal and pAbs. The purification of monoclonal and pAbs was done using a mixed-mode chromatography sorbent, phenylpropylamine HyperCel™. With the purified antibodies, a sandwich ELISA was developed. The sandwich ELISA method was explored to detect and quantify HRP3 of P. falciparum in the spent medium. The generated mAbs could be potentially used for the detection and quantification of P. falciparum HRP3.

International Space Station as Analog of Interplanetary Transit Vehicle For Biomedical Research

NASA Technical Reports Server (NTRS)

Charles, John B.

2012-01-01

Astronaut missions lasting up to six months aboard the International Space Station (ISS) have much in common with interplanetary flights, especially the outbound, Earth-to-Mars transit portion of a Mars mission. Utilization of ISS and other appropriate platforms to prepare for crewed expeditions to planetary destinations including Mars has been the work of NASA's Human Research Program (HRP) since 2005. HRP is charged specifically to understand and reduced the risks to astronaut health and performance in space exploration missions: everything HRP does and has done is directly related to that responsibility. Two major categories of human research have capitalized on ISS capabilities. The first category centers on the biomedical aspects of long-duration exposure to spaceflight factors, including prolonged weightlessness, radiation exposure, isolation and confinement, and actual risk to life and limb. These studies contribute to astronaut safety, health and efficiency on any long-duration missions, whether in low Earth orbit (LEO) or beyond. Qualitatively, weightlessness is weightlessness, whether in LEO or en route to Mars. The HRP sponsors investigations into losses in muscle and bone integrity, cardiovascular function, sensory-motor capability, immune capacity and psychosocial health, and development and demonstration of appropriate treatments and preventative measures. The second category includes studies that are focused on planetary expeditions beyond LEO. For these, ISS offers a high fidelity analog to investigate the combined effects of spaceflight factors (described above) plus the isolation and autonomy associated with simulated increasing distance from Earth. Investigations address crew cohesion, performance and workload, and mission control performance. The behavioral health and performance and space human factors aspects of planetary missions dominate this category. Work has already begun on a new investigation in this category which will examine the effects of a simulated lag in communications (mimicking that expected in transit to Mars) on astronaut performance aboard ISS. Extension of the current ISS increment duration from six months to nine or even twelve months would provide opportunities for expanded research relevant to long duration missions, albeit at the cost of fewer astronauts as subjects for those investigations. Given the possible limited access to ISS after 2020, if ISS is intended to facilitate future exploration missions, then the in-flight human investigations should focus on those that clearly enable future exploration missions.

Integration of multiple stimuli-sensing systems to regulate HrpS and type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Zhao, Youfu

2018-02-01

The bacterial enhancer binding protein (bEBP) HrpS is essential for Erwinia amylovora virulence by activating the type III secretion system (T3SS). However, how the hrpS gene is regulated remains poorly understood in E. amylovora. In this study, 5' rapid amplification of cDNA ends and promoter deletion analyses showed that the hrpS gene contains two promoters driven by HrpX/HrpY and the Rcs phosphorelay system, respectively. Electrophoretic mobility shift and gene expression assays demonstrated that integration host factor IHF positively regulates hrpS expression through directly binding the hrpX promoter and positively regulating hrpX/hrpY expression. Moreover, hrpX expression was down-regulated in the relA/spoT ((p)ppGpp-deficient) mutant and the dksA mutant, but up-regulated when the wild-type strain was treated with serine hydroxamate, which induced (p)ppGpp-mediated stringent response. Furthermore, the csrA mutant showed significantly reduced transcripts of major hrpS activators, including the hrpX/hrpY, rcsA and rcsB genes, indicating that CsrA is required for full hrpS expression. On the other hand, the csrB mutant exhibited up-regulation of the rcsA and rcsB genes, and hrpS expression was largely diminished in the csrB/rcsB mutant, indicating that the Rcs system is mainly responsible for the increased hrpS expression in the csrB mutant. These findings suggest that E. amylovora recruits multiple stimuli-sensing systems, including HrpX/HrpY, the Rcs phosphorelay system and the Gac-Csr system, to regulate hrpS and T3SS gene expression.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall.

PubMed

Brown, I R; Mansfield, J W; Taira, S; Roine, E; Romantschuk, M

2001-03-01

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

PubMed

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determining the Relative Criticality of Diverse Exploration Risks in NASA's Human Research Program

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.; Edwards, J. Michelle; Anton, Wilma; Robotham, Kwesi

2009-01-01

The mission of NASA s Human Research Program (HRP) is to understand and reduce the risk to crew health and performance in exploration missions. The HRP addresses 27 specific risks, primarily in the context of Continuous Risk Management. Each risk is evaluated in terms of two missions (a six month stay on the Moon and a thirty month round trip to Mars) and three types of consequences (in-mission crew health, post-mission crew health, and in-mission performance). The lack of a common metric between the three consequence scales, such as financial costs or quality adjusted life years lost, makes it difficult to compare the relative criticality of the risks. We are, therefore, exploring the use of a ternary scale of criticality based on the common metric of influencing an operational decision. The three levels correspond to the level of concern the risk generates for a "go/no-go" decision to launch a mission: 1) no-go; 2) go with significant reservations; 3) go. The criticality of each of the 27 risks is scored for the three types of consequence in both types of mission. The scores are combined to produce an overall criticality rating for each risk. The overall criticality rating can then be used to guide the prioritization of resources to affect the greatest amount of risk reduction.

Human Research Program Advanced Exercise Concepts (AEC) Overview

NASA Technical Reports Server (NTRS)

Perusek, Gail; Lewandowski, Beth; Nall, Marsha; Norsk, Peter; Linnehan, Rick; Baumann, David

2015-01-01

Exercise countermeasures provide benefits that are crucial for successful human spaceflight, to mitigate the spaceflight physiological deconditioning which occurs during exposure to microgravity. The NASA Human Research Program (HRP) within the Human Exploration and Operations Mission Directorate (HEOMD) is managing next generation Advanced Exercise Concepts (AEC) requirements development and candidate technology maturation to Technology Readiness Level (TRL) 7 (ground prototyping and flight demonstration) for all exploration mission profiles from Multi Purpose Crew Vehicle (MPCV) Exploration Missions (up to 21 day duration) to Mars Transit (up to 1000 day duration) missions. These validated and optimized exercise countermeasures systems will be provided to the ISS Program and MPCV Program for subsequent flight development and operations. The International Space Station (ISS) currently has three major pieces of operational exercise countermeasures hardware: the Advanced Resistive Exercise Device (ARED), the second-generation (T2) treadmill, and the cycle ergometer with vibration isolation system (CEVIS). This suite of exercise countermeasures hardware serves as a benchmark and is a vast improvement over previous generations of countermeasures hardware, providing both aerobic and resistive exercise for the crew. However, vehicle and resource constraints for future exploration missions beyond low Earth orbit will require that the exercise countermeasures hardware mass, volume, and power be minimized, while preserving the current ISS capabilities or even enhancing these exercise capabilities directed at mission specific physiological functional performance and medical standards requirements. Further, mission-specific considerations such as preservation of sensorimotor function, autonomous and adaptable operation, integration with medical data systems, rehabilitation, and in-flight monitoring and feedback are being developed for integration with the exercise countermeasures systems. Numerous technologies have been considered and evaluated against HRP-approved functional device requirements for these extreme mission profiles, and include wearable sensors, exoskeletons, flywheel, pneumatic, and closed-loop microprocessor controlled motor driven systems. Each technology has unique advantages and disadvantages. The Advanced Exercise Concepts project oversees development of candidate next generation exercise countermeasures hardware, performs trade studies of current and state of the art exercise technologies, manages and supports candidate systems physiological evaluations with human test subjects on the ground, in flight analogs and flight. The near term goal is evaluation of candidate systems in flight, culminating in an integrated candidate next generation exercise countermeasures suite on the ISS which coalesces research findings from HRP disciplines in the areas of exercise performance for muscle, bone, cardiovascular, sensorimotor, behavioral health, and nutrition for optimal benefit to the crew.

Humanities Perspectives on Technology Program: Science, Technology & Society Program. Lehigh University, 1977-80.

ERIC Educational Resources Information Center

Cutcliffe, Stephen H., Ed.

Newsletter issues pertaining to Lehigh University's Humanities Perspectives on Technology (HPT) Program, which was renamed the Science, Technology and Society Program, are presented. Additionally, a newsletter article excerpt entitled "Elements of Technology in a Liberal Education" is included. Two 1977 issues of "HRP News,"…

Human Research Program Unique Processes, Criteria, and Guidelines (UPCG). Revision C, July 28, 2011

NASA Technical Reports Server (NTRS)

Chin, Duane

2011-01-01

This document defines the processes, criteria, and guidelines exclusive to managing the Human Research Program (HRP). The intent of this document is to provide instruction to the reader in the form of processes, criteria, and guidelines. Of the three instructional categories, processes contain the most detail because of the need for a systematic series of actions directed to some end. In contrast, criteria have lesser detail than processes with the idea of creating a rule or principle structure for evaluating or testing something. Guidelines are a higher level indication of a course of action typically with the least amount of detail. The lack of detail in guidelines allows the reader flexibility when performing an action or actions.

Use of a Dual-Antigen Rapid Diagnostic Test to Screen Children for Severe Plasmodium falciparum Malaria in a High-Transmission, Resource-Limited Setting.

PubMed

Boyce, Ross; Reyes, Raquel; Matte, Michael; Ntaro, Moses; Mulogo, Edgar; Siedner, Mark J

2017-10-16

In rural areas, many patients with malaria seek care at peripheral health facilities or community case management programs. While this strategy is effective for the management of uncomplicated malaria, severe malaria necessitates prompt detection and referral to facilities with adequate resources. In this prospective, observational cohort study, we assessed the accuracy of a dual-band (histidine-rich protein-2/pan-lactate dehydrogenase [HRP2/pLDH]) rapid diagnostic test (RDT) to differentiate uncomplicated from severe malaria. We included children aged <12 years who presented to a rural clinic in western Uganda with a positive HRP2 or HRP2/pLDH RDT. We estimated the test characteristics of a dual-antigen (HRP2+/pLDH+) band positive RDT compared to World Health Organization-defined clinical and laboratory criteria to detect severe malaria. A total of 2678 children underwent testing for malaria with an RDT, and 83 (9.0%) satisfied criteria for severe malaria. The sensitivity and specificity of a HRP2+/pLDH+ result for severe malaria was 97.6% (95% confidence interval [CI], 90.8%-99.6%) and 75.6% (95% CI, 73.8%-77.4%), respectively. An HRP2+/pLDH+ result was significantly more sensitive (97.6% vs 68.7%, P < .001) for the detection of severe malaria compared to algorithms that incorporate screening for danger signs. A positive dual-antigen (HRP2/pLDH) RDT has higher sensitivity than the use of clinical manifestations to detect severe malaria, making it a promising tool in the triage of children with malaria in low-resource settings. Additional work is needed to operationalize diagnostic and treatment algorithms that include dual-antigen RDTs to avoid over referral. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Management of Service Projects in Support of Space Flight Research

NASA Technical Reports Server (NTRS)

Love, J.

2009-01-01

Goal:To provide human health and performance countermeasures, knowledge, technologies, and tools to enable safe, reliable, and productive human space exploration . [HRP-47051] Specific Objectives: 1) Develop capabilities, necessary countermeasures, and technologies in support of human space exploration, focusing on mitigating the highest risks to human health and performance. 2) Define and improve human spaceflight medical, environmental, and human factors standards. 3) Develop technologies that serve to reduce medical and environmental risks, to reduce human systems resource requirements (mass, volume, power, data, etc.) and to ensure effective human-system integration across exploration systems. 4) Ensure maintenance of Agency core competencies necessary to enable risk reduction in the following areas: A. Space medicine B. Physiological and behavioral effects of long duration spaceflight on the human body C. Space environmental effects, including radiation, on human health and performance D. Space "human factors" [HRP-47051]. Service projects can form integral parts of research-based project-focused programs to provide specialized functions. Traditional/classic project management methodologies and agile approaches are not mutually exclusive paradigms. Agile strategies can be combined with traditional methods and applied in the management of service projects functioning in changing environments. Creative collaborations afford a mechanism for mitigation of constrained resource limitations.

Perspectives on Asthenia in Astronauts and Cosmonauts: Review of the International Research Literature

NASA Technical Reports Server (NTRS)

Sandoval, Luis; Keeton, Kathryn; Shea, Camille; Otto, Christian; Patterson, Holly; Leveton, Lauren

2012-01-01

The Behavioral Health and Performance Element (BHP) is one of the 6 elements in the NASA Human Research Program (HRP) and is responsible for managing 4 of the identified and named risks to human health and performance from human space exploration: a) Risk of Behavioral Conditions (BMed), b) Risk of Psychiatric Disorders (BMed), c) Risk of Performance Decrements due to inadequate Cooperation, Coordination, Communication and Psychological Adaptation within a Team (Team), and d) Risk of Performance Errors due to Sleep Loss, Circadian De-synchronization, Fatigue and Work Overload (Sleep). Each risk is reviewed by a NASA HRP Standing Review Panel (SRP), and recently the Behavioral Medicine Risk of Psychiatric Disorders was reviewed. The aim of this report is to address one of the recommendations made by that panel, specifically the recommendation that the "literature on asthenia should be evaluated (possibly as a psychological or psychosomatic / psycho-physiological analogue of chronic fatigue syndrome)" (SRP p. 4), in addition to General Recommendation 4, which states that "all reviews must include non-English language materials as well as materials appearing in conferences reports, books, and other non-refereed journal outlets" (SRP p. 2).

HrpE3 is a type III effector protein required for full virulence of Xanthomonas oryzae pv. oryzicola in rice.

PubMed

Cui, Yiping; Zou, Lifang; Zou, Huasong; Li, Yurong; Zakria, Muhammad; Chen, Gongyou

2013-09-01

Xanthomonas oryzae pv. oryzicola (Xoc) is the causal agent of bacterial leaf streak, a devastating disease in rice. Xoc uses a type III secretion (T3S) system, which is encoded by the hrp-hrc-hpa (hypersensitive response and pathogenicity, hrp-conserved and hrp-associated) genes, to inject repertoires of T3S effectors (T3Es) into plant cells. Many of the hrp-hrc-hpa genes have roles in pathogenesis, but the role of hrpE3, which shows homology to hpaE in X. campestris pv. vesicatoria (Xcv), is poorly understood. In this study, hrpE3 was shown to be transcribed independent of the hrpD operon, and its expression was dependent on a promoter within hpaB. The expression of hrpE3 was positively regulated by HrpG and HrpX, a finding probably caused by an imperfect plant-inducible promoter (PIP) box (TTCGT-N16 -TTCGA) in the hrpE3 promoter. The secretion of HrpE3 was dependent on T3S, and subcellular localization of HrpE3 was cytoplasmic and nuclear in plant cells. A mutation in hrpE3 reduced the virulence of Xoc by decreasing disease lesion length and bacterial growth in planta. Full virulence was restored to the mutant when Xoc hrpE3, but not Xcv hpaE, was expressed in trans. The differences in transcription, secretion via the T3S system and bacterial virulence in plants were attributed to N-terminal amino acid differences between Xoc HrpE3 and Xcv HpaE. Collectively, the results demonstrate that hrpE3 encodes a T3E protein which is delivered into the plant cell through the T3S system, localizes to the cytoplasm and nucleus, and is required for full virulence in rice. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Cerebrospinal fluid Plasmodium falciparum histidine-rich protein-2 in pediatric cerebral malaria.

PubMed

Thakur, Kiran T; Vareta, Jimmy; Carson, Kathryn A; Kampondeni, Samuel; Potchen, Michael J; Birbeck, Gretchen L; MacCormick, Ian; Taylor, Terrie; Sullivan, David J; Seydel, Karl B

2018-03-23

Cerebral malaria (CM) causes a rapidly developing coma, and remains a major contributor to morbidity and mortality in malaria-endemic regions. This study sought to determine the relationship between cerebrospinal fluid (CSF) Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and clinical, laboratory and radiographic features in a cohort of children with retinopathy-positive CM. Patients included in the study were admitted (2009-2013) to the Pediatric Research Ward (Queen Elizabeth Central Hospital, Blantyre, Malawi) meeting World Health Organization criteria for CM with findings of malarial retinopathy. Enzyme-linked immunosorbent assay was used to determine plasma and CSF PfHRP-2 levels. Wilcoxon rank-sum tests and multivariable logistic regression analysis assessed the association of clinical and radiographic characteristics with the primary outcome of death during hospitalization. In this cohort of 94 patients, median age was 44 (interquartile range 29-62) months, 53 (56.4%) patients were male, 6 (7%) were HIV-infected, and 10 (11%) died during hospitalization. Elevated concentrations of plasma lactate (p = 0.005) and CSF PfHRP-2 (p = 0.04) were significantly associated with death. On multivariable analysis, higher PfHRP-2 in the CSF was associated with death (odds ratio 9.00, 95% confidence interval 1.44-56.42) while plasma PfHRP-2 was not (odds ratio 2.05, 95% confidence interval 0.45-9.35). Elevation of CSF, but not plasma PfHRP-2, is associated with death in this paediatric CM cohort. PfHRP-2 egress into the CSF may represent alteration of blood brain barrier permeability related to the sequestration of parasitized erythrocytes in the cerebral microvasculature.

Identification of the HrpS binding site in the hrpL promoter and effect of the RpoN binding site of HrpS on the regulation of the type III secretion system in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Sundin, George W; Zhao, Youfu

2016-06-01

The type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by an RpoN-HrpL sigma factor cascade, which is activated by the bacterial alarmone (p)ppGpp. In this study, the binding site of HrpS, an enhancer binding protein, was identified for the first time in plant-pathogenic bacteria. Complementation of the hrpL mutant with promoter deletion constructs of the hrpL gene and promoter activity analyses using various lengths of the hrpL promoter fused to a promoter-less green fluorescent protein (gfp) reporter gene delineated the upstream region for HrpS binding. Sequence analysis revealed a dyad symmetry sequence between -138 and -125 nucleotides (TGCAA-N4-TTGCA) as the potential HrpS binding site, which is conserved in the promoter of the hrpL gene among plant enterobacterial pathogens. Results of quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and electrophoresis mobility shift assay coupled with site-directed mutagenesis (SDM) analysis showed that the intact dyad symmetry sequence was essential for HrpS binding, full activation of T3SS gene expression and virulence. In addition, the role of the GAYTGA motif (RpoN binding site) of HrpS in the regulation of T3SS gene expression in E. amylovora was characterized by complementation of the hrpS mutant using mutant variants generated by SDM. Results showed that a Y100F substitution of HrpS complemented the hrpS mutant, whereas Y100A and Y101A substitutions did not. These results suggest that tyrosine (Y) and phenylalanine (F) function interchangeably in the conserved GAYTGA motif of HrpS in E. amylovora. © 2015 BSPP AND JOHN WILEY & SONS LTD.

DCS: A Case Study of Identification of Knowledge and Disposition Gaps Using Principles of Continuous Risk Management

NASA Technical Reports Server (NTRS)

Norcross, Jason; Steinberg, Susan; Kundrot, Craig; Charles, John

2011-01-01

The Human Research Program (HRP) is formulated around the program architecture of Evidence-Risk-Gap-Task-Deliverable. Review of accumulated evidence forms the basis for identification of high priority risks to human health and performance in space exploration. Gaps in knowledge or disposition are identified for each risk, and a portfolio of research tasks is developed to fill them. Deliverables from the tasks inform the evidence base with the ultimate goal of defining the level of risk and reducing it to an acceptable level. A comprehensive framework for gap identification, focus, and metrics has been developed based on principles of continuous risk management and clinical care. Research towards knowledge gaps improves understanding of the likelihood, consequence or timeframe of the risk. Disposition gaps include development of standards or requirements for risk acceptance, development of countermeasures or technology to mitigate the risk, and yearly technology assessment related to watching developments related to the risk. Standard concepts from clinical care: prevention, diagnosis, treatment, monitoring, rehabilitation, and surveillance, can be used to focus gaps dealing with risk mitigation. The research plan for the new HRP Risk of Decompression Sickness (DCS) used the framework to identify one disposition gap related to establishment of a DCS standard for acceptable risk, two knowledge gaps related to DCS phenomenon and mission attributes, and three mitigation gaps focused on prediction, prevention, and new technology watch. These gaps were organized in this manner primarily based on target for closure and ease of organizing interim metrics so that gap status could be quantified. Additional considerations for the knowledge gaps were that one was highly design reference mission specific and the other gap was focused on DCS phenomenon.

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea

PubMed Central

Berhane, Araia; Anderson, Karen; Mihreteab, Selam; Gresty, Karryn; Rogier, Eric; Mohamed, Salih; Hagos, Filmon; Embaye, Ghirmay; Chinorumba, Anderson; Zehaie, Assefash; Dowd, Simone; Waters, Norman C.; Gatton, Michelle L.; Udhayakumar, Venkatachalam; Cunningham, Jane

2018-01-01

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2–based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies. PMID:29460730

The cytoskeleton is disrupted by the bacterial effector HrpZ, but not by the bacterial PAMP flg22, in tobacco BY-2 cells.

PubMed

Guan, Xin; Buchholz, Günther; Nick, Peter

2013-04-01

Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the flagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin filaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a concentration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microfilaments (completed in ~20 min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI.

The cytoskeleton is disrupted by the bacterial effector HrpZ, but not by the bacterial PAMP flg22, in tobacco BY-2 cells

PubMed Central

Guan, Xin; Buchholz, Günther; Nick, Peter

2013-01-01

Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the flagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin filaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a concentration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microfilaments (completed in ~20min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI. PMID:23408828

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea.

PubMed

Berhane, Araia; Anderson, Karen; Mihreteab, Selam; Gresty, Karryn; Rogier, Eric; Mohamed, Salih; Hagos, Filmon; Embaye, Ghirmay; Chinorumba, Anderson; Zehaie, Assefash; Dowd, Simone; Waters, Norman C; Gatton, Michelle L; Udhayakumar, Venkatachalam; Cheng, Qin; Cunningham, Jane

2018-03-01

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2-based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes.

PubMed

Koebnik, Ralf; Krüger, Antje; Thieme, Frank; Urban, Alexander; Bonas, Ulla

2006-11-01

The pathogenicity of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion system which is encoded by the 23-kb hrp (hypersensitive response and pathogenicity) gene cluster. Expression of the hrp operons is strongly induced in planta and in a special minimal medium and depends on two regulatory proteins, HrpG and HrpX. In this study, DNA affinity enrichment was used to demonstrate that the AraC-type transcriptional activator HrpX binds to a conserved cis-regulatory element, the plant-inducible promoter (PIP) box (TTCGC-N(15)-TTCGC), present in the promoter regions of four hrp operons. No binding of HrpX was observed when DNA fragments lacking a PIP box were used. HrpX also bound to a DNA fragment containing an imperfect PIP box (TTCGC-N(8)-TTCGT). Dinucleotide replacements in each half-site of the PIP box strongly decreased binding of HrpX, while simultaneous dinucleotide replacements in both half-sites completely abolished binding. Based on the complete genome sequence of Xanthomonas campestris pv. vesicatoria, putative plant-inducible promoters consisting of a PIP box and a -10 promoter motif were identified in the promoter regions of almost all HrpX-activated genes. Bioinformatic analyses and reverse transcription-PCR experiments revealed novel HrpX-dependent genes, among them a NUDIX hydrolase gene and several genes with a predicted role in the degradation of the plant cell wall. We conclude that HrpX is the most downstream component of the hrp regulatory cascade, which is proposed to directly activate most genes of the hrpX regulon via binding to corresponding PIP boxes.

Electrochemical properties of seamless three-dimensional carbon nanotubes-grown graphene modified with horseradish peroxidase.

PubMed

Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

2016-10-01

Horseradish peroxidase (HRP) was immobilized through sodium dodecyl sulfate (SDS) on the surface of a seamless three-dimensional hybrid of carbon nanotubes grown at the graphene surface (HRP-SDS/CNTs/G) and its electrochemical properties were investigated. Compared with graphene alone electrode modified with HRP via SDS (HRP-SDS/G electrode), the surface coverage of electroactive HRP at the CNTs/G electrode surface was approximately 2-fold greater because of CNTs grown at the graphene surface. Based on the increase in the surface coverage of electroactive HRP, the sensitivity to H2O2 at the HRP-SDS/CNTs/G electrode was higher than that at the HRP-SDS/G electrode. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HRP was also analyzed. Copyright © 2016 Elsevier B.V. All rights reserved.

Immobilization of horseradish peroxidase on silane-modified ceramics and their properties: potential for oily wastewater treatment.

PubMed

Liu, W; Wang, W C; Li, H S; Zhou, X

2011-01-01

Horseradish peroxidase (HRP) is proved being effective in eliminating oil from aqueous solutions, but the elimination is expensive because free HRP can not be reused. In present work, HRP was successfully immobilized on cordierite porous ceramics support with a novel method of N-beta-aminoethyl-gamma-aminopropyl-trimethoxysilane modification and glutaraldehyde activation. Under the optimized immobilized conditions, the actual immobilized HRP was 1.16 mg/g support, the activity of the immobilized HRP could reach as high as 1379.4 U/g support. Experiment results showed that the properties of storage stability, acid-base stability and the tolerance to the pH fluctuation of the immobilized HRP were better than those of the free HRP. The operation stability of the immobilized HRP was also good. The immobilized HRP is suitable for the oily wastewater treatment because of its reusability proved in this work.

Genetic characterization of the HrpL regulon of the fire blight pathogen Erwinia amylovora reveals novel virulence factors.

PubMed

McNally, R Ryan; Toth, Ian K; Cock, Peter J A; Pritchard, Leighton; Hedley, Pete E; Morris, Jenny A; Zhao, Youfu; Sundin, George W

2012-02-01

The bacterial pathogen Erwinia amylovora is the causal agent of fire blight, an economically significant disease of apple and pear. Disease initiation by E. amylovora requires the translocation of effector proteins into host cells via the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS). The alternative sigma factor HrpL positively regulates the transcription of structural and translocated components of the T3SS via hrp promoter elements. To characterize genome-wide HrpL-dependent gene expression in E. amylovora Ea1189, wild-type and Ea1189ΔhrpL strains were cultured in hrp-inducing minimal medium, and total RNA was compared using a custom microarray designed to represent the annotated genes of E. amylovora ATCC 49946. The results revealed 24 genes differentially regulated in Ea1189ΔhrpL relative to Ea1189 with fold-change expression ratios greater than 1.5; of these, 19 genes exhibited decreased transcript abundance and five genes showed increased transcript abundance relative to Ea1189. To expand our understanding of the HrpL regulon and to elucidate direct versus indirect HrpL-mediated effects on gene expression, the genome of E. amylovora ATCC 49946 was examined in silico using a hidden Markov model assembled from known Erwinia spp. hrp promoters. This technique identified 15 putative type III novel hrp promoters, seven of which were validated with quantitative polymerase chain reaction based on expression analyses. It was found that HrpL-regulated genes encode all known components of the hrp T3SS, as well as five putative type III effectors. Eight genes displayed apparent indirect HrpL regulation, suggesting that the HrpL regulon is connected to downstream signalling networks. The construction of deletion mutants of three novel HrpL-regulated genes resulted in the identification of additional virulence factors as well as mutants displaying abnormal motility and biofilm phenotypes. © 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.

ExMC Technology Watch

NASA Technical Reports Server (NTRS)

Krihak, M.; Watkins, S.; Shaw, T.

2014-01-01

The Technology Watch (Tech Watch) project is directed by the NASA Human Research Program's (HRP) Exploration Medical Capability (ExMC) element, and primarily focuses on ExMC technology gaps. The project coordinates the efforts of multiple NASA centers, including the Johnson Space Center (JSC), Glenn Research Center (GRC), Ames Research Center (ARC), and the Langley Research Center (LaRC). The objective of Tech Watch is to identify emerging, high-impact technologies that augment current NASA HRP technology development efforts. Identifying such technologies accelerates the development of medical care and research capabilities for the mitigation of potential health issues encountered during human space exploration missions. The aim of this process is to leverage technologies developed by academia, industry and other government agencies and to identify the effective utilization of NASA resources to maximize the HRP return on investment. The establishment of collaborations with these entities is beneficial to technology development, assessment and/or insertion, and advance NASA's goal to provide a safe and healthy environment for human exploration. In fiscal year 2013, the Tech Watch project maintained student project activity aimed at specific ExMC gaps, completed the gap report review cycle for all gaps through a maturated gap report review process, and revised the ExMC Tech Watch Sharepoint site for enhanced data content and organization. Through site visits, internships and promotions via aerospace journals, several student projects were initiated and completed this past year. Upon project completion, the students presented their results via telecom or WebEx to the ExMC Element as a whole. The upcoming year will continue to forge strategic alliances and student projects in the interest of technology and knowledge gap closure. Through the population of Sharepoint with technologies assessed by the gap owners, the database expansion will develop a more comprehensive technology set for each gap. By placing such data in Sharepoint, the gap report updates in fiscal year 2014 are anticipated to be streamlined since the evaluated technologies will be readily available to the gap owners in a sortable archive, and may be simply exported into the final gap report presentation

ExMC Technology Watch

NASA Technical Reports Server (NTRS)

Krihak, M.; Watkins, S.; Shaw, T.

2014-01-01

The Technology Watch (Tech Watch) project is directed by the NASA Human Research Programs (HRP) Exploration Medical Capability (ExMC) element, and primarily focuses on ExMC technology gaps. The project coordinates the efforts of multiple NASA centers, including the Johnson Space Center (JSC), Glenn Research Center (GRC), Ames Research Center (ARC), and the Langley Research Center (LaRC). The objective of Tech Watch is to identify emerging, high-impact technologies that augment current NASA HRP technology development efforts. Identifying such technologies accelerates the development of medical care and research capabilities for the mitigation of potential health issues encountered during human space exploration missions. The aim of this process is to leverage technologies developed by academia, industry and other government agencies and to identify the effective utilization of NASA resources to maximize the HRP return on investment. The establishment of collaborations with these entities is beneficial to technology development, assessment and/or insertion, and advance NASAs goal to provide a safe and healthy environment for human exploration. In fiscal year 2013, the Tech Watch project maintained student project activity aimed at specific ExMC gaps, completed the gap report review cycle for all gaps through a maturated gap report review process, and revised the ExMC Tech Watch Sharepoint site for enhanced data content and organization. Through site visits, internships and promotions via aerospace journals, several student projects were initiated and completed this past year. Upon project completion, the students presented their results via telecom or WebEx to the ExMC Element as a whole. The upcoming year will continue to forge strategic alliances and student projects in the interest of technology and knowledge gap closure. Through the population of Sharepoint with technologies assessed by the gap owners, the database expansion will develop a more comprehensive technology set for each gap. By placing such data in Sharepoint, the gap report updates in fiscal year 2014 are anticipated to be streamlined since the evaluated technologies will be readily available to the gap owners in a sortable archive, and may be simply exported into the final gap report presentation.

HrpN of Erwinia amylovora functions in the translocation of DspA/E into plant cells.

PubMed

Bocsanczy, Ana M; Nissinen, Riitta M; Oh, Chang-Sik; Beer, Steven V

2008-07-01

The type III secretion system (T3SS) is required by plant pathogenic bacteria for the translocation of certain bacterial proteins to the cytoplasm of plant cells or secretion of some proteins to the apoplast. The T3SS of Erwinia amylovora, which causes fire blight of pear, apple and other rosaceous plants, secretes DspA/E, which is an indispensable pathogenicity factor. Several other proteins, including HrpN, a critical virulence factor, are also secreted by the T3SS. Using a CyaA reporter system, we demonstrated that DspA/E is translocated into the cells of Nicotiana tabacum'Xanthi'. To determine if other T3-secreted proteins are needed for translocation of DspA/E, we examined its translocation in several mutants of E. amylovora strain Ea321. DspA/E was translocated by both hrpW and hrpK mutants, although with some delay, indicating that these two proteins are dispensable in the translocation of DspA/E. Remarkably, translocation of DspA/E was essentially abolished in both hrpN and hrpJ mutants; however, secretion of DspA/E into medium was not affected in any of the mentioned mutants. In contrast to the more virulent strain Ea273, secretion of HrpN was abolished in a hrpJ mutant of strain Ea321. In addition, HrpN was weakly translocated into plant cytoplasm. These results suggest that HrpN plays a significant role in the translocation of DspA/E, and HrpJ affects the translocation of DspA/E by affecting secretion or stability of HrpN. Taken together, these results explain the critical importance of HrpN and HrpJ to the development of fire blight.

Alternative sigma factor RpoN and its modulation protein YhbH are indispensable for Erwinia amylovora virulence.

PubMed

Ancona, Veronica; Li, Wenting; Zhao, Youfu

2014-01-01

In Erwinia amylovora, ECF (extracytoplasmic functions) alternative sigma factor HrpL regulates the transcription of hrp (hypersensitive response and pathogenicity)-type III secretion system (T3SS) genes by binding to a consensus sequence known as the hrp box in hrp gene promoters. In turn, the expression of hrpL has been proposed to be positively controlled by alternative sigma factor 54 (σ(54)) (RpoN) and HrpS, a member of the σ(54) enhancer-binding proteins (EBPs). However, the function of RpoN has not been characterized genetically in E. amylovora. In this study, we investigated the role of RpoN, a nitrogen limitation sigma factor, and its modulation protein YhbH, a novel ribosome-associated protein, in E. amylovora virulence. Our results showed that mutations in hrpS, hrpL, rpoN and yhbH, but not yfiA and rmf3, resulted in a nonpathogenic phenotype on immature pear fruits and apple shoots. Consistently, the expression of T3SS genes, including hrpL, dspE, hrpN and hrpA, was barely detected in hrpS, hrpL, rpoN and yhbH mutants. These mutants were also not capable of eliciting a hypersensitive response (HR) on tobacco; however, the overexpression of hrpL using an inducible promoter rescued the HR-eliciting abilities of these mutants. These results suggest that a sigma factor cascade exists in the regulatory networks of E. amylovora and regulates important virulence factors. On the basis of this study and previously reported data, a model is proposed for the regulation of T3SS in E. amylovora. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Comparative genomics of a cannabis pathogen reveals insight into the evolution of pathogenicity in Xanthomonas

PubMed Central

Jacobs, Jonathan M.; Pesce, Céline; Lefeuvre, Pierre; Koebnik, Ralf

2015-01-01

Pathogenic bacteria in the genus Xanthomonas cause diseases on over 350 plant species, including cannabis (Cannabis sativa L.). Because of regulatory limitations, the biology of the Xanthomonas-cannabis pathosystem remains largely unexplored. To gain insight into the evolution of Xanthomonas strains pathogenic to cannabis, we sequenced the genomes of two geographically distinct Xanthomonas strains, NCPPB 3753 and NCPPB 2877, which were previously isolated from symptomatic plant tissue in Japan and Romania. Comparative multilocus sequence analysis of housekeeping genes revealed that they belong to Group 2, which comprises most of the described species of Xanthomonas. Interestingly, both strains lack the Hrp Type III secretion system and do not contain any of the known Type III effectors. Yet their genomes notably encode two key Hrp pathogenicity regulators HrpG and HrpX, and hrpG and hrpX are in the same genetic organization as in the other Group 2 xanthomonads. Promoter prediction of HrpX-regulated genes suggests the induction of an aminopeptidase, a lipase and two polygalacturonases upon plant colonization, similar to other plant-pathogenic xanthomonads. Genome analysis of the distantly related Xanthomonas maliensis strain 97M, which was isolated from a rice leaf in Mali, similarly demonstrated the presence of HrpG, HrpX, and a HrpX-regulated polygalacturonase, and the absence of the Hrp Type III secretion system and known Type III effectors. Given the observation that some Xanthomonas strains across distinct taxa do not contain hrpG and hrpX, we speculate a stepwise evolution of pathogenicity, which involves (i) acquisition of key regulatory genes and cell wall-degrading enzymes, followed by (ii) acquisition of the Hrp Type III secretion system, which is ultimately accompanied by (iii) successive acquisition of Type III effectors. PMID:26136759

Inhibition mechanism of lanthanum ion on the activity of horseradish peroxidase in vitro

NASA Astrophysics Data System (ADS)

Guo, Shaofen; Wang, Lihong; Lu, Aihua; Lu, Tianhong; Ding, Xiaolan; Huang, Xiaohua

2010-02-01

In order to understand the inhibition mechanism of lanthanum ion (La 3+) on the activity of horseradish peroxidase (HRP), the effects of La 3+ on the activity, electron transfer and conformation of HRP in vitro were investigated by using cyclic voltammetry (CV), atomic force microscopy (AFM), circular dichroism (CD), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and inductively coupled plasma mass spectrometry (ICP-MS). It was found that La 3+ can combine with the amide groups of the polypeptide chain in HRP molecule, forming the complex of La 3+ and HRP (La-HRP). The formation of the La-HRP complex causes the destruction of the native structure of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure extent of active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the direct electrochemical and catalytic activities of HRP are decreased. It is a possible inhibition mechanism of La 3+ on the activity of peroxidase.

Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species.

PubMed

Shen, Yajing; Zhang, Ye; Zhang, Xiang; Zhou, Xiuhong; Teng, Xiyao; Yan, Manqing; Bi, Hong

2015-02-21

Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles (MMSNs-HRP) have been synthesized by a NHS/EDC coupling between the amino groups of horseradish peroxidase (HRP) and the carboxyl groups on the MMSNs surface. It is found that the immobilized HRP on MMSNs still retain high activity and the MMSNs-HRP can eliminate the reactive oxygen species (ROS) in Chinese hamster ovary (CHO) cells induced by the addition of H2O2 aqueous solution. Further, the fluorescent MMSN-HRP-CD nanoparticles have been prepared by attaching biocompatible, fluorescent carbon dots (CDs) to MMSNs-HRP. We have also investigated the effect of an applied magnetic field on cellular uptake of MMSNs-HRP-CDs and found that the internalization of MMSNs-HRP-CDs by CHO cells could be enhanced within 2 hours under the magnetic field. This work provides us with a novel and efficient method to eliminate ROS in living cells by using HRP-immobilized nanoparticles.

Size-dependent tuning of horseradish peroxidase bioreactivity by gold nanoparticles

NASA Astrophysics Data System (ADS)

Wu, Haohao; Liu, Yi; Li, Meng; Chong, Yu; Zeng, Mingyong; Lo, Y. Martin; Yin, Jun-Jie

2015-02-01

Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates.Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr07056a

NASA Habitat Demonstration Unit (HDU) Deep Space Habitat Analog

NASA Technical Reports Server (NTRS)

Howe, A. Scott; Kennedy, Kriss J.; Gill, Tracy

2013-01-01

The NASA Habitat Demonstration Unit (HDU) vertical cylinder habitat was established as a exploration habitat testbed platform for integration and testing of a variety of technologies and subsystems that will be required in a human-occupied planetary surface outpost or Deep Space Habitat (DSH). The HDU functioned as a medium-fidelity habitat prototype from 2010-2012 and allowed teams from all over NASA to collaborate on field analog missions, mission operations tests, and system integration tests to help shake out equipment and provide feedback for technology development cycles and crew training. This paper documents the final 2012 configuration of the HDU, and discusses some of the testing that took place. Though much of the higher-fidelity functionality has 'graduated' into other NASA programs, as of this writing the HDU, renamed Human Exploration Research Analog (HERA), will continue to be available as a volumetric and operational mockup for NASA Human Research Program (HRP) research from 2013 onward.

False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission.

PubMed

Kozycki, Christina T; Umulisa, Noella; Rulisa, Stephen; Mwikarago, Emil I; Musabyimana, Jean Pierre; Habimana, Jean Pierre; Karema, Corine; Krogstad, Donald J

2017-03-20

Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub-Saharan Africa, these results suggest that combination HRP2/pLDH-based RDTs could reduce the impact of false-negative HRP2-based RDTs for detection of symptomatic P. falciparum malaria.

10 CFR 712.2 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Applicability. 712.2 Section 712.2 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Establishment of and Procedures for the Human Reliability Program General Provisions § 712.2 Applicability. The HRP applies to all applicants for, or current employees of...

A comparative approach to recombinantly produce the plant enzyme horseradish peroxidase in Escherichia coli.

PubMed

Gundinger, Thomas; Spadiut, Oliver

2017-04-20

Horseradish peroxidase (HRP) is used in various biotechnological and medical applications. Since its isolation from plant provides several disadvantages, the bacterium Escherichia coli was tested as recombinant expression host in former studies. However, neither production from refolded inclusion bodies nor active enzyme expression in the periplasm exceeded final titres of 10mg per litre cultivation broth. Thus, the traditional way of production of HRP from plant still prevails. In this study, we revisited the recombinant production of HRP in E. coli and investigated and compared both strategies, (a) the production of HRP as inclusion bodies (IBs) and subsequent refolding and (b) the production of active HRP in the periplasm. In fact, we were able to produce HRP in E. coli either way. We obtained a refolding yield of 10% from IBs giving a final titre of 100mgL -1 cultivation broth, and were able to produce 48mg active HRP per litre cultivation broth in the periplasm. In terms of biochemical properties, soluble HRP showed a highly reduced catalytic activity and stability which probably results from the fusion partner DsbA used in this study. Refolded HRP showed similar substrate affinity, an 11-fold reduced catalytic efficiency and 2-fold reduced thermal stability compared to plant HRP. In conclusion, we developed a toolbox for HRP engineering and production. We propose to engineer HRP by directed evolution or semi-rational protein design, express HRP in the periplasm of E. coli allowing straight forward screening for improved variants, and finally produce these variants as IB in high amounts, which are then refolded. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Assessing the Likelihood of Rare Medical Events in Astronauts

NASA Technical Reports Server (NTRS)

Myers, Jerry G., Jr.; Leandowski, Beth E.; Brooker, John E.; Weaver, Aaron S.

2011-01-01

Despite over half a century of manned space flight, the space flight community is only now coming to fully assess the short and long term medical dangers of exposure to reduced gravity environments. Further, as new manned spacecraft are designed and with the advent of commercial flight capabilities to the general public, a full understanding of medical risk becomes even more critical for maintaining and understanding mission safety and crew health. To address these critical issues, the National Aeronautics and Space Administration (NASA) Human Research Program (HRP) has begun to address the medical hazards with a formalized risk management approach by effectively identifying and attempting to mitigate acute and chronic medical risks to manned space flight. This paper describes NASA Glenn Research Center?s (GRC) efforts to develop a systematic methodology to assess the likelihood of in-flight medical conditions. Using a probabilistic approach, medical risks are assessed using well established and accepted biomedical and human performance models in combination with fundamentally observed data that defines the astronauts? physical conditions, environment and activity levels. Two different examples of space flight risk are used to show the versatility of our approach and how it successfully integrates disparate information to provide HRP decision makers with a valuable source of information which is otherwise lacking.

An Overview of NASA's Risk of Cardiovascular Disease from Radiation Exposure

NASA Technical Reports Server (NTRS)

Patel, Zarana S.; Huff, Janice L.; Simonsen, Lisa C.

2015-01-01

The association between high doses of radiation exposure and cardiovascular damage is well established. Patients that have undergone radiotherapy for primary cancers of the head and neck and mediastinal regions have shown increased risk of heart and vascular damage and long-term development of radiation-induced heart disease [1]. In addition, recent meta-analyses of epidemiological data from atomic bomb survivors and nuclear industry workers has also shown that acute and chronic radiation exposures is strongly correlated with an increased risk of circulatory disease at doses above 0.5 Sv [2]. However, these analyses are confounded for lower doses by lifestyle factors, such as drinking, smoking, and obesity. The types of radiation found in the space environment are significantly more damaging than those found on Earth and include galactic cosmic radiation (GCR), solar particle events (SPEs), and trapped protons and electrons. In addition to the low-LET data, only a few studies have examined the effects of heavy ion radiation on atherosclerosis, and at lower, space-relevant doses, the association between exposure and cardiovascular pathology is more varied and unclear. Understanding the qualitative differences in biological responses produced by GCR compared to Earth-based radiation is a major focus of space radiation research and is imperative for accurate risk assessment for long duration space missions. Other knowledge gaps for the risk of radiation-induced cardiovascular disease include the existence of a dose threshold, low dose rate effects, and potential synergies with other spaceflight stressors. The Space Radiation Program Element within NASA's Human Research Program (HRP) is managing the research and risk mitigation strategies for these knowledge gaps. In this presentation, we will review the evidence and present an overview of the HRP Risk of Cardiovascular Disease and Other Degenerative Tissue Effects from Radiation Exposure.

Thermometric sensing of peroxide in organic media. Application to monitor the stability of RBP-retinol-HRP complex.

PubMed

Ramanathan, K; Jönsson, B R; Danielsson, B

2000-08-01

The stability of horseradish peroxidase (HRP) in aqueous and organic solvents is applied to develop a simple thermometric procedure to detect the binding of retinoic acid-HRP conjugate to retinol binding protein (RBP). Butanone peroxide (BP) in organic phase and hydrogen peroxide in aqueous phase is detected thermometrically on a HRP column, immobilized by cross-linking with glutaraldehyde on controlled pore glass (CPG). Acetone, acetonitrile, methanol, and 2-butanol are used for detection of BP, in the flow injection analysis (FIA) mode. A linear range between 1 and 50 mM BP is obtained in all the organic solvents with a precision of 5-7% (CV%). The magnitude and nature of the thermometric response is significantly different in each organic solvent. The stability of HRP in the organic phase is used to study the stability of a retinoic acid-HRP conjugate bound to immobilized RBP. The response of HRP (to 20 mM BP) in the retinoic acid-HRP conjugate is used as an indicator of the stability of the RBP-retinoic acid-HRP complex, after challenges with various organic/aqueous solvents. Both immobilized HRP and RBP are stable at least for 6 months. The effect of o-phenylene diamine on the thermometric response of HRP is also investigated. A scheme for the design of a thermometric retinol (vitamin A) biosensor is proposed.

HrpG and HrpV proteins from the Type III secretion system of Erwinia amylovora form a stable heterodimer.

PubMed

Gazi, Anastasia D; Charova, Spyridoula; Aivaliotis, Michalis; Panopoulos, Nicholas J; Kokkinidis, Michael

2015-01-01

Bacterial type III secretion systems (T3SSs) are specialized multicomponent nanomachines that mediate the transport of proteins either to extracellular locations or directly into eukaryotic host cell cytoplasm. Erwinia amylovora, the main agent of rosaceous plants fireblight disease, employs an Hrp/Hrc1 T3SS to accomplish its pathogenesis. The regulatory network that controls the activation of this T3SS is largely unknown in E. amylovora. However, in Pseudomonas syringae pathovars, the HrpG/HrpV complex has been shown to directly regulate the activity of transcription factor HrpS and consequently the upregulation of the Hrp/Hrc1 T3SS related genes. In this work, we report the successful recombinant production and purification of a stable E. amylovora HrpG/HrpV complex, using pPROpET, a bicistronic expression vector. Furthermore, we present the first solution structure of this complex based on small-angle X-ray scattering data. © FEMS 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Combined experimental and theoretical study on the reactivity of compounds I and II in horseradish peroxidase biomimetics.

PubMed

Ji, Li; Franke, Alicja; Brindell, Małgorzata; Oszajca, Maria; Zahl, Achim; van Eldik, Rudi

2014-10-27

For the exploration of the intrinsic reactivity of two key active species in the catalytic cycle of horseradish peroxidase (HRP), Compound I (HRP-I) and Compound II (HRP-II), we generated in situ [Fe(IV) O(TMP(+.) )(2-MeIm)](+) and [Fe(IV) O(TMP)(2-MeIm)](0) (TMP=5,10,15,20-tetramesitylporphyrin; 2-MeIm=2-methylimidazole) as biomimetics for HRP-I and HRP-II, respectively. Their catalytic activities in epoxidation, hydrogen abstraction, and heteroatom oxidation reactions were studied in acetonitrile at -15 °C by utilizing rapid-scan UV/Vis spectroscopy. Comparison of the second-order rate constants measured for the direct reactions of the HRP-I and HRP-II mimics with the selected substrates clearly confirmed the outstanding oxidizing capability of the HRP-I mimic, which is significantly higher than that of HRP-II. The experimental study was supported by computational modeling (DFT calculations) of the oxidation mechanism of the selected substrates with the involvement of quartet and doublet HRP-I mimics ((2,4) Cpd I) and the closed-shell triplet spin HRP-II model ((3) Cpd II) as oxidizing species. The significantly lower activation barriers calculated for the oxidation systems involving (2,4) Cpd I than those found for (3) Cpd II are in line with the much higher oxidizing efficiency of the HRP-I mimic proven in the experimental part of the study. In addition, the DFT calculations show that all three reaction types catalyzed by HRP-I occur on the doublet spin surface in an effectively concerted manner, whereas these reactions may proceed in a stepwise mechanism with the HRP-II mimic as oxidant. However, the high desaturation or oxygen rebound barriers during CH bond activation processes by the HRP-II mimic predict a sufficient lifetime for the substrate radical formed through hydrogen abstraction. Thus, the theoretical calculations suggest that the dissociation of the substrate radical may be a more favorable pathway than desaturation or oxygen rebound processes. Importantly, depending on the electronic nature of the oxidizing species, that is, (2,4) Cpd I or (3) Cpd II, an interesting region-selective conversion phenomenon between sulfoxidation and H-atom abstraction was revealed in the course of the oxidation reaction of dimethylsulfide. The combined experimental and theoretical study on the elucidation of the intrinsic reactivity patterns of the HRP-I and HRP-II mimics provides a valuable tool for evaluating the particular role of the HRP active species in biological systems. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

EEG-Informed fMRI Reveals a Disturbed Gamma-Band-Specific Network in Subjects at High Risk for Psychosis.

PubMed

Leicht, Gregor; Vauth, Sebastian; Polomac, Nenad; Andreou, Christina; Rauh, Jonas; Mußmann, Marius; Karow, Anne; Mulert, Christoph

2016-01-01

Abnormalities of oscillatory gamma activity are supposed to reflect a core pathophysiological mechanism underlying cognitive disturbances in schizophrenia. The auditory evoked gamma-band response (aeGBR) is known to be reduced across all stages of the disease. The present study aimed to elucidate alterations of an aeGBR-specific network mediated by gamma oscillations in the high-risk state of psychosis (HRP) by means of functional magnetic resonance imaging (fMRI) informed by electroencephalography (EEG). EEG and fMRI were simultaneously recorded from 27 HRP individuals and 26 healthy controls (HC) during performance of a cognitively demanding auditory reaction task. We used single trial coupling of the aeGBR with the corresponding blood oxygen level depending response (EEG-informed fMRI). A gamma-band-specific network was significantly lower active in HRP subjects compared with HC (random effects analysis, P < .01, Bonferroni-corrected for multiple comparisons) accompanied by a worse task performance. This network involved the bilateral auditory cortices, the thalamus and frontal brain regions including the anterior cingulate cortex, as well as the bilateral dorsolateral prefrontal cortex. For the first time we report a reduced activation of an aeGBR-specific network in HRP subjects brought forward by EEG-informed fMRI. Because the HRP reflects the clinical risk for conversion to psychotic disorders including schizophrenia and the aeGBR has repeatedly been shown to be altered in patients with schizophrenia the results of our study point towards a potential applicability of aeGBR disturbances as a marker for the prediction of transition of HRP subjects to schizophrenia. © The Author 2015. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center.

Retest of a Principal Components Analysis of Two Household Environmental Risk Instruments.

PubMed

Oneal, Gail A; Postma, Julie; Odom-Maryon, Tamara; Butterfield, Patricia

2016-08-01

Household Risk Perception (HRP) and Self-Efficacy in Environmental Risk Reduction (SEERR) instruments were developed for a public health nurse-delivered intervention designed to reduce home-based, environmental health risks among rural, low-income families. The purpose of this study was to test both instruments in a second low-income population that differed geographically and economically from the original sample. Participants (N = 199) were recruited from the Women, Infants, and Children (WIC) program. Paper and pencil surveys were collected at WIC sites by research-trained student nurses. Exploratory principal components analysis (PCA) was conducted, and comparisons were made to the original PCA for the purpose of data reduction. Instruments showed satisfactory Cronbach alpha values for all components. HRP components were reduced from five to four, which explained 70% of variance. The components were labeled sensed risks, unseen risks, severity of risks, and knowledge. In contrast to the original testing, environmental tobacco smoke (ETS) items was not a separate component of the HRP. The SEERR analysis demonstrated four components explaining 71% of variance, with similar patterns of items as in the first study, including a component on ETS, but some differences in item location. Although low-income populations constituted both samples, differences in demographics and risk exposures may have played a role in component and item locations. Findings provided justification for changing or reducing items, and for tailoring the instruments to population-level risks and behaviors. Although analytic refinement will continue, both instruments advance the measurement of environmental health risk perception and self-efficacy. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Implications of Parasites Lacking Plasmodium falciparum Histidine-Rich Protein 2 on Malaria Morbidity and Control When Rapid Diagnostic Tests Are Used for Diagnosis.

PubMed

Gatton, Michelle L; Dunn, Jessica; Chaudhry, Alisha; Ciketic, Sadmir; Cunningham, Jane; Cheng, Qin

2017-04-01

Rapid diagnostic tests (RDTs) are an important tool for malaria diagnosis, with most using antibodies against Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Reports of P. falciparum lacking this protein are increasing, creating a problem for diagnosis of falciparum malaria in locations without quality-assured microscopy. An agent-based stochastic simulation model of P. falciparum transmission was used to investigate the selective pressure exerted on parasite populations by use of RDTs for diagnosis of symptomatic cases. The model considered parasites with normal, reduced, or no PfHRP2, and diagnosis using PfHRP2-only or combination RDTs. Use of PfHRP2-only RDTs in communities where a PfHRP2-negative parasite was introduced during the simulation resulted in transmission of the parasite in >80% of cases, compared with <30% for normal or PfHRP2-reduced parasites. Using PfHRP2-only RDTs in the presence of PfHRP2-negative parasites caused an increase in prevalence, reduced RDT positivity within symptomatic patients but no change in the number of antimalarial treatments due to false-negative RDT results. Diagnosis with PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs did not select for PfHRP2-negative parasites. The use of PfHRP2-only RDTs is sufficient to select P. falciparum parasites lacking this protein, thus posing a significant public health problem, which could be moderated by using PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Identification of 17 HrpX-Regulated Proteins Including Two Novel Type III Effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola

PubMed Central

Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

2014-01-01

The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9≤X≤25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism. PMID:24675748

Identification of 17 HrpX-regulated proteins including two novel type III effectors, XOC_3956 and XOC_1550, in Xanthomonas oryzae pv. oryzicola.

PubMed

Xue, Xiao-bo; Zou, Li-fang; Ma, Wen-xiu; Liu, Zhi-yang; Chen, Gong-you

2014-01-01

The function of some hypothetical proteins, possibly regulated by key hrp regulators, in the pathogenicity of phytopathogenic bacteria remains largely unknown. In the present study, in silicon microarray data demonstrated that the expression of 17 HrpX-regulated protein (Xrp) genes of X. oryzae pv. oryzicola (Xoc), which causes bacterial leaf streak in rice, were either positively or negatively regulated by HrpX or/and HrpG. Bioinformatics analysis demonstrated that five Xrps possess a putative type III secretion (T3S) signal in the first 50 N-terminal amino acids, six xrp genes contain a PIP-box-like sequence (TTCGB-NX-TTCGB, 9 ≤ X ≤ 25) in the promoter regions, and two Xrps have both motifs. Twelve Xrps are widely conserved in Xanthomonas spp., whereas four are specific for X. oryzae (Xrp6) or Xoc (Xrp8, Xrp14 and Xrp17). In addition to the regulation by HrpG/HrpX, some of the 17 genes were also modulated by another hrp regulator HrpD6. Mutagenesis of these 17 genes indicated that five Xrps (Xrp1, Xrp2, Xrp5, Xrp8 and Xrp14) were required for full virulence and bacterial growth in planta. Immunoblotting assays and fusion with N-terminally truncated AvrXa10 indicated that Xrp3 and Xrp5 were secreted and translocated into rice cells through the type-III secretion system (T3S), suggesting they are novel T3S effectors. Our results suggest that Xoc exploits an orchestra of proteins that are regulated by HrpG, HrpX and HrpD6, and these proteins facilitate both infection and metabolism.

Erwinia amylovora Expresses Fast and Simultaneously hrp/dsp Virulence Genes during Flower Infection on Apple Trees

PubMed Central

Pester, Doris; Milčevičová, Renáta; Schaffer, Johann; Wilhelm, Eva; Blümel, Sylvia

2012-01-01

Background Pathogen entry through host blossoms is the predominant infection pathway of the Gram-negative bacterium Erwinia amylovora leading to manifestation of the disease fire blight. Like in other economically important plant pathogens, E. amylovora pathogenicity depends on a type III secretion system encoded by hrp genes. However, timing and transcriptional order of hrp gene expression during flower infections are unknown. Methodology/Principal Findings Using quantitative real-time PCR analyses, we addressed the questions of how fast, strong and uniform key hrp virulence genes and the effector dspA/E are expressed when bacteria enter flowers provided with the full defense mechanism of the apple plant. In non-invasive bacterial inoculations of apple flowers still attached to the tree, E. amylovora activated expression of key type III secretion genes in a narrow time window, mounting in a single expression peak of all investigated hrp/dspA/E genes around 24–48 h post inoculation (hpi). This single expression peak coincided with a single depression in the plant PR-1 expression at 24 hpi indicating transient manipulation of the salicylic acid pathway as one target of E. amylovora type III effectors. Expression of hrp/dspA/E genes was highly correlated to expression of the regulator hrpL and relative transcript abundances followed the ratio: hrpA>hrpN>hrpL>dspA/E. Acidic conditions (pH 4) in flower infections led to reduced virulence/effector gene expression without the typical expression peak observed under natural conditions (pH 7). Conclusion/Significance The simultaneous expression of hrpL, hrpA, hrpN, and the effector dspA/E during early floral infection indicates that speed and immediate effector transmission is important for successful plant invasion. When this delicate balance is disturbed, e.g., by acidic pH during infection, virulence gene expression is reduced, thus partly explaining the efficacy of acidification in fire blight control on a molecular level. PMID:22412891

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila

PubMed Central

Ji, Yingbiao

2016-01-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3′ untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3′ UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3′ UTR, increasing the translation in vivo and in vitro. hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. PMID:27402862

Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

PubMed Central

Ellinger, Isabella; Fuchs, Renate

2010-01-01

Horseradish peroxidase (HRP) is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs) on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport. PMID:20168981

Poly(ADP-Ribosyl)ation of hnRNP A1 Protein Controls Translational Repression in Drosophila.

PubMed

Ji, Yingbiao; Tulin, Alexei V

2016-10-01

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) regulates the posttranscriptional fate of RNA during development. Drosophila hnRNP A1, Hrp38, is required for germ line stem cell maintenance and oocyte localization. The mRNA targets regulated by Hrp38 are mostly unknown. We identified 428 Hrp38-associated gene transcripts in the fly ovary, including mRNA of the translational repressor Nanos. We found that Hrp38 binds to the 3' untranslated region (UTR) of Nanos mRNA, which contains a translation control element. We have demonstrated that translation of the luciferase reporter bearing the Nanos 3' UTR is enhanced by dsRNA-mediated Hrp38 knockdown as well as by mutating potential Hrp38-binding sites. Our data show that poly(ADP-ribosyl)ation inhibits Hrp38 binding to the Nanos 3' UTR, increasing the translation in vivo and in vitro hrp38 and Parg null mutants showed an increased ectopic Nanos translation early in the embryo. We conclude that Hrp38 represses Nanos translation, whereas its poly(ADP-ribosyl)ation relieves the repression effect, allowing restricted Nanos expression in the posterior germ plasm during oogenesis and early embryogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: toward a functional halophenol bioremediation catalyst.

PubMed

Sumithran, Suganya; Sono, Masanori; Raner, Gregory M; Dawson, John H

2012-12-01

Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217-222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H(2)O(2)) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst. Copyright © 2012 Elsevier Inc. All rights reserved.

Tailor-made biocatalysts based on scarcely studied acidic horseradish peroxidase for biodegradation of reactive dyes.

PubMed

Janović, Barbara S; Mićić Vićovac, Milica Lj; Vujčić, Zoran M; Vujčić, Miroslava T

2017-02-01

Peroxidases (EC 1.11.1.7) have enormous biotechnological applications. Usage of more abundant, basic isoforms of peroxidases in diagnostic kits and/or in immunochemistry has led to under exploitation and disregard of horseradish peroxidase (HRP) acidic isoforms. Therefore, acidic horseradish peroxidase (HRP-A) isoenzyme was used for the preparation of a biocatalyst with improved ability in dye decolorization. Ten biocatalysts were prepared by covalent binding of enzyme to chitosan and alginate, adsorption followed by cross-linking on inorganic support (aluminum oxide), and encapsulation in spherical calcium alginate beads via polyethylene glycol. Model dyes of 50 to 175 mg l -1 were removed by the biocatalysts. Among the tested biocatalysts, the three with the highest specific activity and biodegradation rate were further studied (Chitosan-HRP, Al-Gel-HRP and Al-HRP-Gel). The impact of hydrogen peroxide concentration on dye decolorization was examined on the Chitosan-HRP biocatalyst, since the HRP is susceptible to inhibition/inactivation by high H 2 O 2 . On the other hand, H 2 O 2 is needed as a co-substrate for the HRP, and the H 2 O 2 /dye ratio can greatly influence decolorization efficiency. Concentrations of H 2 O 2 ranging from 0.22 to 4.4 mM showed no difference in terms of impact on the biocatalyst decolorization efficiency. The high decolorization efficiency of the biocatalysts was validated by the removal of 25 and 100 mg l -1 anthraquinone (Remazol Brilliant Blue R (RBBR)), triphenylmethane (Coomassie Brilliant Blue (CBB)), acridine (Acridine Orange (AO)), and formazan metal complex dye (Reactive Blue 52 (RB52)). After the seven consecutive decolorization cycles, the decolorization was still 53, 78, and 67% of the initial dye for the Al-HRP-Gel, Al-Gel-HRP, and Chitosan-HRP immobilizate, respectively. The results obtained showed potential of otherwise neglected acidic HRP isoforms as a cost-effective biocatalyst with significant potential in wastewater dyestuff treatment.

Resveratrol-Loaded Albumin Nanoparticles with Prolonged Blood Circulation and Improved Biocompatibility for Highly Effective Targeted Pancreatic Tumor Therapy

NASA Astrophysics Data System (ADS)

Geng, Tao; Zhao, Xia; Ma, Meng; Zhu, Gang; Yin, Ling

2017-06-01

Human serum albumin (HSA) is an intrinsic protein and important carrier that transports endogenous as well as exogenous substances across cell membranes. Herein, we have designed and prepared resveratrol (RV)-loaded HSA nanoparticles conjugating RGD (arginine-glycine-aspartate) via a polyethylene glycol (PEG) "bridge" (HRP-RGD NPs) for highly effective targeted pancreatic tumor therapy. HRP-RGD NPs possess an average size of 120 ± 2.6 nm with a narrow distribution, a homodisperse spherical shape, a RV encapsulation efficiency of 62.5 ± 4.21%, and a maximum RV release ratio of 58.4.2 ± 2.8% at pH 5.0 and 37 °C. In vitro biocompatibility of RV is improved after coating with HSA and PEG. Confocal fluorescence images show that HRP-RGD NPs have the highest cellular uptake ratio of 47.3 ± 4.6% compared to HRP NPs and HRP-RGD NPs with free RGD blocking, attributing to an RGD-mediated effect. A cell counting kit-8 (CCK-8) assay indicates that HRP-RGD NPs without RV (HP-RGD NPs) have nearly no cytotoxicity, but HRP-RGD NPs are significantly more cytotoxic to PANC-1 cells compared to free RV and HRP NPs in a concentration dependent manner, showing apoptotic morphology. Furthermore, with a formulated PEG and HSA coating, HRP-RGD NPs prolong the blood circulation of RV, increasing approximately 5.43-fold (t1/2). After intravenous injection into tumor-bearing mice, the content of HRP-RGD NPs in tumor tissue was proven to be approximately 3.01- and 8.1-fold higher than that of HRP NPs and free RV, respectively. Based on these results, HRP-RGD NPs were used in an in vivo anti-cancer study and demonstrated the best tumor growth suppression effect of all tested drugs with no relapse, high in vivo biocompatibility, and no significant systemic toxicity over 35 days treatment. These results demonstrate that HRP-RGD NPs with prolonged blood circulation and improved biocompatibility have high anti-cancer effects with promising future applications in cancer therapy.

The Post-transcriptional Regulator rsmA/csrA Activates T3SS by Stabilizing the 5′ UTR of hrpG, the Master Regulator of hrp/hrc Genes, in Xanthomonas

PubMed Central

Andrade, Maxuel O.; Farah, Chuck S.; Wang, Nian

2014-01-01

The RsmA/CsrA family of the post-transcriptional regulators of bacteria is involved in the regulation of many cellular processes, including pathogenesis. In this study, we demonstrated that rsmA not only is required for the full virulence of the phytopathogenic bacterium Xanthomonas citri subsp. citri (XCC) but also contributes to triggering the hypersensitive response (HR) in non-host plants. Deletion of rsmA resulted in significantly reduced virulence in the host plant sweet orange and a delayed and weakened HR in the non-host plant Nicotiana benthamiana. Microarray, quantitative reverse-transcription PCR, western-blotting, and GUS assays indicated that RsmA regulates the expression of the type 3 secretion system (T3SS) at both transcriptional and post-transcriptional levels. The regulation of T3SS by RsmA is a universal phenomenon in T3SS-containing bacteria, but the specific mechanism seems to depend on the interaction between a particular bacterium and its hosts. For Xanthomonads, the mechanism by which RsmA activates T3SS remains unknown. Here, we show that RsmA activates the expression of T3SS-encoding hrp/hrc genes by directly binding to the 5′ untranslated region (UTR) of hrpG, the master regulator of the hrp/hrc genes in XCC. RsmA stabilizes hrpG mRNA, leading to increased accumulation of HrpG proteins and subsequently, the activation of hrp/hrc genes. The activation of the hrp/hrc genes by RsmA via HrpG was further supported by the observation that ectopic overexpression of hrpG in an rsmA mutant restored its ability to cause disease in host plants and trigger HR in non-host plants. RsmA also stabilizes the transcripts of another T3SS-associated hrpD operon by directly binding to the 5′ UTR region. Taken together, these data revealed that RsmA primarily activates T3SS by acting as a positive regulator of hrpG and that this regulation is critical to the pathogenicity of XCC. PMID:24586158

ExMC Technology Watch

NASA Technical Reports Server (NTRS)

Krihak, M.; Watkins, S.; Fung, Paul P.

2013-01-01

The Technology Watch (Tech Watch) project is a NASA project that is operated under the Human Research Programs (HRP) Exploration Medical Capability (ExMC) element, and focuses on ExMC technology gaps. The project coordinates the efforts of several NASA centers, including the Johnson Space Center (JSC), Glenn Research Center (GRC), Ames Research Center (ARC), and the Langley Research Center (LaRC). The objective of Tech Watch is to identify emerging, high-impact technologies that augment current NASA HRP technology development efforts. Identifying such technologies accelerates the development of medical care and research capabilities for the mitigation of potential health issues encountered during human space exploration missions. The aim of this process is to leverage technologies developed by academia, industry and other government agencies and to identify the effective utilization of NASA resources to maximize the HRP return on investment. The establishment of collaborations with these entities is beneficial to technology development, assessment and/or insertion and further NASAs goal to provide a safe and healthy environment for human exploration. In 2012, the Tech Watch project expanded the scope of activities to cultivate student projects targeted at specific ExMC gaps, generate gap reports for a majority of the ExMC gaps and maturate a gap report review process to optimize the technical and managerial aspects of ExMC gap status. Through numerous site visits and discussions with academia faculty, several student projects were initiated and/or completed this past year. A key element to these student projects was the ability of the project to align with a specific ExMC technology or knowledge gap. These projects were mentored and reviewed by Tech Watch leads at the various NASA centers. Another result of the past years efforts was the population of the ExMC wiki website that now contains more the three quarters of the ExMC gap reports. The remaining gap reports will be completed in FY13. Finally, the gap report review process for all ExMC gaps was initiated. This review process was instrumental in ensuring that each gap report was thoroughly reviewed for accuracy and relevant content prior to its public release. In the upcoming year, the gap report review process will be refined such that in addition to the gap report update, programmatic information related to gap closure will also be emphasized.

Advancing Translational Space Research Through Biospecimen Sharing: Amplifying the Impact of Ground-Based Studies

NASA Technical Reports Server (NTRS)

Ronca, A.; Lewis, L.; Staten, B.; Moyer, E.; Vizir, V.; Gompf, H.; Hoban-Higgins, T.; Fuller, C. A.

2017-01-01

Biospecimen Sharing Programs (BSPs) have been organized by NASA Ames Research Center since the 1960s with the goal of maximizing utilization and scientific return from rare, complex and costly spaceflight experiments. BSPs involve acquiring otherwise unused biological specimens from primary space research experiments for distribution to secondary experiments. Here we describe a collaboration leveraging Ames expertise in biospecimen sharing to magnify the scientific impact of research informing astronaut health funded by the NASA Human Research Program (HRP) Human Health Countermeasures (HHC) Element. The concept expands biospecimen sharing to one-off ground-based studies utilizing analogue space platforms (e.g., Hind limb Unloading (HLU), Artificial Gravity) for rodent experiments, thereby significantly broadening the range of research opportunities with translational relevance for protecting human health in space and on Earth. In this presentation, we will report on biospecimens currently being acquired from HHC Award Head-Down Tilt as a Model for Intracranial and Intraocular Pressures, and Retinal Changes during Spaceflight, and their availability. The BSP add-on to the project described herein has already yielded for HHC-funded investigators more than 4,700 additional tissues that would otherwise have been discarded as waste, with additional tissues available for analysis. Young (3-mo old) male and female rats and Older (9-mo old) male rats are being exposed to HLU for either 7, 14, 28, or 90 days. Additional groups are exposed to 90 days of unloading followed by either 7, 14, 28 days or 90 days of recovery (normal loading). Comparisons are made with non-suspended controls. Unused tissues are: Skin, Lungs, Thymus, Adrenals, Kidneys, Spleen, Hindlimb Muscles (Soleus, Extensor Digitorum Longus, Tibialis Anterior, Plantaris Gastrocnemius), Fat Pads, Reproductive Organs, and Intestines. Tissues are harvested, weighed, preserved then archived (with metadata) using a sample tracking system (CryoTrack). Preservation techniques include snap-freezing and RNALatersnap-freezing. Specimens were weighed at the time of dissection, and organ mass: body mass ratios analyzed to determine unloading effects across conditions and durations. The results corroborate previously reported effects of short-term exposure to microgravity or unloading exposure on various organs, and provide new insights into adaptation to long-duration unloading relevant to sustained spaceflight exposures on ISS. Supported by the Human Research Program (HRP) Human Health Countermeasures (HHC) Element and NASA Grant NNX13AD94G (CAF).

Polymerization reactivity of sulfomethylated alkali lignin modified with horseradish peroxidase.

PubMed

Yang, Dongjie; Wu, Xiaolei; Qiu, Xueqing; Chang, Yaqi; Lou, Hongming

2014-03-01

Alkali lignin (AL) was employed as raw materials in the present study. Sulfomethylation was conducted to improve the solubility of AL, while sulfomethylated alkali lignin (SAL) was further polymerized by horseradish peroxidase (HRP). HRP modification caused a significant increase in molecular weight of SAL which was over 20 times. It was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. The adsorption quantity of self-assembled SAL film was improved after HRP modification. Sulfonation and HRP modification were mutually promoted. The polymerization reactivity of SAL in HRP modification was increased with its sulfonation degree. Meanwhile, HRP modification facilitated SAL's radical-sulfonation reaction. Copyright © 2014. Published by Elsevier Ltd.

2014 Space Human Factors Engineering Standing Review Panel

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

The 2014 Space Human Factors Engineering (SHFE) Standing Review Panel (from here on referred to as the SRP) participated in a WebEx/teleconference with members of the Space Human Factors and Habitability (SHFH) Element, representatives from the Human Research Program (HRP), the National Space Biomedical Research Institute (NSBRI), and NASA Headquarters on November 17, 2014 (list of participants is in Section XI of this report). The SRP reviewed the updated research plans for the Risk of Incompatible Vehicle/Habitat Design (HAB Risk) and the Risk of Performance Errors Due to Training Deficiencies (Train Risk). The SRP also received a status update on the Risk of Inadequate Critical Task Design (Task Risk), the Risk of Inadequate Design of Human and Automation/Robotic Integration (HARI Risk), and the Risk of Inadequate Human-Computer Interaction (HCI Risk).

Psychosocial Characteristics of Optimum Performance in Isolated and Confined Environments (ICE)

NASA Technical Reports Server (NTRS)

Palinkas, Lawrence A.; Keeton, Kathryn E.; Shea, Camille; Leveton, Lauren B.

2010-01-01

The Behavioral Health and Performance (BHP) Element addresses human health risks in the NASA Human Research Program (HRP), including the Risk of Adverse Behavioral Conditions and the Risk of Psychiatric Disorders. BHP supports and conducts research to help characteristics and mitigate the Behavioral Medicine risk for exploration missions, and in some instances, current Flight Medical Operations. The Behavioral Health and Performance (BHP) Element identified research gaps within the Behavioral Medicine Risk, including Gap BMed6: What psychosocial characteristics predict success in an isolated, confined environment (ICE)? To address this gap, we conducted an extensive and exhaustive literature review to identify the following: 1) psychosocial characteristics that predict success in ICE environments; 2) characteristics that are most malleable; and 3) specific countermeasures that could enhance malleable characteristics.

Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination

PubMed Central

Sun, Huihui; Liu, Zhuang; Wu, Chao; Xu, Ping; Wang, Xia

2016-01-01

As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1–40 μM in sulfide detection with a high sensitivity of 1720 μA mM−1 cm−2 and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported. PMID:27515253

Amperometric inhibitive biosensor based on horseradish peroxidase-nanoporous gold for sulfide determination

NASA Astrophysics Data System (ADS)

Sun, Huihui; Liu, Zhuang; Wu, Chao; Xu, Ping; Wang, Xia

2016-08-01

As a well-known toxic pollutant, sulfide is harmful to human health. In this study, a simple and sensitive amperometric inhibitive biosensor was developed for the determination of sulfide in the environment. By immobilizing nanoporous gold (NPG) on glassy carbon electrode (GCE), and encapsulating horseradish peroxidase (HRP) onto NPG, a HRP/NPG/GCE bioelectrode for sulfide detection was successfully constructed based on the inhibition of sulfide on HRP activity with o-Phenylenediamine (OPD) as a substrate. The resulted HRP/NPG/GCE bioelectrode achieved a wide linear range of 0.1-40 μM in sulfide detection with a high sensitivity of 1720 μA mM-1 cm-2 and a low detection limit of 0.027 μM. Additionally, the inhibition of sulfide on HRP is competitive inhibition with OPD as a substrate by Michaelis-Menten analysis. Notably, the recovery of HRP activity was quickly achieved by washing the HRP/NPG/GCE bioelectrode using differential pulse voltammetry (DPV) technique in deaerated PBS (50 mM, pH 7.0) for only 60 s. Furthermore, the real sample analysis of sulfide by the HRP/NPG/GCE bioelectrode was achieved. Based on above results, the HRP/NPG/GCE bioelectrode could be a better choice for the real determination of sulfide compared to inhibitive biosensors previously reported.

Implication of an Aldehyde Dehydrogenase Gene and a Phosphinothricin N-Acetyltransferase Gene in the Diversity of Pseudomonas cichorii Virulence

PubMed Central

Tanaka, Masayuki; Wali, Ullah Md; Nakayashiki, Hitoshi; Fukuda, Tatsuya; Mizumoto, Hiroyuki; Ohnishi, Kouhei; Kiba, Akinori; Hikichi, Yasufumi

2011-01-01

Pseudomonas cichorii harbors the hrp genes. hrp-mutants lose their virulence on eggplant but not on lettuce. A phosphinothricin N-acetyltransferase gene (pat) is located between hrpL and an aldehyde dehydrogenase gene (aldH) in the genome of P. cichorii. Comparison of nucleotide sequences and composition of the genes among pseudomonads suggests a common ancestor of hrp and pat between P. cichorii strains and P. viridiflava strains harboring the single hrp pathogenicity island. In contrast, phylogenetic diversification of aldH corresponded to species diversification amongst pseudomonads. In this study, the involvement of aldH and pat in P. cichorii virulence was analyzed. An aldH-deleted mutant (ΔaldH) and a pat-deleted mutant (Δpat) lost their virulence on eggplant but not on lettuce. P. cichorii expressed both genes in eggplant leaves, independent of HrpL, the transcriptional activator for the hrp. Inoculation into Asteraceae species susceptible to P. cichorii showed that the involvement of hrp, pat and aldH in P. cichorii virulence is independent of each other and has no relationship with the phylogeny of Asteraceae species based on the nucleotide sequences of ndhF and rbcL. It is thus thought that not only the hrp genes but also pat and aldH are implicated in the diversity of P. cichorii virulence on susceptible host plant species. PMID:24704843

PecS and PecT coregulate the synthesis of HrpN and pectate lyases, two virulence determinants in Erwinia chrysanthemi 3937.

PubMed

Nasser, William; Reverchon, Sylvie; Vedel, Regine; Boccara, Martine

2005-11-01

Erwinia chrysanthemi strain 3937 is a necrotrophic bacterial plant pathogen. Pectinolytic enzymes and, in particular, pectate lyases play a key role in soft rot symptoms; however, the efficient colonization of plants by E. chrysanthemi requires additional factors. These factors include HrpN (harpin), a heat-stable, glycine-rich hydrophilic protein, which is secreted by the type III secretion system. We investigated the expression of hrpN in E. chrysanthemi 3937 in various environmental conditions and different regulatory backgrounds. Using lacZ fusions, hrpN expression was markedly influenced by the carbon source, osmolarity, growth phase, and growth substrate. hrpN was repressed when pectinolysis started and negatively regulated by the repressors of pectate lyase synthesis, PecS and PecT. Primer extension data and in vitro DNA-protein interaction experiments support a model whereby PecS represses hrpN expression by binding to the hrpN regulatory region and inhibiting transcript elongation. The results suggest coordinated regulation of HrpN and pectate lyases by PecS and PecT. A putative model of the synthesis of these two virulence factors in E. chrysanthemi during pathogenesis is presented.

Integrated Extravehicular Activity Human Research Plan: 2017

NASA Technical Reports Server (NTRS)

Abercromby, Andrew

2017-01-01

Multiple organizations within NASA as well as industry and academia fund and participate in research related to extravehicular activity (EVA). In October 2015, representatives of the EVA Office, the Crew and Thermal Systems Division (CTSD), and the Human Research Program (HRP) at NASA Johnson Space Center agreed on a formal framework to improve multi-year coordination and collaboration in EVA research. At the core of the framework is an Integrated EVA Human Research Plan and a process by which it will be annually reviewed and updated. The over-arching objective of the collaborative framework is to conduct multi-disciplinary cost-effective research that will enable humans to perform EVAs safely, effectively, comfortably, and efficiently, as needed to enable and enhance human space exploration missions. Research activities must be defined, prioritized, planned and executed to comprehensively address the right questions, avoid duplication, leverage other complementary activities where possible, and ultimately provide actionable evidence-based results in time to inform subsequent tests, developments and/or research activities. Representation of all appropriate stakeholders in the definition, prioritization, planning and execution of research activities is essential to accomplishing the over-arching objective. A formal review of the Integrated EVA Human Research Plan will be conducted annually. Coordination with stakeholders outside of the EVA Office, CTSD, and HRP is already in effect on a study-by-study basis; closer coordination on multi-year planning with other EVA stakeholders including academia is being actively pursued. Details of the preliminary Integrated EVA Human Research Plan are presented including description of ongoing and planned research activities in the areas of: physiological and performance capabilities; suit design parameters; EVA human health and performance modeling; EVA tasks and concepts of operations; EVA informatics; human-suit sensors; suit sizing and fit; and EVA injury risk and mitigation. This paper represents the 2017 update to the Integrated EVA Human Research Plan.

Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests

PubMed Central

2013-01-01

Background An accurate diagnosis is essential for the rapid and appropriate treatment of malaria. The accuracy of the histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT) Palutop+4® was assessed here. One possible factor contributing to the failure to detect malaria by this test is the diversity of the parasite PfHRP2 antigens. Methods PfHRP2 detection with the Palutop+4® RDT was carried out. The pfhrp2 and pfhrp3 genes were amplified and sequenced from 136 isolates of Plasmodium falciparum that were collected in Dakar, Senegal from 2009 to 2011. The DNA sequences were determined and statistical analyses of the variation observed between these two genes were conducted. The potential impact of PfHRP2 and PfHRP3 sequence variation on malaria diagnosis was examined. Results Seven P. falciparum isolates (5.9% of the total isolates, regardless of the parasitaemia; 10.7% of the isolates with parasitaemia ≤0.005% or ≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT. Low parasite density is not sufficient to explain the PfHRP2 detection failure. Three of these seven samples showed pfhrp2 deletion (2.4%). The pfhrp3 gene was deleted in 12.8%. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. Of the 109 PfHRP3 sequences, 64 unique sequences were identified. Using the Baker’s regression model, at least 7.4% of the P. falciparum isolates in Dakar were likely to be undetected by PfHRP2 at a parasite density of ≤250 parasites/μl (slightly lower than the evaluated prevalence of 10.7%). This predictive prevalence increased significantly between 2009 and 2011 (P = 0.0046). Conclusion In the present work, 10.7% of the isolates with a parasitaemia ≤0.005% (≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT (7.4% by the predictive Baker’model). In addition, all of the parasites with pfhrp2 deletion (2.4% of the total samples) and 2.1% of the parasites with parasitaemia >0.005% and presence of pfhrp2 were not detected by PfHRP2 RDT. PfHRP2 is highly polymorphic in Senegal. Efforts should be made to more accurately determine the prevalence of non-sensitive parasites to pfHRP2. PMID:23347727

NH2Fe3O4@SiO2 supported peroxidase catalyzed H2O2 for degradation of endocrine disrupter from aqueous solution: Roles of active radicals and NOMs.

PubMed

Ai, Jing; Zhang, Weijun; Liao, Guiying; Xia, Hua; Wang, Dongsheng

2017-11-01

In this work, magnetic Fe 3 O 4 was utilized to immobilize horseradish peroxidase (IM-HRP) in order to improve its stability and reusability by crosslinking method process with glutaraldehyde. The physicochemical properties of NH 2 Fe 3 O 4 @SiO 2 and IM-HRP were characterized by powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Thermo-gravimetric Analysis (TGA) and Transmission electron microscopy (TEM). The thermal stability of immobilized-HRP was considerably improved in comparison with free counterpart. The catalytic performance of IM-HRP for estrogens removal from aqueous solution was evaluated, it was found that the presence of natural organic matters (NOM) have no significant effects on E2 removal and the E2 enzyme-degradation reached around 80% when pH = 7.0 with 0.552 × 10 -3 ratio of IM-HRP/H 2 O 2. In addition, the active radicals responsible for estrogens degradation were identified with electro-spin resonance spectra (ESR). It was found that immobilization process on Fe 3 O 4 showed no adverse effects on catalytic performance on HRP, estrogens degradation could be fitted well with pseudo-second kinetic equation. Estrogens degradation efficiency was reduced in the presence of humic substances. Both O 2 - and OH were detected in IM-HRP catalyzed H 2 O 2 system and radicals quenching test indicated O 2 - played a more important role in estrogens removal. IM-HRP exhibited excellent stability and E2 removal efficiency could reach 45.41% after use seven times. Therefore, HRP enzymes immobilized on NH 2 Fe 3 O 4 @SiO 2 by cross-linking method in glutaraldehyde solutions was an effective way to improve stability and reusability of HRP, and which could avoid potential secondary pollution in water environment caused by free HRP after treatment. Copyright © 2017 Elsevier Ltd. All rights reserved.

ExMC Technology Watch

NASA Technical Reports Server (NTRS)

Krihak, M.; Barr, Y.; Watkins, S.; Fung, P.; McGrath, T.; Baumann, D.

2012-01-01

The Technology Watch (Tech Watch) project is a NASA endeavor conducted under the Human Research Program's (HRP) Exploration Medical Capability (ExMC) element, and focusing on ExMC technology gaps. The project involves several NASA centers, including the Johnson Space Center (JSC), Glenn Research Center (GRC), Ames Research Center (ARC), and the Langley Research Center (LaRC). The objective of Tech Watch is to identify emerging, high-impact technologies that augment current NASA HRP technology development efforts. Identifying such technologies accelerates the development of medical care and research capabilities for the mitigation of potential health issues encountered during human space exploration missions. The aim of this process is to leverage technologies developed by academia, industry and other government agencies and to identify the effective utilization of NASA resources to maximize the HRP return on investment. The establishment of collaborations with these entities is beneficial to technology development, assessment and/or insertion and further NASA's goal to provide a safe and healthy environment for human exploration. In 2011, the major focus areas for Tech Watch included information dissemination, education outreach and public accessibility to technology gaps and gap reports. The dissemination of information was accomplished through site visits to research laboratories and/or companies, and participation at select conferences where Tech Watch objectives and technology gaps were presented. Presentation of such material provided researchers with insights on NASA ExMC needs for space exploration and an opportunity to discuss potential areas of common interest. The second focus area, education outreach, was accomplished via two mechanisms. First, several senior student projects, each related to an ExMC technology gap, were sponsored by the various NASA centers. These projects presented ExMC related technology problems firsthand to collegiate laboratories. Second, a RASC-AL (Revolutionary Aerospace Systems Concepts - Academic Linkage) topic for FY12 was developed for medical systems and astronaut health under the Human-Focused Mars Mission Systems and Technologies theme. Announcement of the competition was made to the public in August 2011. Finally, critical Tech Watch information was prepared for public release in the form of gap reports. Complementing the ExMC technology gaps in the public domain, gap reports were generated, reviewed and revised through a series of technical, medical and subject matter expert reviews before approval for public release. An important vehicle for the public release of such documents was development of the ExMC wiki website, which will continue to be populated with gap reports and relevant documents throughout the upcoming year.

ExMC Technology Watch

NASA Technical Reports Server (NTRS)

Krihak, M.; Barr, Y.; Watkins, S.; Fung, P.; McGrath, T.; Baumann, D.

2012-01-01

The Technology Watch (Tech Watch) project is a NASA endeavor conducted under the Human Research Programs (HRP) Exploration Medical Capability (ExMC) element, and focusing on ExMC technology gaps. The project involves several NASA centers, including the Johnson Space Center (JSC), Glenn Research Center (GRC), Ames Research Center (ARC), and the Langley Research Center (LaRC). The objective of Tech Watch is to identify emerging, high-impact technologies that augment current NASA HRP technology development efforts. Identifying such technologies accelerates the development of medical care and research capabilities for the mitigation of potential health issues encountered during human space exploration missions. The aim of this process is to leverage technologies developed by academia, industry and other government agencies and to identify the effective utilization of NASA resources to maximize the HRP return on investment. The establishment of collaborations with these entities is beneficial to technology development, assessment and/or insertion and further NASAs goal to provide a safe and healthy environment for human exploration. In 2011, the major focus areas for Tech Watch included information dissemination, education outreach and public accessibility to technology gaps and gap reports. The dissemination of information was accomplished through site visits to research laboratories and/or companies, and participation at select conferences where Tech Watch objectives and technology gaps were presented. Presentation of such material provided researchers with insights on NASA ExMC needs for space exploration and an opportunity to discuss potential areas of common interest. The second focus area, education outreach, was accomplished via two mechanisms. First, several senior student projects, each related to an ExMC technology gap, were sponsored by the various NASA centers. These projects presented ExMC related technology problems firsthand to collegiate laboratories. Second, a RASC-AL (Revolutionary Aerospace Systems Concepts Academic Linkage) topic for FY12 was developed for medical systems and astronaut health under the Human-Focused Mars Mission Systems and Technologies theme. Announcement of the competition was made to the public in August 2011. Finally, critical Tech Watch information was prepared for public release in the form of gap reports. Complementing the ExMC technology gaps in the public domain, gap reports were generated, reviewed and revised through a series of technical, medical and subject matter expert reviews before approval for public release. An important vehicle for the public release of such documents was development of the ExMC wiki website, which will continue to be populated with gap reports and relevant documents throughout the upcoming year.

Bead-based immunoassay allows sub-picogram detection of histidine-rich protein 2 from Plasmodium falciparum and estimates reliability of malaria rapid diagnostic tests

PubMed Central

Rogier, Eric; Plucinski, Mateusz; Lucchi, Naomi; Mace, Kimberly; Chang, Michelle; Lemoine, Jean Frantz; Candrinho, Baltazar; Colborn, James; Dimbu, Rafael; Fortes, Filomeno; Udhayakumar, Venkatachalam; Barnwell, John

2017-01-01

Detection of histidine-rich protein 2 (HRP2) from the malaria parasite Plasmodium falciparum provides evidence for active or recent infection, and is utilized for both diagnostic and surveillance purposes, but current laboratory immunoassays for HRP2 are hindered by low sensitivities and high costs. Here we present a new HRP2 immunoassay based on antigen capture through a bead-based system capable of detecting HRP2 at sub-picogram levels. The assay is highly specific and cost-effective, allowing fast processing and screening of large numbers of samples. We utilized the assay to assess results of HRP2-based rapid diagnostic tests (RDTs) in different P. falciparum transmission settings, generating estimates for true performance in the field. Through this method of external validation, HRP2 RDTs were found to perform well in the high-endemic areas of Mozambique and Angola with 86.4% and 73.9% of persons with HRP2 in their blood testing positive by RDTs, respectively, and false-positive rates of 4.3% and 0.5%. However, in the low-endemic setting of Haiti, only 14.5% of persons found to be HRP2 positive by the bead assay were RDT positive. Additionally, 62.5% of Haitians showing a positive RDT test had no detectable HRP2 by the bead assay, likely indicating that these were false positive tests. In addition to RDT validation, HRP2 biomass was assessed for the populations in these different settings, and may provide an additional metric by which to estimate P. falciparum transmission intensity and measure the impact of interventions. PMID:28192523

Fructose-Bisphophate Aldolase Exhibits Functional Roles between Carbon Metabolism and the hrp System in Rice Pathogen Xanthomonas oryzae pv. oryzicola

PubMed Central

Li, Yu-rong; Cui, Yi-ping; Ji, Zhi-yuan; Cai, Lu-lu; Zou, Hua-song; Hutchins, William C.; Yang, Ching-hong; Chen, Gong-you

2012-01-01

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator. PMID:22384086

Novel characteristics of horseradish peroxidase immobilized onto the polyvinyl alcohol-alginate beads and its methyl orange degradation potential.

PubMed

Bilal, Muhammad; Rasheed, Tahir; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2017-12-01

Herein, we report the immobilization of in-house isolated horseradish peroxidase (HRP) from Armoracia rusticana with novel characteristics. The HRP was immobilized onto the self-fabricated polyvinyl alcohol-alginate (PVA-alginate) beads using sodium nitrate as a cross-linker. The PVA-alginate beads (2.0mm size) developed using 10% PVA and 1.5% sodium alginate showed maximal immobilization yield. The surface morphologies of the PVA-alginate (control) and immobilized-HRP were characterized by scanning electron microscopy (SEM). The immobilized-HRP retained 64.14% of its initial activity after 10 consecutive substrate-oxidation cycles as compared to the free counterpart. Simultaneously, the thermal stability of the immobilized-HRP was significantly enhanced as compared to the free HRP. The enzyme leakage (E L ) assay was performed by storing the immobilized-HRP in phosphate buffer solution for 30days. Evidently, the leakage of immobilized-HRP was recorded to be 6.98% and 14.82% after 15 and 30days of incubation, respectively. Finally, the immobilized-HRP was used for methyl orange (MO) dye degradation in a batch mode. A noticeable decline in spectral shift accompanied by no appearance of a new peak demonstrated the complete degradation of MO. The degraded fragments of MO were scrutinized by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A plausible degradation pathway for MO was proposed based on the identified intermediates. In conclusion, the study portrays the PVA-alginate-immobilized-HRP as a cost-effective and industrially desirable green catalyst, for biotechnological at large and industrial in particular, especially for the treatment of textile dyes or dye-containing industrial waste effluents. Copyright © 2017 Elsevier B.V. All rights reserved.

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III).

PubMed

Guo, Shaofen; Cao, Rui; Lu, Aihua; Zhou, Qing; Lu, Tianhong; Ding, Xiaolan; Li, Chaojun; Huang, Xiaohua

2008-05-01

One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb-HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb-HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb-HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb-HRP is due to the destruction (unfolding) of the conformation in Tb-HRP. The planarity of the heme active center in the Tb-HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb-HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.

Genetic diversity of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and its effect on the performance of PfHRP2-based rapid diagnostic tests.

PubMed

Baker, Joanne; McCarthy, James; Gatton, Michelle; Kyle, Dennis E; Belizario, Vicente; Luchavez, Jennifer; Bell, David; Cheng, Qin

2005-09-01

Rising costs of antimalarial agents are increasing the demand for accurate diagnosis of malaria. Rapid diagnostic tests (RDTs) offer great potential to improve the diagnosis of malaria, particularly in remote areas. Many RDTs are based on the detection of Plasmodium falciparum histidine-rich protein (PfHRP) 2, but reports from field tests have questioned their sensitivity and reliability. We hypothesize that the variability in the results of PfHRP2-based RDTs is related to the variability in the target antigen. We tested this hypothesis by examining the genetic diversity of PfHRP2, which includes numerous amino acid repeats, in 75 P. falciparum lines and isolates originating from 19 countries and testing a subset of parasites by use of 2 PfHRP2-based RDTs. We observed extensive diversity in PfHRP2 sequences, both within and between countries. Logistic regression analysis indicated that 2 types of repeats were predictive of RDT detection sensitivity (87.5% accuracy), with predictions suggesting that only 84% of P. falciparum parasites in the Asia-Pacific region are likely to be detected at densities < or = 250 parasites/microL. Our data also indicated that PfHRP3 may play a role in the performance of PfHRP2-based RDTs. These findings provide an alternative explanation for the variable sensitivity in field tests of malaria RDTs that is not due to the quality of the RDTs.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

PubMed

Huddy, Suzanne M; Hitzeroth, Inga I; Meyers, Ann E; Weber, Brandon; Rybicki, Edward P

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish ( Armoracia rusticana ); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana

PubMed Central

Huddy, Suzanne M.; Hitzeroth, Inga I.; Weber, Brandon; Rybicki, Edward P.

2018-01-01

Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish (Armoracia rusticana); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens-mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme. PMID:29301255

Estimating the Added Utility of Highly Sensitive Histidine-Rich Protein 2 Detection in Outpatient Clinics in Sub-Saharan Africa.

PubMed

Plucinski, Mateusz M; Rogier, Eric; Dimbu, Pedro Rafael; Fortes, Filomeno; Halsey, Eric S; Aidoo, Michael

2017-10-01

Most malaria testing is by rapid diagnostic tests (RDTs) that detect Plasmodium falciparum histidine-rich protein 2 (HRP2). Recently, several RDT manufacturers have developed highly sensitive RDTs (hsRDTs), promising a limit of detection (LOD) orders of magnitude lower than conventional RDTs. To model the added utility of hsRDTs, HRP2 concentration in Angolan outpatients was measured quantitatively using an ultrasensitive bead-based assay. The distribution of HRP2 concentration was bimodal in both afebrile and febrile patients. The conventional RDT was able to detect 81% of all HRP2-positive febrile patients and 52-77% of HRP2-positive afebrile patients. The added utility of hsRDTs was estimated to be greater in afebrile patients, where an hsRDT with a LOD of 200 pg/mL would detect an additional 50-60% of HRP2-positive persons compared with a conventional RDT with a LOD of 3,000 pg/mL. In febrile patients, the hsRDT would detect an additional 10-20% of cases. Conventional RDTs already capture the vast majority of symptomatic HRP2-positive individuals, and hsRDTs would have to reach a sufficiently low LOD approaching 200 pg/mL to provide added utility in identifying HRP2-positive, asymptomatic individuals.

Production and characterization of specific monoclonal antibodies binding the Plasmodium falciparum diagnostic biomarker, histidine-rich protein 2.

PubMed

Leow, Chiuan Herng; Jones, Martina; Cheng, Qin; Mahler, Stephen; McCarthy, James

2014-07-18

Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10(-11) M, followed by control mAb C1-13 at 1.03 × 10(-10) M and mAb D2 at 3.05 × 10(-10) M. Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria.

Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

PubMed

Romero, Lucía Virginia; Targovnik, Alexandra Marisa; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, María Victoria

2011-05-01

A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots. © Springer Science+Business Media B.V. 2011

[Rapid diagnostic test for malaria].

PubMed

Houzé, S

2017-02-01

The rapid diagnostic tests (RDTs) whose main interest lies in their implementation without special equipment by unskilled personnel have grown significantly over the past fifteen years to diagnose malaria. They rely on the detection of specific Plasmodium proteins, PfHRP2, pLDH and aldolase. If the detection of PfHRP2 has very good sensitivity for the diagnosis of Plasmodium falciparum malaria, the detection of pLDH or aldolase is less efficient for other species, leaving its place to the reference microscopic diagnosis. RDT could not generally be used to monitor therapeutic efficacy because they can remain positive after clinical and parasitological cure. Furthermore, the development of the use of these tests has highlighted the need for quality assurance programs to monitor their production as their use.

Ultra-High-Throughput Screening of an In Vitro-Synthesized Horseradish Peroxidase Displayed on Microbeads Using Cell Sorter

PubMed Central

Zhu, Bo; Mizoguchi, Takuro; Kojima, Takaaki; Nakano, Hideo

2015-01-01

The C1a isoenzyme of horseradish peroxidase (HRP) is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro) DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence) via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT) and inactive mutant (MUT) genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT) library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases. PMID:25993095

Different Perspectives on Asthenia in Astronauts and Cosmonauts: International Research Literature

NASA Technical Reports Server (NTRS)

Sandoval, Luis; Shea, Camille; Otto, Christian; Leventon, Lauren

2010-01-01

The Behavioral Health and Performance (BHP) Element is one of the six elements within the NASA Human Research Program (HRP) and is responsible for managing four risks: a) The Risk of Performance Decrements due to inadequate Cooperation, Coordination, Communication and Psychological Adaptation within a Team (Team), b) the Risk of Performance Errors due to Sleep Loss, Circadian De-synchronization, Fatigue and Work Overload (Sleep), c) Risk of Behavioral Conditions (BMed), and d) the Risk of Psychiatric Disorders (BMed). The aim of this report is to address some of the recommendations made by the recent NASA HRP Standing Review Panel for the Behavioral Medicine Risk of Psychiatric Disorders. Such recommendations included: a) the inclusion of important national and international literature in English and non-English language materials; including journals, books, magazines, conference reports and b) an extensive literature review of certain types of psychological states to predict, detect, and assess adverse mental states that may negatively affect the psychological well being of the astronauts, specifically asthenia. This report was a collaborative international work effort focused on the evaluation and determination of the importance of continuing research on asthenia as a possible psychological problem that might affect the optimal psychological functioning among crewmembers during long-duration space flight missions. Russian medical personnel (flight surgeons and psychologist) have observed symptoms of asthenia (weakness, increased fatigue, irritability, and attention and memory disorders) in cosmonauts after four months in space (Myasnikov& Zamaleddinov1996; Grigorieve, 1996 ) and believe that asthenia is one of the greater risks that will affect crews? optimal psychological functioning.

Studies on the oxidation reaction of tyrosine (Tyr) with H2O2 catalyzed by horseradish peroxidase (HRP) in alcohol-water medium by spectrofluorimetry and differential spectrophotometry.

PubMed

Tang, Bo; Wang, Yan; Liang, Huiling; Chen, Zhenzhen; He, Xiwen; Shen, Hanxi

2006-03-01

An oxidation reaction of tyrosine (Tyr) with H(2)O(2) catalyzed by horseradish peroxidase (HRP) was studied by spectrofluorimetry and differential spectrophotometry in the alcohol(methanol, ethanol, 1-propanol and isopropanol)-water mutual solubility system. Compared with the enzymatic-catalyzed reaction in the water medium, the fluorescence intensities of the product weakened, even extinguished. Because the addition of alcohols made the conformation of HRP change, the catalytic reaction shifted to the side of polymerization and the polymer (A(n)H(2), n>or=3) exhibited no fluorescence. The four alcohols cannot deactivate HRP. Moreover isopropanol activated HRP remarkably.

Visual Impairment/Intracranial Pressure Risk Assessment

NASA Technical Reports Server (NTRS)

Fogarty, Jennifer A.; Durham, T.; Otto, C.; Grounds, D.; Davis, J. R.

2010-01-01

Since 2006 there have been 6 reported cases of altered visual acuity and intracranial pressure (ICP) in long duration astronauts. In order to document this risk and develop an integrated approach to its mitigation, the NASA Space Life Sciences Directorate (SLSD) and Human Research Program (HRP) have chosen to use the Human System Risk Board (HSRB) and the risk management analysis tool (RMAT). The HSRB is the venue in which the stakeholders and customers discuss and vet the evidence and the RMAT is the tool that facilitates documentation and comparison of the evidence across mission profiles as well as identification of risk factors, and documentation of mitigation strategies. This process allows for information to be brought forward and dispositioned so that it may be properly incorporated into the RMAT and contribute to the design of the research and mitigation plans. The evidence thus far has resulted in the identification of a visual impairment/intracranial pressure (VIIP) project team, updating of both short and long duration medical requirements designed to assess visual acuity, and a research plan to characterize this issue further. In order to understand this issue more completely, a plan to develop an Accelerated Research Collaboration (ARC) has been approved by the HSRB. The ARC is a novel research model pioneered by the Myelin Repair Foundation. It is a patient centered research model that brings together researchers and clinicians, under the guidance of a scientific advisory panel, to collaborate and produce results much quickly than accomplished through traditional research models. The data and evidence from the updated medical requirements and the VIIP ARC will be reviewed at the HSRB on a regular basis. Each review package presented to the HSRB will include an assessment and recommendation with respect to continuation of research, countermeasure development, occupational surveillance modalities, selection criteria, etc. This process will determine the course of the VIIP project and ultimately how SLSD and HRP mitigate this emerging human health and performance risk.

Effectiveness of sequential automatic-manual home respiratory polygraphy scoring.

PubMed

Masa, Juan F; Corral, Jaime; Pereira, Ricardo; Duran-Cantolla, Joaquin; Cabello, Marta; Hernández-Blasco, Luis; Monasterio, Carmen; Alonso-Fernandez, Alberto; Chiner, Eusebi; Vázquez-Polo, Francisco-José; Montserrat, Jose M

2013-04-01

Automatic home respiratory polygraphy (HRP) scoring functions can potentially confirm the diagnosis of sleep apnoea-hypopnoea syndrome (SAHS) (obviating technician scoring) in a substantial number of patients. The result would have important management and cost implications. The aim of this study was to determine the diagnostic cost-effectiveness of a sequential HRP scoring protocol (automatic and then manual for residual cases) compared with manual HRP scoring, and with in-hospital polysomnography. We included suspected SAHS patients in a multicentre study and assigned them to home and hospital protocols at random. We constructed receiver operating characteristic (ROC) curves for manual and automatic scoring. Diagnostic agreement for several cut-off points was explored and costs for two equally effective alternatives were calculated. Of 366 randomised patients, 348 completed the protocol. Manual scoring produced better ROC curves than automatic scoring. There was no sensitive automatic or subsequent manual HRP apnoea-hypopnoea index (AHI) cut-off point. The specific cut-off points for automatic and subsequent manual HRP scorings (AHI >25 and >20, respectively) had a specificity of 93% for automatic and 94% for manual scorings. The costs of manual protocol were 9% higher than sequential HRP protocol; these were 69% and 64%, respectively, of the cost of the polysomnography. A sequential HRP scoring protocol is a cost-effective alternative to polysomnography, although with limited cost savings compared to HRP manual scoring.

The hrp23 Protein in the Balbiani Ring Pre-mRNP Particles Is Released Just before or at the Binding of the Particles to the Nuclear Pore Complex

PubMed Central

Sun, Xin; Alzhanova-Ericsson, Alla T.; Visa, Neus; Aissouni, Youssef; Zhao, Jian; Daneholt, Bertil

1998-01-01

Balbiani ring (BR) pre-mRNP particles reside in the nuclei of salivary glands of the dipteran Chironomus tentans and carry the message for giant-sized salivary proteins. In the present study, we identify and characterize a new protein component in the BR ribonucleoprotein (RNP) particles, designated hrp23. The protein with a molecular mass of 20 kD has a single RNA-binding domain and a glycine-arginine-serine–rich auxiliary domain. As shown by immunoelectron microscopy, the hrp23 protein is added to the BR transcript concomitant with transcription, is still present in the BR particles in the nucleoplasm, but is absent from the BR particles that are bound to the nuclear pore complex or are translocating through the central channel of the complex. Thus, hrp23 is released just before or at the binding of the particles to the nuclear pore complex. It is noted that hrp23 behaves differently from two other BR RNP proteins earlier studied: hrp36 and hrp45. These proteins both reach the nuclear pore complex, and hrp36 even accompanies the RNA into the cytoplasm. It is concluded that each BR RNA-binding protein seems to have a specific flow pattern, probably related to the particular role of the protein in gene expression. PMID:9732280

Biotechnological advances towards an enhanced peroxidase production in Pichia pastoris.

PubMed

Krainer, Florian W; Gerstmann, Michaela A; Darnhofer, Barbara; Birner-Gruenberger, Ruth; Glieder, Anton

2016-09-10

Horseradish peroxidase (HRP) is a high-demand enzyme for applications in diagnostics, bioremediation, biocatalysis and medicine. Current HRP preparations are isolated from horseradish roots as mixtures of biochemically diverse isoenzymes. Thus, there is a strong need for a recombinant production process enabling a steady supply with enzyme preparations of consistent high quality. However, most current recombinant production systems are limited at titers in the low mg/L range. In this study, we used the well-known yeast Pichia pastoris as host for recombinant HRP production. To enhance recombinant enzyme titers we systematically evaluated engineering approaches on the secretion process, coproduction of helper proteins, and compared expression from the strong methanol-inducible PAOX1 promoter, the strong constitutive PGAP promoter, and a novel bidirectional promoter PHTX1. Ultimately, coproduction of HRP and active Hac1 under PHTX1 control yielded a recombinant HRP titer of 132mg/L after 56h of cultivation in a methanol-independent and easy-to-do bioreactor cultivation process. With regard to the many versatile applications for HRP, the establishment of a microbial host system suitable for efficient recombinant HRP production was highly overdue. The novel HRP production platform in P. pastoris presented in this study sets a new benchmark for this medically relevant enzyme. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Probing horseradish peroxidase catalyzed degradation of azo dye from tannery wastewater.

PubMed

Preethi, Sadhanandam; Anumary, Ayyappan; Ashokkumar, Meiyazhagan; Thanikaivelan, Palanisamy

2013-01-01

Biocatalysis based effluent treatment has outclassed the presently favored physico-chemical treatments due to nil sludge production and monetary savings. Azo dyes are commonly employed in the leather industry and pose a great threat to the environment. Here, we show the degradation of C. I. Acid blue 113 using horseradish peroxidase (HRP) assisted with H2O2 as a co-substrate. It was observed that 0.08 U HRP can degrade 3 mL of 30 mg/L dye up to 80% within 45 min with the assistance of 14 μL of H2O2 at pH 6.6 and 30°C. The feasibility of using the immobilized HRP for dye degradation was also examined and the results show up to 76% dye degradation under similar conditions to that of free HRP with the exception of longer contact time of 240 min. Recycling studies reveal that the immobilized HRP can be recycled up to 3 times for dye degradation. Kinetics drawn for the free HRP catalyzed reaction marked a lower K m and higher V max values, which denotes a proper and faster affinity of the enzyme towards the dye, when compared to the immobilized HRP. The applicability of HRP for treating the actual tannery dye-house wastewater was also demonstrated.

10 CFR 712.30 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Applicability. 712.30 Section 712.30 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Medical Standards § 712.30 Applicability. This subpart establishes standards and procedures for conducting medical assessments of DOE and DOE contractor individuals in HRP...

10 CFR 712.15 - Management evaluation.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Management evaluation. 712.15 Section 712.15 Energy... Program Procedures § 712.15 Management evaluation. (a) Evaluation components. An evaluation by the HRP management official is required before an individual can be considered for initial certification or...

Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.

PubMed

Schwartz, R S; Rybicki, A C; Nagel, R L

1997-10-15

We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton.

Horseradish peroxidase immobilized on copper surfaces and applications in selective electrocatalysis of p-dihydroxybenzene

NASA Astrophysics Data System (ADS)

Wang, Chuntao; Luo, Xiaoxiao; Jia, Zehui; Shi, Qinghua; Zhu, Ritao

2017-06-01

Horseradish Peroxidase (HRP) was immobilized on copper surfaces with the linker of L-Cysteine (L-Cys) self-assembled films to form Cu/L-Cys/HRP electrodes. The activity of HRP can be preserved by the Cu/L-Cys self-assembled films. The Cu/L-Cys/HRP electrodes can be used for the selective electrocatalytic oxidase of p-dihydroxybenzen in absent of H2O2. The optimum pH for electrocatalyzing p-dihydroxybenzen was 5.5 or 7.0, which corresponds to the isoelectric points of L-Cys and HRP, respectively. X-ray photoelectron spectroscopy (XPS) provided the evidence that L-Cys linked with Cu surface by the Cusbnd S bond. Fourier transform infrared spectroscopy (FTIR) analyses indicated that aromatic plane of p-dihydroxybenzen was connected parallel to porphyrin ring of heme in HRP. Quantum chemical calculation of density functional theory (DFT) revealed that symmetry of molecular structure and minimum space steric hindrance for p-dihydroxybenzen were benefit to combination with HRP. Moreover, the lowest energy of LUMO and most negative charges of oxygen atom on hydroxyl group of p-dihydroxybenzen were advantage to lose the hydrogen atom of hydroxyl group to be oxided.

Bulk-modified modified screen-printing carbon electrodes with both lactate oxidase (LOD) and horseradish peroxide (HRP) for the determination of L-lactate in flow injection analysis mode.

PubMed

Ghamouss, Fouad; Ledru, Sophie; Ruillé, Nadine; Lantier, Françoise; Boujtita, Mohammed

2006-06-16

A screen-printed carbon electrode modified with both HRP and LOD (SPCE-HRP/LOD) has been developed for the determination of L-lactate concentration in real samples. The resulting SPCE-HRP/LOD was prepared in a one-step procedure, and was then optimised as an amperometric biosensor operating at [0, -100]mV versus Ag/AgCl for L-lactate determination in flow injection mode. A significant improvement in the reproducibility (coefficient variation of about 10%) of the preparation of the biosensors was obtained when graphite powder was modified with LOD in the presence of HRP previously oxidised by periodate ion (IO4-). Optimisation studies were performed by examining the effects of LOD loading, periodation step and rate of the binder on analytical performances of SPCE-HRP/LOD. The sensitivity of the optimised SPCE-HRP/LOD to L-lactate was 0.84 nAL micromol(-1) in a detection range between 10 and 180 microMol. The possibility of using the developed biosensor to determine L-lactate concentrations in various dairy products was also evaluated.

Luminol, horseradish peroxidase, and glucose oxidase ternary functionalized graphene oxide for ultrasensitive glucose sensing.

PubMed

Li, Fang; Ma, Wenjing; Liu, Jiachang; Wu, Xiang; Wang, Yan; He, Jianbo

2018-01-01

Luminol, horseradish peroxidase (HRP), and glucose oxidase (GOx) ternary functionalized graphene oxide (HRP/GOx-luminol-GO) with excellent chemiluminescence (CL) activity and specific enzymatic property was prepared via a simple and general strategy for the first time. In this approach, luminol functionalized GO (luminol-GO) was prepared by gently stirring GO with luminol. Then HRP and GOx were further co-immobilized onto the surface of luminol-GO by storing HRP and GOx with luminol-GO at 4 °C overnight, to form HRP/GOx-luminol-GO bionanocomposites. The synthesized HRP/GOx-luminol-GO could react with H 2 O 2 generated from GOx catalyzed glucose oxidization reaction, to produce strong CL emission in the presence of co-immobilized HRP. Thus, we developed an ultrasensitive, homogeneous, reagentless, selective, and simple CL sensing system for glucose detection. The resulting biosensors exhibited ultra-wide linear range from 5.0 nM to 5.0 mM, and an ultra-low detection limit of 1.2 nM, which was more than 3 orders of magnitude lower than previously reported methods. Furthermore, the sensing system was successfully applied for the detection of glucose in human blood samples.

Fabrication of an electrochemical platform based on the self-assembly of graphene oxide-multiwall carbon nanotube nanocomposite and horseradish peroxidase: direct electrochemistry and electrocatalysis

NASA Astrophysics Data System (ADS)

Zhang, Qian; Yang, Shaojun; Zhang, Jing; Zhang, Ling; Kang, Pingli; Li, Jinghong; Xu, Jingwei; Zhou, Hua; Song, Xi-Ming

2011-12-01

A novel hybrid nanomaterial (GO-MWNTs) was explored based on the self-assembly of multiwall carbon nanotubes (MWNTs) and graphene oxide (GO). Compared with pristine MWNTs, such a nanocomposite could be well dispersed in aqueous solution and exhibit a negative charge. Driven by the electrostatic interaction, positively charged horseradish peroxidase (HRP) could then be immobilized onto GO-MWNTs at the surface of a glassy carbon (GC) electrode to form a HRP/GO-MWNT/GC electrode under mild conditions. TEM was used to characterize the morphology of the GO-MWNT nanocomposite. UV-vis and FTIR spectra suggested that HRP was immobilized onto the hybrid matrix without denaturation. Furthermore, the immobilized HRP showed enhanced direct electron transfer for the HRP-Fe(III)/Fe(II) redox center. Based on the direct electron transfer of the immobilized HRP, the HRP/GO-MWNT/GC electrode exhibited excellent electrocatalytic behavior to the reduction of H2O2 and NaNO2, respectively. Therefore, GO-MWNTs could provide a novel and efficient platform for the immobilization and biosensing of redox enzymes, and thus may find wide potential applications in the fabrication of biosensors, biomedical devices, and bioelectronics.

Pharmaceuticals Exposed to the Space Environment: Problems and Prospects

NASA Technical Reports Server (NTRS)

Jaworske, Donald A.; Myers, Jerry G.

2016-01-01

The NASA Human Research Program (HRP) Health Countermeasures Element maintains ongoing efforts to inform detailed risks, gaps, and further questions associated with the use of pharmaceuticals in space. Most recently, the Pharmacology Risk Report, released in 2010, illustrates the problems associated with maintaining pharmaceutical efficacy. Since the report, one key publication includes evaluation of pharmaceutical products stored on the International Space Station (ISS). This study shows that selected pharmaceuticals on ISS have a shorter shelf-life in space than corresponding terrestrial controls. The HRP Human Research Roadmap for planetary exploration identifies the risk of ineffective or toxic medications due to long-term storage during missions to Mars. The roadmap also identifies the need to understand and predict how pharmaceuticals will behave when exposed to radiation for long durations. Terrestrial studies of returned samples offer a start for predictive modeling. This paper shows that pharmaceuticals returned to Earth for post-flight analyses are amenable to a Weibull distribution analysis in order to support probabilistic risk assessment modeling. The paper also considers the prospect of passive payloads of key pharmaceuticals on sample return missions outside of Earth's magnetic field to gather additional statistics. Ongoing work in radiation chemistry suggests possible mitigation strategies where future work could be done at cryogenic temperatures to explore methods for preserving the strength of pharmaceuticals in the space radiation environment, perhaps one day leading to an architecture where pharmaceuticals are cached on the Martian surface and preserved cryogenically.

The cDNA sequence of a neutral horseradish peroxidase.

PubMed

Bartonek-Roxå, E; Eriksson, H; Mattiasson, B

1991-02-16

A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

Human System Risk Management for Space Flight

NASA Technical Reports Server (NTRS)

Davis, Jeffrey

2015-01-01

This brief abstract reviews the development of the current day approach to human system risk management for space flight and the development of the critical components of this process over the past few years. The human system risk management process now provides a comprehensive assessment of each human system risk by design reference mission (DRM) and is evaluated not only for mission success but also for long-term health impacts for the astronauts. The discipline of bioastronautics is the study of the biological and medical effects of space flight on humans. In 1997, the Space Life Sciences Directorate (SLSD) initiated the Bioastronautics Roadmap (Roadmap) as the "Critical Path Roadmap", and in 1998 participation in the roadmap was expanded to include the National Space Biomedical Research Institute (NSBRI) and the external community. A total of 55 risks and 250 questions were identified and prioritized and in 2000, the Roadmap was base-lined and put under configuration control. The Roadmap took into account several major advisory committee reviews including the Institute of Medicine (IOM) "Safe Passage: Astronaut care for Exploration Missions", 2001. Subsequently, three collaborating organizations at NASA HQ (Chief Health and Medical Officer, Office of Space Flight and Office of Biological & Physical Research), published the Bioastronautics Strategy in 2003, that identified the human as a "critical subsystem of space flight" and noted that "tolerance limits and safe operating bands must be established" to enable human space flight. These offices also requested a review by the IOM of the Roadmap and that review was published in October 2005 as "A Risk Reduction Strategy for Human Exploration of Space: A Review of NASA's Bioastronautics Roadmap", that noted several strengths and weaknesses of the Roadmap and made several recommendations. In parallel with the development of the Roadmap, the Office of the Chief Health and Medical Officer (OCHMO) began a process in 2004 of evaluating the tolerance limits and safe operating bands called for in the Bioastronautics Strategy. Over the next several years, the concept of the "operating bands" were turned into Space Flight Human System Standards (SFHSS), developed by the technical resources of the SLSD at the NASA Johnson Space Center (JSC). These standards were developed and reviewed at the SLSD and then presented to the OCHMO for acceptance. The first set of standards was published in 2007 as the NASA-STD-3001, Volume 1, Crew Health that elaborated standards for several physiological areas such as cardiovascular, musculoskeletal, radiation exposure and nutrition. Volume 2, Human Factors, Habitability and Human Health was published in 2011, along with development guidance in the Human Integration Design Handbook (HIDH). Taken together, the SFHSS Volumes 1 and 2, and the HIDH replaced the NASA-STD-3000 with new standards and revisions of the older document. Three other changes were also taking place that facilitated the development of the human system risk management approach. In 2005, the life sciences research and development portfolio underwent a comprehensive review through the Exploration Systems Architecture Study (ESAS) that resulted in the reformulation of the Bioastronautics Program into Human Research Program (HRP) that was focused on appropriate mitigation results for high priority human health risks. The baseline HRP budget was established in August 2005. In addition, the OCHMO formulated the Health and Medical Technical Authority (HMTA) in 2006 that established the position of the Chief Medical Officer (CMO) at the NASA JSC along with other key technical disciplines, and the OCHMO became the responsible office for the SFHSS as noted above. The final change was the establishment in 2008 of the Human System Risk Board (HSRB), chaired by the CMO with representation from the HRP, SLSD management and technical experts. The HSRB then began to review all human system risks, established a comprehensive risk management and configuration management plan and data sharing policy. These major developments of standards, the HRP, the HMTA and a forum for review of human system risks (HSRB) facilitated the integration of human research, medical operations, systems engineering and many other disciplines in the comprehensive review of human system risks. The HSRB began a comprehensive review of all potential inflight medical conditions and events and over the course of several reviews consolidated the number of human system risks to 30 where the greatest emphasis is placed for investing program dollars for risk mitigation. The HSRB considers all available evidence from human research, medical operations and occupational surveillance in assessing the risks for appropriate mitigation and future work. All applicable DRMs (low earth orbit 6 and 12 months, deep space sortie for 30 days and 1 year, a one year lunar mission, and a planetary mission for 3 years) are considered as human system risks are modified by the hazards associated with space flight such as microgravity, exposure to radiation, distance from the earth, isolation and a closed environment. Each risk has a summary assessment representing the state of knowledge/evidence base for that risk, the available risk mitigations, traceability to the SFHSS and program requirements, and future work required. These data then can drive coordinated budgets across the HRP, the International Space Station, Crew Health and Safety and Advanced Exploration System budgets. These risk assessments were completed for 6 DRMs in December of 2014 and serve as the baseline for which subsequent research and technology development and crew health care portfolios can be assessed. The HSRB will review each risk at least annually and especially when new information is available that must be considered for effective risk mitigation. The current status of each risk can be reported to program management for operations, budget reviews and general oversight of the human system risk management program.

Integration Host Factor Is Required for RpoN-Dependent hrpL Gene Expression and Controls Motility by Positively Regulating rsmB sRNA in Erwinia amylovora.

PubMed

Lee, Jae Hoon; Zhao, Youfu

2016-01-01

Erwinia amylovora requires an hrp-type III secretion system (T3SS) to cause disease. It has been reported that HrpL, the master regulator of T3SS, is transcriptionally regulated by sigma factor 54 (RpoN), YhbH, and HrpS. In this study, the role of integration host factor (IHF) in regulating hrpL and T3SS gene expression was investigated. IHF is a nucleoid-associated protein that regulates gene expression by influencing nucleoid structure and DNA bending. Our results showed that both ihfA and ihfB mutants of E. amylovora did not induce necrotic lesions on pear fruits. Growth of both mutants was greatly reduced, and expression of the hrpL and T3SS genes was significantly down-regulated as compared with those of the wild type. In addition, expression of the ihfA, but not the ihfB gene, was under auto-suppression by IHF. Furthermore, both ihfA and ihfB mutants were hypermotile, due to significantly reduced expression of small RNA (sRNA) rsmB. Electrophoresis mobility shift assay further confirmed that IHF binds to the promoters of the hrpL and ihfA genes, as well as the rsmB sRNA gene. These results indicate that IHF is required for RpoN-dependent hrpL gene expression and virulence, and controls motility by positively regulating the rsmB sRNA in E. amylovora.

Capillary electrophoresis-chemiluminescence detection for carcino-embryonic antigen based on aptamer/graphene oxide structure.

PubMed

Zhou, Zi-Ming; Feng, Zhe; Zhou, Jun; Fang, Bi-Yun; Qi, Xiao-Xiao; Ma, Zhi-Ya; Liu, Bo; Zhao, Yuan-Di; Hu, Xue-Bin

2015-02-15

A new strategy is proposed for determination of carcino-embryonic antigen (CEA) based on aptamer/graphene oxide (Apt/GO) by capillary electrophoresis-chemiluminescence (CE-CL) detection system. CEA aptamer conjugated with horseradish peroxidase (HRP) firstly mixes with GO, and the CL will be quenched because the stack of HRP-Apt on GO leads to chemiluminescence resonance energy transfer (CRET). When CEA exists, the specific combination of HRP-Apt and CEA can form HRP-Apt-CEA complex, which dissociates from GO. Then, the CL catalyzed by HRP-Apt-CEA complex can be detected without any CRET, and the content of CEA can be estimated by the CL intensity. It has been proved that the interference issue resulted from free HRP-Apt is solved well by mixing GO firstly with HRP-Apt, which blocks the free HRP-Apt's CL signal due to CL quenching effect of GO; and the interference resulted from GO to CL is also solved by CE, then the sensitivity and accuracy can be greatly improved. Results also showed that the CL intensity had a linear relationship with the concentration of CEA in the range from 0.0654 to 6.54 ng/mL, and the limit of detection was approximately 4.8 pg/mL (S/N = 3). This proposed method with high specificity offers a new way for separation and determination of biomolecule, and has good potential in application of biochemistry and bioanalysis. Copyright © 2014 Elsevier B.V. All rights reserved.

THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

PubMed Central

Steinman, Ralph M.; Cohn, Zanvil A.

1972-01-01

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 106 cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-125I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-125I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response. PMID:4347251

Ultrasensitive colorimetric immunoassay for hCG detection based on dual catalysis of Au@Pt core-shell nanoparticle functionalized by horseradish peroxidase

NASA Astrophysics Data System (ADS)

Wang, Weiguo; Zou, Yake; Yan, Jinwu; Liu, Jing; Chen, Huixiong; Li, Shan; Zhang, Lei

2018-03-01

In this paper, an ultrasensitive colorimetric biosensor for human chorionic gonadotrophin (hCG) detection was designed from bottom-up method based on the dual catalysis of the horseradish peroxidase (HRP) and Au@Pt nanoparticles (NPs) relative to H2O2-TEM system. HRP and monoclonal mouse anti-hCG antibody (β-submit, mAb1) were co-immobilized onto the Au@Pt NP surface to improve catalytic efficiency and specificity, which formed a dual functionalized Au@Pt-HRP probe with the mean size of 42.8 nm (D50). The colorimetric immunoassay was developed for the hCG detection, and the Au@Pt-HRP probe featured a higher sensitivity in the concentration range of 0.4-12.8 IU L- 1 with a low limit of detection (LOD) of 0.1 IU L- 1 compared with the LODs of 0.8 IU L- 1 for BA-ELISA and of 2.0 IU L- 1 for Au@Pt, which indicated that the Au@Pt-HRP probe possessed higher catalytic efficiency with 2.8-fold increase over Au@Pt and 33.8-fold increase over HRP. Also, the Au@Pt-HRP probe exhibited good precision and reproducibility, high specificity and acceptable accuracy with CV being less than 15%. The dual functionalized Au@Pt-HRP probe as a type of signal amplified method was firstly applied in the colorimetric immunoassay for the hCG detection.

The special programme of research in human reproduction: forty years of activities to achieve reproductive health for all.

PubMed

Benagiano, Giuseppe; d'Arcangues, Catherine; Harris Requejo, Jennifer; Schafer, Alessandra; Say, Lale; Merialdi, Mario

2012-01-01

The Special Programme of Research in Human Reproduction (HRP), co-sponsored by the UNDP, UNFPA, WHO, and the World Bank, is celebrating 40 years of activities with an expansion of its mandate and new co-sponsors. When it began, in 1972, the main focus was on evaluating the acceptability, effectiveness, and safety of existing fertility-regulating methods, as well as developing new, improved modalities for family planning. In 1994, HRP not only made major contributions to the Plan of Action of the International Conference on Population and Development (ICPD); it also broadened its scope of work to include other aspects of health dealing with sexuality and reproduction, adding a specific perspective on gender issues and human rights. In 2002, HRP's mandate was once again broadened to include sexually transmitted infections and HIV/AIDS and in 2003 it was further expanded to research activities on preventing violence against women and its many dire health consequences. Today, the work of the Programme includes research on: the sexual and reproductive health of adolescents, women, and men; maternal and perinatal health; reproductive tract and sexually transmitted infections (including HIV/AIDS); family planning; infertility; unsafe abortion; sexual health; screening for cancer of the cervix in developing countries, and gender and reproductive rights. Additional activities by the Programme have included: fostering international cooperation in the field of human reproduction; the elaboration of WHO's first Global Reproductive Health Strategy; work leading to the inclusion of ICPD's goal 'reproductive health for all by 2015' into the Millennium Development Goal framework; the promotion of critical interagency statements on the public health, legal, and human rights implications of female genital mutilation and gender-biased sex selection. Finally, HRP has been involved in the creation of guidelines and tools, such as the 'Medical eligibility criteria for contraceptive use', the 'Global handbook for family planning providers', the 'Definition of core competencies in primary health care', and designing tools for operationalizing a human rights approach to sexual and reproductive health programmes. Copyright © 2012 S. Karger AG, Basel.

Crew Exploration Vehicle (CEV) (Orion) Occupant Protection

NASA Technical Reports Server (NTRS)

Currie-Gregg, Nancy J.; Gernhardt, Michael L.; Lawrence, Charles; Somers, Jeffrey T.

2016-01-01

Dr. Nancy J. Currie, of the NASA Engineering and Safety Center (NESC), Chief Engineer at Johnson Space Center (JSC), requested an assessment of the Crew Exploration Vehicle (CEV) occupant protection as a result of issues identified by the Constellation Program and Orion Project. The NESC, in collaboration with the Human Research Program (HRP), investigated new methods associated with occupant protection for the Crew Exploration Vehicle (CEV), known as Orion. The primary objective of this assessment was to investigate new methods associated with occupant protection for the CEV, known as Orion, that would ensure the design provided minimal risk to the crew during nominal and contingency landings in an acceptable set of environmental and spacecraft failure conditions. This documents contains the outcome of the NESC assessment. NASA/TM-2013-217380, "Application of the Brinkley Dynamic Response Criterion to Spacecraft Transient Dynamic Events." supercedes this document.

Molecular genetics of Erwinia amylovora involved in the development of fire blight.

PubMed

Oh, Chang-Sik; Beer, Steven V

2005-12-15

The bacterial plant pathogen, Erwinia amylovora, causes the devastating disease known as fire blight in some Rosaceous plants like apple, pear, quince, raspberry and several ornamentals. Knowledge of the factors affecting the development of fire blight has mushroomed in the last quarter century. On the molecular level, genes encoding a Hrp type III secretion system, genes encoding enzymes involved in synthesis of extracellular polysaccharides and genes facilitating the growth of E. amylovora in its host plants have been characterized. The Hrp pathogenicity island, delimited by genes suggesting horizontal gene transfer, is composed of four distinct regions, the hrp/hrc region, the HEE (Hrp effectors and elicitors) region, the HAE (Hrp-associated enzymes) region, and the IT (Island transfer) region. The Hrp pathogenicity island encodes a Hrp type III secretion system (TTSS), which delivers several proteins from bacteria to plant apoplasts or cytoplasm. E. amylovora produces two exopolysaccharides, amylovoran and levan, which cause the characteristic fire blight wilting symptom in host plants. In addition, other genes, and their encoded proteins, have been characterized as virulence factors of E. amylovora that encode enzymes facilitating sorbitol metabolism, proteolytic activity and iron harvesting. This review summarizes our understanding of the genes and gene products of E. amylovora that are involved in the development of the fire blight disease.

Reagentless biosensor based on layer-by-layer assembly of functional multiwall carbon nanotubes and enzyme-mediator biocomposite.

PubMed

Zhou, Xing-Hua; Xi, Feng-Na; Zhang, Yi-Ming; Lin, Xian-Fu

2011-06-01

A simple and controllable layer-by-layer (LBL) assembly method was proposed for the construction of reagentless biosensors based on electrostatic interaction between functional multiwall carbon nanotubes (MWNTs) and enzyme-mediator biocomposites. The carboxylated MWNTs were wrapped with polycations poly(allylamine hydrochloride) (PAH) and the resulting PAH-MWNTs were well dispersed and positively charged. As a water-soluble dye methylene blue (MB) could mix well with horseradish peroxidase (HRP) to form a biocompatible and negatively-charged HRP-MB biocomposite. A (PAH-MWNTs/HRP-MB)(n) bionanomultilayer was then prepared by electrostatic LBL assembly of PAH-MWNTs and HRP-MB on a polyelectrolyte precursor film-modified Au electrode. Due to the excellent biocompatibility of HRP-MB biocomposite and the uniform LBL assembly, the immobilized HRP could retain its natural bioactivity and MB could efficiently shuttle electrons between HRP and the electrode. The incorporation of MWNTs in the bionanomultilayer enhanced the surface coverage concentration of the electroactive enzyme and increased the catalytic current response of the electrode. The proposed biosensor displayed a fast response (2 s) to hydrogen peroxide with a low detection limit of 2.0×10⁻⁷ mol/L (S/N=3). This work provided a versatile platform in the further development of reagentless biosensors.

Automated Screening of Children With Obstructive Sleep Apnea Using Nocturnal Oximetry: An Alternative to Respiratory Polygraphy in Unattended Settings

PubMed Central

Álvarez, Daniel; Alonso-Álvarez, María L.; Gutiérrez-Tobal, Gonzalo C.; Crespo, Andrea; Kheirandish-Gozal, Leila; Hornero, Roberto; Gozal, David; Terán-Santos, Joaquín; Del Campo, Félix

2017-01-01

Study Objectives: Nocturnal oximetry has become known as a simple, readily available, and potentially useful diagnostic tool of childhood obstructive sleep apnea (OSA). However, at-home respiratory polygraphy (HRP) remains the preferred alternative to polysomnography (PSG) in unattended settings. The aim of this study was twofold: (1) to design and assess a novel methodology for pediatric OSA screening based on automated analysis of at-home oxyhemoglobin saturation (SpO2), and (2) to compare its diagnostic performance with HRP. Methods: SpO2 recordings were parameterized by means of time, frequency, and conventional oximetric measures. Logistic regression models were optimized using genetic algorithms (GAs) for three cutoffs for OSA: 1, 3, and 5 events/h. The diagnostic performance of logistic regression models, manual obstructive apnea-hypopnea index (OAHI) from HRP, and the conventional oxygen desaturation index ≥ 3% (ODI3) were assessed. Results: For a cutoff of 1 event/h, the optimal logistic regression model significantly outperformed both conventional HRP-derived ODI3 and OAHI: 85.5% accuracy (HRP 74.6%; ODI3 65.9%) and 0.97 area under the receiver operating characteristics curve (AUC) (HRP 0.78; ODI3 0.75) were reached. For a cutoff of 3 events/h, the logistic regression model achieved 83.4% accuracy (HRP 85.0%; ODI3 74.5%) and 0.96 AUC (HRP 0.93; ODI3 0.85) whereas using a cutoff of 5 events/h, oximetry reached 82.8% accuracy (HRP 85.1%; ODI3 76.7) and 0.97 AUC (HRP 0.95; ODI3 0.84). Conclusions: Automated analysis of at-home SpO2 recordings provide accurate detection of children with high pretest probability of OSA. Thus, unsupervised nocturnal oximetry may enable a simple and effective alternative to HRP and PSG in unattended settings. Citation: Álvarez D, Alonso-Álvarez ML, Gutiérrez-Tobal GC, Crespo A, Kheirandish-Gozal L, Hornero R, Gozal D, Terán-Santos J, Del Campo F. Automated screening of children with obstructive sleep apnea using nocturnal oximetry: an alternative to respiratory polygraphy in unattended settings. J Clin Sleep Med. 2017;13(5):693–702. PMID:28356177

Coordination of International Risk-Reduction Investigations by the Multilateral Human Research Panel for Exploration

NASA Technical Reports Server (NTRS)

Charles, John B.; Bogomolov, Valery V.

2015-01-01

Effective use of the unique capabilities of the International Space Station (ISS) for risk reduction on future deep space missions involves preliminary work in analog environments to identify and evaluate the most promising techniques, interventions and treatments. This entails a consolidated multinational approach to biomedical research both on ISS and in ground analogs. The Multilateral Human Research Panel for Exploration (MHRPE) was chartered by the five ISS partners to recommend the best combination of partner investigations on ISS for risk reduction in the relatively short time available for ISS utilization. MHRPE will also make recommendations to funding agencies for appropriate preparatory analog work. In 2011, NASA's Human Research Program (HRP) and the Institute of Biomedical Problems (IBMP) of the Russian Academy of Science, acting for MHRPE, developed a joint US-Russian biomedical program for the 2015 one-year ISS mission (1YM) of American and Russian crewmembers. This was to evaluate the possibilities for multilateral research on ISS. An overlapping list of 16 HRP, 9 IBMP, 3 Japanese, 3 European and 1 Canadian investigations were selected to address risk-reduction goals in 7 categories: Functional Performance, Behavioral Health, Visual Impairment, Metabolism, Physical Capacity, Microbial and Human Factors. MHRPE intends to build on this bilateral foundation to recommend more fully-integrated multilateral investigations on future ISS missions commencing after the 1YM. MHRPE has also endorsed an on-going program of coordinated research on 6-month, one-year and 6-week missions ISS expeditions that is now under consideration by ISS managers. Preparatory work for these missions will require coordinated and collaborative campaigns especially in the psychological and psychosocial areas using analog isolation facilities in Houston, Köln and Moscow, and possibly elsewhere. The multilateral Human Analogs research working group (HANA) is the focal point of those planning discussions, with MHRPE coordinating between the national programs and then supporting implementation on ISS. Experience gained during preparations for the 1YM has identified improvements in both American and Russian processes to enable well-integrated investigations on all subsequent ISS expeditions. Among those is that the greatest efficiency is to be gained with investigations that are fully integrated from their conception, with co-principal investigators, a consolidated proposal and integrated plans for crewmember time and other flight-related resources. Analog investigations preceding future ISS expeditions will employ these lessons in efficiency to evaluate the techniques and tools to be validated aboard ISS. In this way, the resources and capabilities of ISS can be applied most efficiently to solving the problems facing astronauts of all nations in missions deep into the solar system.

10 CFR 712.36 - Medical assessment process.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Medical assessment process. 712.36 Section 712.36 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Medical Standards § 712.36 Medical assessment process. (a) The Designated Physician, under the supervision of the SOMD, is responsible for the medical assessment of HRP...

10 CFR 712.36 - Medical assessment process.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Medical assessment process. 712.36 Section 712.36 Energy DEPARTMENT OF ENERGY HUMAN RELIABILITY PROGRAM Medical Standards § 712.36 Medical assessment process. (a) The Designated Physician, under the supervision of the SOMD, is responsible for the medical assessment of HRP...

The strategic approach to contraceptive introduction.

PubMed

Simmons, R; Hall, P; Díaz, J; Díaz, M; Fajans, P; Satia, J

1997-06-01

The introduction of new contraceptive technologies has great potential for expanding contraceptive choice, but in practice, benefits have not always materialized as new methods have been added to public-sector programs. In response to lessons from the past, the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction (HRP) has taken major steps to develop a new approach and to support governments interested in its implementation. After reviewing previous experience with contraceptive introduction, the article outlines the strategic approach and discusses lessons from eight countries. This new approach shifts attention from promotion of a particular technology to an emphasis on the method mix, the capacity to provide services with quality of care, reproductive choice, and users' perspectives and needs. It also suggests that technology choice should be undertaken through a participatory process that begins with an assessment of the need for contraceptive introduction and is followed by research and policy and program development. Initial results from Bolivia, Brazil, Burkina Faso, Chile, Myanmar, South Africa, Vietnam, and Zambia confirm the value of the new approach.

The hrpZ Gene of Pseudomonas syringae pv. phaseolicola Enhances Resistance to Rhizomania Disease in Transgenic Nicotiana benthamiana and Sugar Beet

PubMed Central

Pavli, Ourania I.; Kelaidi, Georgia I.; Tampakaki, Anastasia P.; Skaracis, George N.

2011-01-01

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZPsph). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZPsph was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZPsph showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZPsph developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed. PMID:21394206

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet.

PubMed

Pavli, Ourania I; Kelaidi, Georgia I; Tampakaki, Anastasia P; Skaracis, George N

2011-03-04

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.

Eicosapentaenoic acid to arachidonic acid (EPA/AA) ratio as an associated factor of high risk plaque on coronary computed tomography in patients without coronary artery disease.

PubMed

Nagahara, Yasuomi; Motoyama, Sadako; Sarai, Masayoshi; Ito, Hajime; Kawai, Hideki; Takakuwa, Yoko; Miyagi, Meiko; Shibata, Daisuke; Takahashi, Hiroshi; Naruse, Hiroyuki; Ishii, Junichi; Ozaki, Yukio

2016-07-01

Coronary computed tomography angiography (CCTA)-verified high risk plaque (HRP) characteristics including positive remodeling and low attenuation plaque have been associated with acute coronary syndromes. Several studies reported that the n-3 polyunsaturated fatty acids have been associated with cardiovascular events. However, the relationship between serum eicosapentaenoic acid to arachidonic acid (EPA/AA) ratio and CCTA-verified HRP in patients without known coronary artery disease (CAD) is unclear. We aimed at investigating the relation between EPA/AA and CCTA-verified HRP in patients without known CAD. We included 193 patients undergoing CCTA without known CAD (65.5 ± 12.0 years, 55.0% male). No patient has been treated with EPA. The relation of coronary risk factors, lipid profile, high-sensitivity C-reactive protein, coronary artery calcification score (CACS), number of vessel disease, plaque burden, and EPA/AA with the presence of HRP was evaluated by logistic regression analysis. Incremental value of EPA/AA to predict HRP was also analyzed by C-index, NRI, and IDI. A Cox proportional hazards model was used to estimate the time to cardiovascular event. HRP was observed in 37 (19%) patients. Multivariable logistic regression analysis revealed that current smoking (OR 2.58; p=0.046), number of vessel disease (OR 1.87; p=0.031), and EPA/AA ratio (OR 0.65; p=0.0006) were independent associated factors of HRP on CCTA. Although the addition of EPA/AA to the baseline model did not significantly improve C-index, both NRI (0.60, p=0.0049) and IDI (0.054, p=0.0072) were significantly improved. Patients with HRP had significantly higher rate of events compared with patients without HRP (14% vs. 3%, Logrank p=0.0004). On multivariable Cox hazard analysis, baseline EPA/AA ratio was an independent predictor (HR 0.57, p=0.047). Low EPA/AA was an associated factor of HRP on CCTA in patients without CAD. In addition to conventional coronary risk factors and CACS, EPA/AA and CCTA might be useful for risk stratification of CAD. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Conformation and activity alteration of horseradish peroxidase induced by the interaction with gene carrier polyethyleneimines

NASA Astrophysics Data System (ADS)

Huang, Aimin; Wei, Bangzhi; Mo, Junyong; Wang, Yajing; Ma, Lin

2018-01-01

Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carriers. However the molecular basis of the cytotoxicity of PEI is poorly understood. Little is known about the effects of PEI on the structure and functions of biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism spectroscopy were conducted to investigate the influence of PEI of average molecular weight 25, 10 and 1.8 kDa (denoted as PEI25k, PEI10k and PEI1.8k) on the conformation of horseradish peroxidase (HRP) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the mechanism of the interaction between PEIs and HRP. PEIs were found to bind onto the surface of HRP predominantly via hydrophobic interaction and hydrogen bond or van der Waals interaction. The complex formation between HRP and PEI induced a more compact conformation of the enzyme and an increased hydrophobicity of the microenvironment surrounding heme pocket. The conformational change of HRP had little impact on the affinity towards H2O2 and phenol. However, the increase in the non-planarity of porphyrin ring in the heme group led to an increase in the exposure degree of the active center and thus an enhancement of catalytic efficiency of HRP in the presence of high molecular weight PEIs (PEI25k and PEI10k). The polymer size played an important role in PEI-HRP interaction. PEI of low molecular weight (PEI1.8k) was less efficient to alter the conformation and catalytic activity of HRP in aqueous solutions.

Expressed Glycosylphosphatidylinositol-Anchored Horseradish Peroxidase Identifies Co-Clustering Molecules in Individual Lipid Raft Domains

PubMed Central

Miyagawa-Yamaguchi, Arisa; Kotani, Norihiro; Honke, Koichi

2014-01-01

Lipid rafts that are enriched in glycosylphosphatidylinositol (GPI)-anchored proteins serve as a platform for important biological events. To elucidate the molecular mechanisms of these events, identification of co-clustering molecules in individual raft domains is required. Here we describe an approach to this issue using the recently developed method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. GPI-anchored HRP fusion proteins (HRP-GPIs), in which the GPI attachment signals derived from human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these different HRP-GPIs had differences in glycosylation and localization and formed distinct clusters. This novel approach distinguished molecular clusters associated with individual GPI-anchored proteins, suggesting that it can identify co-clustering molecules in individual raft domains. PMID:24671047

Production and purification of the multifunctional enzyme horseradish peroxidase

PubMed Central

Spadiut, Oliver; Herwig, Christoph

2014-01-01

The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing. PMID:24683473

Oxidative degradation of alkylphenols by horseradish peroxidase.

PubMed

Sakuyama, Hisae; Endo, Yasushi; Fujimoto, Kenshiro; Hatana, Yasuhiko

2003-01-01

Alkylphenols such as bisphenol A (2,2-bis(4-hydroxyphenyl)propane; BPA), p-nonylphenol (p-NP), and p-octylphenol (p-OP) that are known as endocrine disrupters were oxidized by horseradish (Armoracia rusticana) peroxidase (HRP) with H2O2. The optimal pHs for BPA, p-NP, and p-OP were 8.0, 7.0, and 5.0, respectively. The optimal temperature for BPA was 20 degrees C. Although BPA was rapidly degraded by HRP, its degradation depended on the concentration of HRP. Most of the oxidation products of BPA were polymers, although some 4-isopropenylphenol was produced. When male Japanese medaka (Oryzias latipes) were exposed to BPA, vitellogenin in the blood increased. However, no increased vitellogenin was observed in medaka exposed to HRP-oxidized BPA. The enzymatic oxidation of BPA using HRP was able to eliminate its estrogen-like activity.

Mode of bindings of zinc oxide nanoparticles to myoglobin and horseradish peroxidase: A spectroscopic investigations

NASA Astrophysics Data System (ADS)

Mandal, Gopa; Bhattacharya, Sudeshna; Ganguly, Tapan

2011-07-01

The interactions between two heme proteins myoglobin (HMb) and horseradish peroxidase (HRP) with zinc oxide (ZnO) nanoparticles are investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR, atomic force microscopy (AFM) and circular dichroism (CD) techniques under physiological condition of pH˜7.4. The presence of mainly static mode in fluorescence quenching mechanism of HMb and HRP by ZnO nanoparticle indicates the possibility of formation of ground state complex. The processes of bindings of ZnO nanoparticles with the two proteins are spontaneous molecular interaction procedures. In both cases hydrogen bonding plays a major role. The circular dichroism (CD) spectra reveal that a helicity of the proteins is reduced by increasing ZnO nanoparticle concentration although the α-helical structures of HMb and HRP retain their identity. On binding to the ZnO nanoparticles the secondary structure of HRP molecules (or HMb molecules) remains unchanged while there is a substantial change in the environment of the tyrosin active site in case of HRP molecules and tryptophan active site in case of HMb molecules. Tapping mode atomic force microscopy (AFM) was applied for the investigation the structure of HRP adsorbed in the environment of nanoparticles on the silicon and on the bare silicon. HRP molecules adsorb and aggregate on the mica with ZnO nanoparticle. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed on the bare silicon wafer. The adsorption of HRP in the environment of ZnO nanoparticle changes drastically the domains due to a strong interaction between HRP and ZnO nanoparticles. Similar situation is observed in case of HMb molecules. These findings demonstrate the efficacy of biomedical applications of ZnO nanoparticles as well as in elucidating their mechanisms of action as drugs in both human and plant systems.

Study on Enhancement Principle and Stabilization for the Luminol-H2O2-HRP Chemiluminescence System

PubMed Central

Yang, Lihua; Jin, Maojun; Du, Pengfei; Chen, Ge; Zhang, Chan; Wang, Jian; Jin, Fen; Shao, Hua; She, Yongxin; Wang, Shanshan; Zheng, Lufei; Wang, Jing

2015-01-01

A luminol-H2O2-HRP chemiluminescence system with high relative luminescent intensity (RLU) and long stabilization time was investigated. First, the comparative study on the enhancement effect of ten compounds as enhancers to the luminol-H2O2-HRP chemiluminescence system was carried out, and the results showed that 4-(imidazol-1-yl)phenol (4-IMP), 4-iodophenol (4-IOP), 4-bromophenol (4-BOP) and 4-hydroxy-4’-iodobiphenyl (HIOP) had the best performance. Based on the experiment, the four enhancers were dissolved in acetone, acetonitrile, methanol, and dimethylformamide (DMF) with various concentrations, the results indicated that 4-IMP, 4-IOP, 4-BOP and HIOP dissolved in DMF with the concentrations of 0.2%, 3.2%, 1.6% and 3.2% could get the highest RLU values. Subsequently, the influences of pH, ionic strength, HRP, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol on the stabilization of the luminol-H2O2-HRP chemiluminescence system were studied, and we found that pH value, ionic strength, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol have little influence on luminescent stabilization, while HRP has a great influence. In different ranges of HRP concentration, different enhancers should be selected. When the concentration is within the range of 0~6 ng/mL, 4-IMP should be selected. When the concentration of HRP ranges from 6 to 25ng/mL, 4-IOP was the best choice. And when the concentration is within the range of 25~80 ng/mL, HIOP should be selected as the enhancer. Finally, the three well-performing chemiluminescent enhanced solutions (CESs) have been further optimized according to the three enhancers (4-IMP, 4-IOP and HIOP) in their utilized HRP concentration ranges. PMID:26154162

Different effect of handle region peptide on β-cell function in different sexes of rats neonatally treated with sodium L-glutamate.

PubMed

Wu, Yi-xi; Sun, Ru-qiong; Yin, Guo-shu; Xu, Dong-chuan; Wang, Ping; Lin, Kun; Lin, Chu-jia; Lin, Shao-da

2015-03-17

BACKGROUND The (pro)renin receptor ((P)RR) was reported to be expressed in various tissues including the pancreas, and handle region peptide (HRP) is believed to block the function of (P)RR. This study aimed to investigate the effect of HRP on the glucose tolerance status and β-cell function of female rats, neonatally treated with sodium L-glutamate (MSG) and to compare with the previously reported HRP effect on male rats. MATERIAL AND METHODS Female MSG rats aged 8 weeks were divided into MSG control group and HRP treated group and the normal SD rats served as control. The MSG rats were treated with HRP by osmotic minipumps with dose of 1 mg/kg per day for total 28 days. Glucose tolerance status was evaluated at the end of the study. Islets α-cell and β-cell were marked with insulin antibody and glucagon antibody respectively. The proliferation of islet cells and expression of subunit of NADPH oxidase P22phox were marked by PCNA and P22phox antibody. Picrosirius red staining was performed for evaluating fibrosis of islets. RESULTS HRP improved the glucose status tolerance with decreasing α-cell mass, islets PCNA-positive cells, expression of P22phox and picrosirius red stained areas, and increasing β-cell mass in female MSG rats. The indexes with obviously interacted effect of sexes and HRP for the MSG rats were the AUC of blood glucose concentration (P<0.01), α-cell mass (P<0.05), proliferation of islet cells (P<0.01) and area of picrosirius red staining (P<0.01). CONCLUSIONS HRP improved the glucose tolerance status in the females although it was previously reported to worsen the glucose tolerance in male MSG rats. Different levels of sex hormones may partly account for the disparate effects observed for HRP in different sexes.

Different Effect of Handle Region Peptide on β-Cell Function in Different Sexes of Rats Neonatally Treated with Sodium L-Glutamate

PubMed Central

Wu, Yi-xi; Sun, Ru-qiong; Yin, Guo-shu; Xu, Dong-chuan; Wang, Ping; Lin, Kun; Lin, Chu-jia; Lin, Shao-da

2015-01-01

Background The (pro)renin receptor ((P)RR) was reported to be expressed in various tissues including the pancreas, and handle region peptide (HRP) is believed to block the function of (P)RR. This study aimed to investigate the effect of HRP on the glucose tolerance status and β-cell function of female rats, neonatally treated with sodium L-glutamate (MSG) and to compare with the previously reported HRP effect on male rats. Material/Methods Female MSG rats aged 8 weeks were divided into MSG control group and HRP treated group and the normal SD rats served as control. The MSG rats were treated with HRP by osmotic minipumps with dose of 1 mg/kg per day for total 28 days. Glucose tolerance status was evaluated at the end of the study. Islets α-cell and β-cell were marked with insulin antibody and glucagon antibody respectively. The proliferation of islet cells and expression of subunit of NADPH oxidase P22phox were marked by PCNA and P22phox antibody. Picrosirius red staining was performed for evaluating fibrosis of islets. Results HRP improved the glucose status tolerance with decreasing α-cell mass, islets PCNA-positive cells, expression of P22phox and picrosirius red stained areas, and increasing β-cell mass in female MSG rats. The indexes with obviously interacted effect of sexes and HRP for the MSG rats were the AUC of blood glucose concentration (P<0.01), α-cell mass (P<0.05), proliferation of islet cells (P<0.01) and area of picrosirius red staining (P<0.01). Conclusions HRP improved the glucose tolerance status in the females although it was previously reported to worsen the glucose tolerance in male MSG rats. Different levels of sex hormones may partly account for the disparate effects observed for HRP in different sexes. PMID:25783768

Sorting of endocytosed transferrin and asialoglycoprotein occurs immediately after internalization in HepG2 cells

PubMed Central

1987-01-01

After receptor-mediated uptake, asialoglycoproteins are routed to lysosomes, while transferrin is returned to the medium as apotransferrin. This sorting process was analyzed using 3,3'- diaminobenzidine (DAB) cytochemistry, followed by Percoll density gradient cell fractionation. A conjugate of asialoorosomucoid (ASOR) and horseradish peroxidase (HRP) was used as a ligand for the asialoglycoprotein receptor. Cells were incubated at 0 degree C in the presence of both 131I-transferrin and 125I-ASOR/HRP. Endocytosis of prebound 125I-ASOR/HRP and 131I-transferrin was monitored by cell fractionation on Percoll density gradients. Incubation of the cell homogenate in the presence of DAB and H2O2 before cell fractionation gave rise to a density shift of 125I-ASOR/HRP-containing vesicles due to HRP-catalyzed DAB polymerization. An identical change in density for 125I-transferrin and 125I-ASOR/HRP, induced by DAB cytochemistry, is taken as evidence for the concomitant presence of both ligands in the same compartment. At 37 degrees C, sorting of the two ligands occurred with a half-time of approximately 2 min, and was nearly completed within 10 min. The 125I-ASOR/HRP-induced shift of 131I-transferrin was completely dependent on the receptor-mediated uptake of 125I-ASOR/HRP in the same compartment. In the presence of a weak base (0.3 mM primaquine), the recycling of transferrin receptors was blocked. The cell surface transferrin receptor population was decreased within 6 min to 15% of its original size. DAB cytochemistry showed that sorting between endocytosed 131I-transferrin and 125I-ASOR/HRP was also blocked in the presence of primaquine. These results indicate that transferrin and asialoglycoprotein are taken up via the same compartments and that segregation of the transferrin-receptor complex and asialoglycoprotein occurs very efficiently soon after uptake. PMID:3032986

Study on Enhancement Principle and Stabilization for the Luminol-H2O2-HRP Chemiluminescence System.

PubMed

Yang, Lihua; Jin, Maojun; Du, Pengfei; Chen, Ge; Zhang, Chan; Wang, Jian; Jin, Fen; Shao, Hua; She, Yongxin; Wang, Shanshan; Zheng, Lufei; Wang, Jing

2015-01-01

A luminol-H2O2-HRP chemiluminescence system with high relative luminescent intensity (RLU) and long stabilization time was investigated. First, the comparative study on the enhancement effect of ten compounds as enhancers to the luminol-H2O2-HRP chemiluminescence system was carried out, and the results showed that 4-(imidazol-1-yl)phenol (4-IMP), 4-iodophenol (4-IOP), 4-bromophenol (4-BOP) and 4-hydroxy-4'-iodobiphenyl (HIOP) had the best performance. Based on the experiment, the four enhancers were dissolved in acetone, acetonitrile, methanol, and dimethylformamide (DMF) with various concentrations, the results indicated that 4-IMP, 4-IOP, 4-BOP and HIOP dissolved in DMF with the concentrations of 0.2%, 3.2%, 1.6% and 3.2% could get the highest RLU values. Subsequently, the influences of pH, ionic strength, HRP, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol on the stabilization of the luminol-H2O2-HRP chemiluminescence system were studied, and we found that pH value, ionic strength, 4-IMP, 4-IOP, 4-BOP, HIOP, H2O2 and luminol have little influence on luminescent stabilization, while HRP has a great influence. In different ranges of HRP concentration, different enhancers should be selected. When the concentration is within the range of 0~6 ng/mL, 4-IMP should be selected. When the concentration of HRP ranges from 6 to 25 ng/mL, 4-IOP was the best choice. And when the concentration is within the range of 25~80 ng/mL, HIOP should be selected as the enhancer. Finally, the three well-performing chemiluminescent enhanced solutions (CESs) have been further optimized according to the three enhancers (4-IMP, 4-IOP and HIOP) in their utilized HRP concentration ranges.

Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

NASA Astrophysics Data System (ADS)

Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

2012-08-01

Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections.

PubMed

Das, Smita; Jang, Ihn Kyung; Barney, Becky; Peck, Roger; Rek, John C; Arinaitwe, Emmanuel; Adrama, Harriet; Murphy, Maxwell; Imwong, Mallika; Ling, Clare L; Proux, Stephane; Haohankhunnatham, Warat; Rist, Melissa; Seilie, Annette M; Hanron, Amelia; Daza, Glenda; Chang, Ming; Nakamura, Tomoka; Kalnoky, Michael; Labarre, Paul; Murphy, Sean C; McCarthy, James S; Nosten, Francois; Greenhouse, Bryan; Allauzen, Sophie; Domingo, Gonzalo J

2017-11-01

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum -induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections

PubMed Central

Das, Smita; Jang, Ihn Kyung; Barney, Becky; Peck, Roger; Rek, John C.; Arinaitwe, Emmanuel; Adrama, Harriet; Murphy, Maxwell; Imwong, Mallika; Ling, Clare L.; Proux, Stephane; Haohankhunnatham, Warat; Rist, Melissa; Seilie, Annette M.; Hanron, Amelia; Daza, Glenda; Chang, Ming; Nakamura, Tomoka; Kalnoky, Michael; Labarre, Paul; Murphy, Sean C.; McCarthy, James S.; Nosten, Francois; Greenhouse, Bryan; Allauzen, Sophie; Domingo, Gonzalo J.

2017-01-01

Abstract. Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies. PMID:28820709

Monitoring fetal maturation—objectives, techniques and indices of autonomic function*

PubMed Central

Hoyer, Dirk; Żebrowski, Jan; Cysarz, Dirk; Gonçalves, Hernâni; Pytlik, Adelina; Amorim-Costa, Célia; Bernardes, João; Ayres-de-Campos, Diogo; Witte, Otto W; Schleußner, Ekkehard; Stroux, Lisa; Redman, Christopher; Georgieva, Antoniya; Payne, Stephen; Clifford, Gari; Signorini, Maria G; Magenes, Giovanni; Andreotti, Fernando; Malberg, Hagen; Zaunseder, Sebastian; Lakhno, Igor; Schneider, Uwe

2017-01-01

Objective Monitoring the fetal behavior does not only have implications for acute care but also for identifying developmental disturbances that burden the entire later life. The concept, of ‘fetal programming’, also known as ‘developmental origins of adult disease hypothesis’, e.g. applies for cardiovascular, metabolic, hyperkinetic, cognitive disorders. Since the autonomic nervous system is involved in all of those systems, cardiac autonomic control may provide relevant functional diagnostic and prognostic information. Approach The fetal heart rate patterns (HRP) are one of the few functional signals in the prenatal period that relate to autonomic control and, therefore, is key to fetal autonomic assessment. The development of sensitive markers of fetal maturation and its disturbances requires the consideration of physiological fundamentals, recording technology and HRP parameters of autonomic control. Main Results Based on the ESGCO2016 special session on monitoring the fetal maturation we herein report the most recent results on: (i) functional fetal autonomic brain age score (fABAS), Recurrence Quantitative Analysis and Binary Symbolic Dynamics of complex HRP resolve specific maturation periods, (ii) magnetocardiography (MCG) based fABAS was validated for cardiotocography (CTG), (iii) 30 min recordings are sufficient for obtaining episodes of high variability, important for intrauterine growth restriction (IUGR) detection in handheld Doppler, (iv) novel parameters from PRSA to identify Intra IUGR fetuses, (v) evaluation of fetal electrocardiographic (ECG) recordings, (vi) correlation between maternal and fetal HRV is disturbed in pre-eclampsia. Significance The reported novel developments significantly extend the possibilities for the established CTG methodology. Novel HRP indices improve the accuracy of assessment due to their more appropriate consideration of complex autonomic processes across the recording technologies (CTG, handheld Doppler, MCG, ECG). The ultimate objective is their dissemination into routine practice and studies of fetal developmental disturbances with implications for programming of adult diseases. PMID:28186000

A Drosophila receptor tyrosine phosphatase expressed in the embryonic CNS and larval optic lobes is a member of the set of proteins bearing the "HRP" carbohydrate epitope.

PubMed

Desai, C J; Popova, E; Zinn, K

1994-12-01

Recent studies have defined several cell surface glycoproteins expressed in the developing nervous system of insect embryos that may be involved in axon outgrowth and guidance processes. These glycoproteins include the fasciclins and a group of receptor-linked protein tyrosine phosphatases (R-PTPs). In embryos, the fasciclins are localized to axonal subsets, while the R-PTPs appear to be expressed on most or all CNS axons. To identify other neuronal cell surface glycoproteins in the Drosophila embryo, we have taken a biochemical approach. This is based on the observation that antisera against horseradish peroxidase (HRP) recognize a carbohydrate epitope that is selectively expressed in the insect nervous system. A large number of neuronal glycoproteins (denoted "HRP proteins") apparently bear the HRP carbohydrate epitope. We have used polyclonal anti-HRP antibodies to purify these proteins from Drosophila embryos, and have obtained protein sequences from seven HRP protein bands. These data define three major HRP proteins as neurotactin, fasciclin I, and an R-PTP, DPTP69D. Western blotting data suggest that fasciclin II, neuroglian, DPTP10D, and DPTP99A are also HRP proteins. We show that DPTP69D, like the previously characterized R-PTPs, is localized to CNS axons in the embryo. In third instar larvae, DPTP69D expression is restricted to subsets of neuronal processes in the brain, ventral nerve cord, and eye disk. In the optic lobes, DPTP69D is localized to the neuropils of the lamina and medulla, and to an array of parallel thick bundles that may be the transmedullary fibers of the developing lobula complex.

Humans vs Hardware: The Unique World of NASA Human System Risk Assessment

NASA Technical Reports Server (NTRS)

Anton, W.; Havenhill, M.; Overton, Eric

2016-01-01

Understanding spaceflight risks to crew health and performance is a crucial aspect of preparing for exploration missions in the future. The research activities of the Human Research Program (HRP) provide substantial evidence to support most risk reduction work. The Human System Risk Board (HSRB), acting on behalf of the Office of Chief Health and Medical Officer (OCHMO), assesses these risks and assigns likelihood and consequence ratings to track progress. Unfortunately, many traditional approaches in risk assessment such as those used in the engineering aspects of spaceflight are difficult to apply to human system risks. This presentation discusses the unique aspects of risk assessment from the human system risk perspective and how these limitations are accommodated and addressed in order to ensure that reasonable inputs are provided to support the OCHMO's overall risk posture for manned exploration missions.

2014 Sensorimotor Risk Standing Review Panel

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

The Sensorimotor Risk Standing Review Panel (from here on referred to as the SRP) met on December 17 - 18, 2014 in Houston, TX to review the current status of the Risk of Impaired Control of Spacecraft, Associated Systems and Immediate Vehicle Egress due to Vestibular/Sensorimotor Alteration Associated with Space Flight (Sensorimotor Risk) in the Integrated Research Plan (IRP). During the meeting, the SRP received an in-depth briefing of the current status of the Sensorimotor Risk from Dr. Jacob Bloomberg, the Human Research Program (HRP) Sensorimotor Discipline Lead Scientist and Dr. Millard Reschke, the Chief Scientist of the Neuroscience Laboratories at the NASA Johnson Space Center (JSC). The SRP was impressed with the information that Dr. Bloomberg and Dr. Reschke presented and think that the in-person meeting (instead of WebEx/teleconference) allowed for more interactive and thoughtful conversations.

Modifying sulfomethylated alkali lignin by horseradish peroxidase to improve the dispersibility and conductivity of polyaniline

NASA Astrophysics Data System (ADS)

Yang, Dongjie; Huang, Wenjing; Qiu, Xueqing; Lou, Hongming; Qian, Yong

2017-12-01

Pine and wheat straw alkali lignin (PAL and WAL) were sulfomethylated to improve water solubility, polymerized with horseradish peroxidase (HRP) to improve the molecular weight (Mw) and applied to dope and disperse polyaniline (PANI). The structural effect of lignin from different origins on the reactivities of sulfomethylation and HRP polymerization was investigated. The results show that WAL with less methoxyl groups and lower Mw have higher reactivity in sulfomethylation (SWAL). More phenolic hydroxyl groups and lower Mw benefit the HRP polymerization of sulfomethylated PAL (SPAL). Due to the natural three-dimensional aromatic structure and introduced sulfonic groups, SPAL and SWAL could effectively dope and disperse PANI in water by π-π stacking and electrostatic interaction. HRP modified SPAL (HRP-SPAL) with much higher sulfonation degree and larger Mw significantly increased the conductivity and dispersibility of lignin/PANI composites.

Techno-economic analysis of horseradish peroxidase production using a transient expression system in Nicotiana benthamiana.

PubMed

Walwyn, David Richard; Huddy, Suzanne M; Rybicki, Edward P

2015-01-01

Despite the advantages of plant-based transient expression systems relative to microbial or mammalian cell systems, the commercial production of recombinant proteins using plants has not yet been achieved to any significant extent. One of the challenges has been the lack of published data on the costs of manufacture for products other than biopharmaceuticals. In this study, we report on the techno-economic analysis of the production of a standard commercial enzyme, namely, horseradish peroxidase (HRP), using a transient expression system in Nicotiana benthamiana. Based on the proven plant yield of 240 mg HRP/kg biomass, a biomass productivity of 15-kg biomass/m(2)/year and a process yield of 54 % (mg HRP product/mg HRP in biomass), it is apparent that HRP can be manufactured economically via transient expression in plants in a large-scale facility (>5 kg HRP/year). At this level, the process is competitive versus the existing technology (extraction of the enzyme from horseradish), and the product is of comparable or improved activity, containing only the preferred isoenzyme C. Production scale, protein yield and biomass productivity are found to be the most important determinants of overall viability.

What Contributes Most to High Health Care Costs? Health Care Spending in High Resource Patients.

PubMed

Pritchard, Daryl; Petrilla, Allison; Hallinan, Shawn; Taylor, Donald H; Schabert, Vernon F; Dubois, Robert W

2016-02-01

U.S. health care spending nearly doubled in the decade from 2000-2010. Although the pace of increase has moderated recently, the rate of growth of health care costs is expected to be higher than the growth in the economy for the near future. Previous studies have estimated that 5% of patients account for half of all health care costs, while the top 1% of spenders account for over 27% of costs. The distribution of health care expenditures by type of service and the prevalence of particular health conditions for these patients is not clear, and is likely to differ from the overall population. To examine health care spending patterns and what contributes to costs for the top 5% of managed health care users based on total expenditures. This retrospective observational study employed a large administrative claims database analysis of health care claims of managed care enrollees across the full age and care spectrum. Direct health care expenditures were compared during calendar year 2011 by place of service (outpatient, inpatient, and pharmacy), payer type (commercially insured, Medicare Advantage, and Medicaid managed care), and therapy area between the full population and high resource patients (HRP). The mean total expenditure per HRP during calendar year 2011 was $43,104 versus $3,955 per patient for the full population. Treatment of back disorders and osteoarthritis contributed the largest share of expenditures in both HRP and the full study population, while chronic renal failure, heart disease, and some oncology treatments accounted for disproportionately higher expenditures in HRP. The share of overall expenditures attributed to inpatient services was significantly higher for HRP (40.0%) compared with the full population (24.6%), while the share of expenditures attributed to pharmacy (HRP = 18.1%, full = 21.4%) and outpatient services (HRP = 41.9%, full = 54.1%) was reduced. This pattern was observed across payer type. While the use of physician-administered pharmaceuticals was slightly higher in HRP, their use did not alter this spending pattern. Overall, expenditures in the HRP population are more than 10-fold higher compared with the full population. Managed care pharmacy can benefit from understanding what contributes to these higher costs, and managed care directors should consider an appropriately balanced assessment of the share of total spend by service and therapeutic category in HRP when devising drug usage and related cost-management strategies.

Crew Exploration Vehicle (CEV) (Orion) Occupant Protection. Part 1; Appendices

NASA Technical Reports Server (NTRS)

Currie-Gregg, Nancy J.; Gernhardt, Michael L.; Lawrence, Charles; Somers, Jeffrey T.

2016-01-01

Dr. Nancy J. Currie, of the NASA Engineering and Safety Center (NESC), Chief Engineer at Johnson Space Center (JSC), requested an assessment of the Crew Exploration Vehicle (CEV) occupant protection as a result of issues identified by the Constellation Program and Orion Project. The NESC, in collaboration with the Human Research Program (HRP), investigated new methods associated with occupant protection for the Crew Exploration Vehicle (CEV), known as Orion. The primary objective of this assessment was to investigate new methods associated with occupant protection for the CEV, known as Orion, that would ensure the design provided minimal risk to the crew during nominal and contingency landings in an acceptable set of environmental and spacecraft failure conditions. This documents contains the appendices to the NESC assessment report. NASA/TM-2013-217380, Application of the Brinkley Dynamic Response Criterion to Spacecraft Transient Dynamic Events supersedes this document.

Coronary Plaque Characterization in Psoriasis Reveals High-Risk Features That Improve After Treatment in a Prospective Observational Study.

PubMed

Lerman, Joseph B; Joshi, Aditya A; Chaturvedi, Abhishek; Aberra, Tsion M; Dey, Amit K; Rodante, Justin A; Salahuddin, Taufiq; Chung, Jonathan H; Rana, Anshuma; Teague, Heather L; Wu, Jashin J; Playford, Martin P; Lockshin, Benjamin A; Chen, Marcus Y; Sandfort, Veit; Bluemke, David A; Mehta, Nehal N

2017-07-18

Psoriasis, a chronic inflammatory disease associated with an accelerated risk of myocardial infarction, provides an ideal human model to study inflammatory atherogenesis in vivo. We hypothesized that the increased cardiovascular risk observed in psoriasis would be partially attributable to an elevated subclinical coronary artery disease burden composed of noncalcified plaques with high-risk features. However, inadequate efforts have been made to directly measure coronary artery disease in this vulnerable population. As such, we sought to compare total coronary plaque burden and noncalcified coronary plaque burden (NCB) and high-risk plaque (HRP) prevalence between patients with psoriasis (n=105), patients with hyperlipidemia eligible for statin therapy under National Cholesterol Education Program-Adult Treatment Panel III guidelines (n=100) who were ≈10 years older, and healthy volunteers without psoriasis (n=25). Patients underwent coronary computed-tomography angiography for total coronary plaque burden and NCB quantification and HRP identification, defined as low attenuation (<30 hounsfield units), positive remodeling (>1.10), and spotty calcification. A consecutive sample of the first 50 patients with psoriasis was scanned again 1 year after therapy. Despite being younger and at lower traditional risk than patients with hyperlipidemia, patients with psoriasis had increased NCB (mean±SD: 1.18±0.33 versus 1.11±0.32, P =0.02) and similar HRP prevalence ( P =0.58). Furthermore, compared to healthy volunteers, patients with psoriasis had increased total coronary plaque burden (1.22±0.31 versus 1.04±0.22, P =0.001), NCB (1.18±0.33 versus 1.03±0.21, P =0.004), and HRP prevalence beyond traditional risk (odds ratio, 6.0; 95% confidence interval, 1.1-31.7; P =0.03). Last, among patients with psoriasis followed for 1 year, improvement in psoriasis severity was associated with improvement in total coronary plaque burden (β=0.45, 0.23-0.67; P <0.001) and NCB (β=0.53, 0.32-0.74; P <0.001) beyond traditional risk factors. Patients with psoriasis had greater NCB and increased HRP prevalence than healthy volunteers. In addition, patients with psoriasis had elevated NCB and equivalent HRP prevalence as older patients with hyperlipidemia. Last, modulation of target organ inflammation (eg, skin) was associated with an improvement in NCB at 1 year, suggesting that control of remote sites of inflammation may translate into reduced coronary artery disease risk. © 2017 American Heart Association, Inc.

Iodide oxidation and iodine reduction mediated by horseradish peroxidase in the presence of ethylenediaminetetraacetic acid (EDTA): the superoxide effect.

PubMed

Chang, H C; Bumpus, J A

2001-04-01

Ethylenediaminetetraacetic acid (EDTA) is an inhibitor of iodide (I-) oxidation that is catalyzed by horseradish peroxidase (HRP). HRP-mediated iodine (I2) reduction and triiodide (I3+) disappearance occur in the presence of this inhibitor. It is interesting that in the presence of EDTA, HRP produces superoxide radical, a reactive oxygen species that is required for iodine reduction. Substitution of potassium superoxide (KO2) or a biochemical superoxide generating system (xanthine/xanthine oxidase) for HRP and H2O2 in the reaction mixture also can reduce iodine to iodide. Thus, iodine reduction mediated by HRP occurs because HRP is able to mediate the formation of superoxide in the presence of EDTA and H2O2. Although superoxide is able to mediate iodine reduction directly, other competing reactions appear to be more important. For example, high concentrations (mM range) of EDTA are required for efficient iodine reduction in this system. Under such conditions, the concentration (microM range) of contaminating EDTA-Fe(III) becomes catalytically important. In the presence of superoxide, EDTA-Fe(III) is reduced to EDTA-Fe(II), which is able to reduce iodine and form triiodide rapidly. Also of importance is the fact that EDTA-Fe(II) reacts with hydrogen peroxide to form hydroxyl radical. Hydroxyl radical involvement is supported by the fact that a wide variety of hydroxyl radical (OH) scavengers can inhibit HRP dependent iodine reduction in the presence of EDTA and hydrogen peroxide.

Sequential Actions of Rab5 and Rab7 Regulate Endocytosis in the Xenopus Oocyte

PubMed Central

Mukhopadhyay, Amitabha; Barbieri, Alejandro M.; Funato, Kouichi; Roberts, Richard; Stahl, Philip D.

1997-01-01

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes. PMID:9087439

Development of Methodologies, Metrics, and Tools for Investigating Human-Robot Interaction in Space Robotics

NASA Technical Reports Server (NTRS)

Ezer, Neta; Zumbado, Jennifer Rochlis; Sandor, Aniko; Boyer, Jennifer

2011-01-01

Human-robot systems are expected to have a central role in future space exploration missions that extend beyond low-earth orbit [1]. As part of a directed research project funded by NASA s Human Research Program (HRP), researchers at the Johnson Space Center have started to use a variety of techniques, including literature reviews, case studies, knowledge capture, field studies, and experiments to understand critical human-robot interaction (HRI) variables for current and future systems. Activities accomplished to date include observations of the International Space Station s Special Purpose Dexterous Manipulator (SPDM), Robonaut, and Space Exploration Vehicle (SEV), as well as interviews with robotics trainers, robot operators, and developers of gesture interfaces. A survey of methods and metrics used in HRI was completed to identify those most applicable to space robotics. These methods and metrics included techniques and tools associated with task performance, the quantification of human-robot interactions and communication, usability, human workload, and situation awareness. The need for more research in areas such as natural interfaces, compensations for loss of signal and poor video quality, psycho-physiological feedback, and common HRI testbeds were identified. The initial findings from these activities and planned future research are discussed. Human-robot systems are expected to have a central role in future space exploration missions that extend beyond low-earth orbit [1]. As part of a directed research project funded by NASA s Human Research Program (HRP), researchers at the Johnson Space Center have started to use a variety of techniques, including literature reviews, case studies, knowledge capture, field studies, and experiments to understand critical human-robot interaction (HRI) variables for current and future systems. Activities accomplished to date include observations of the International Space Station s Special Purpose Dexterous Manipulator (SPDM), Robonaut, and Space Exploration Vehicle (SEV), as well as interviews with robotics trainers, robot operators, and developers of gesture interfaces. A survey of methods and metrics used in HRI was completed to identify those most applicable to space robotics. These methods and metrics included techniques and tools associated with task performance, the quantification of human-robot interactions and communication, usability, human workload, and situation awareness. The need for more research in areas such as natural interfaces, compensations for loss of signal and poor video quality, psycho-physiological feedback, and common HRI testbeds were identified. The initial findings from these activities and planned future research are discussed.

ExMC Work Prioritization Process

NASA Technical Reports Server (NTRS)

Simon, Matthew

2015-01-01

Last year, NASA's Human Research Program (HRP) introduced the concept of a "Path to Risk Reduction" (PRR), which will provide a roadmap that shows how the work being done within each HRP element can be mapped to reducing or closing exploration risks. Efforts are currently underway within the Exploration Medical Capability (ExMC) Element to develop a structured, repeatable process for prioritizing work utilizing decision analysis techniques and risk estimation tools. The goal of this effort is to ensure that the work done within the element maximizes risk reduction for future exploration missions in a quantifiable way and better aligns with the intent and content of the Path to Risk Reduction. The Integrated Medical Model (IMM) will be used to identify those conditions that are major contributors of medical risk for a given design reference mission. For each of these conditions, potential prevention, screening, diagnosis, and treatment methods will be identified. ExMC will then aim to prioritize its potential investments in these mitigation methods based upon their potential for risk reduction and other factors such as vehicle performance impacts, near term schedule needs, duplication with external efforts, and cost. This presentation will describe the process developed to perform this prioritization and inform investment discussions in future element planning efforts. It will also provide an overview of the required input information, types of process participants, figures of merit, and the expected outputs of the process.

Protective effects of sea buckthorn polysaccharide extracts against LPS/d-GalN-induced acute liver failure in mice via suppressing TLR4-NF-κB signaling.

PubMed

Liu, Huan; Zhang, Wei; Dong, Shichao; Song, Liang; Zhao, Shimin; Wu, Chunyan; Wang, Xue; Liu, Fang; Xie, Jiming; Wang, Jinling; Wang, Yuzhen

2015-12-24

Sea buckthorn (Hippophae rhamnoides L.) berries have been traditionally used to treat gastric disorders, cardiovascular problems, and liver injuries in oriental medicinal system. This study aimed to explore the protective effects and mechanisms of the polysaccharide extracts of Sea buckthorn (HRP) berries against lipopolysaccharide (LPS) and d-galactosamine hydrochloride (d-GalN)-induced acute liver failure in mice. HRP was isolated by hot-water extraction and characterized by HPLC and infrared spectrum analysis. The total carbohydrate, uronic acid and protein contents of HRP were measured by a spectrophotometric method. Mice were orally administrated with HRP (50, 100, 200mg/kg) once daily for 14 consecutive days prior to the challenge with LPS (50 μg/kg) and d-GalN (300 mg/kg). Animals of positive control group were intraperitoneally injected with dexamethasone (10mg/kg). Mice were sacrificed at 8h after LPS/d-GalN injection. Pretreatment with HRP significantly inhibited LPS/d-GalN-induced increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, which were accompanied by alleviated liver injuries and reduced production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). HRP was also found to reduce malondialdehyde (MDA) content and to restore superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities. Furthermore, HRP supplementation dose-dependently inhibited the expression of Toll-like receptor 4 (TLR4), phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated mitogen activated protein kinase 38 (p-p38 MAPK) in the liver of LPS/d-GalN challenged mice. Pretreatment with HRP also inhibited LPS/d-GalN-induced activation and translocation of nuclear factor-κB (NF-κB). This study indicates that pretreatment with HRP protects against LPS/d-GalN-induced liver injury in mice via suppressing the TLR4-NF-κB signaling pathway. Sea buckthorn may be a hopeful drug for prevention of acute live injury. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Improving the Specificity of Plasmodium falciparum Malaria Diagnosis in High-Transmission Settings with a Two-Step Rapid Diagnostic Test and Microscopy Algorithm.

PubMed

Murungi, Moses; Fulton, Travis; Reyes, Raquel; Matte, Michael; Ntaro, Moses; Mulogo, Edgar; Nyehangane, Dan; Juliano, Jonathan J; Siedner, Mark J; Boum, Yap; Boyce, Ross M

2017-05-01

Poor specificity may negatively impact rapid diagnostic test (RDT)-based diagnostic strategies for malaria. We performed real-time PCR on a subset of subjects who had undergone diagnostic testing with a multiple-antigen (histidine-rich protein 2 and pan -lactate dehydrogenase pLDH [HRP2/pLDH]) RDT and microscopy. We determined the sensitivity and specificity of the RDT in comparison to results of PCR for the detection of Plasmodium falciparum malaria. We developed and evaluated a two-step algorithm utilizing the multiple-antigen RDT to screen patients, followed by confirmatory microscopy for those individuals with HRP2-positive (HRP2 + )/pLDH-negative (pLDH - ) results. In total, dried blood spots (DBS) were collected from 276 individuals. There were 124 (44.9%) individuals with an HRP2 + /pLDH + result, 94 (34.1%) with an HRP2 + /pLDH - result, and 58 (21%) with a negative RDT result. The sensitivity and specificity of the RDT compared to results with real-time PCR were 99.4% (95% confidence interval [CI], 95.9 to 100.0%) and 46.7% (95% CI, 37.7 to 55.9%), respectively. Of the 94 HRP2 + /pLDH - results, only 32 (34.0%) and 35 (37.2%) were positive by microscopy and PCR, respectively. The sensitivity and specificity of the two-step algorithm compared to results with real-time PCR were 95.5% (95% CI, 90.5 to 98.0%) and 91.0% (95% CI, 84.1 to 95.2), respectively. HRP2 antigen bands demonstrated poor specificity for the diagnosis of malaria compared to that of real-time PCR in a high-transmission setting. The most likely explanation for this finding is the persistence of HRP2 antigenemia following treatment of an acute infection. The two-step diagnostic algorithm utilizing microscopy as a confirmatory test for indeterminate HRP2 + /pLDH - results showed significantly improved specificity with little loss of sensitivity in a high-transmission setting. Copyright © 2017 American Society for Microbiology.

Performance of an ultra-sensitive Plasmodium falciparum HRP2-based rapid diagnostic test with recombinant HRP2, culture parasites, and archived whole blood samples.

PubMed

Das, Smita; Peck, Roger B; Barney, Rebecca; Jang, Ihn Kyung; Kahn, Maria; Zhu, Meilin; Domingo, Gonzalo J

2018-03-17

As malaria endemic countries shift from control to elimination, the proportion of low density Plasmodium falciparum infections increases. Current field diagnostic tools, such as microscopy and rapid diagnostic tests (RDT), with detection limits of approximately 100-200 parasites/µL (p/µL) and 800-1000 pg/mL histidine-rich protein 2 (HRP2), respectively, are unable to detect these infections. A novel ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT) was evaluated in laboratory conditions to define the test's performance against recombinant HRP2 and native cultured parasites. The uRDT detected dilutions of P. falciparum recombinant GST-W2 and FliS-W2, as well as cultured W2 and ITG, diluted in whole blood down to 10-40 pg/mL HRP2, depending on the protein tested. uRDT specificity was 100% against 123 archived frozen whole blood samples. Rapid test cross-reactivity with HRP3 was investigated using pfhrp2 gene deletion strains D10 and Dd2, pfhrp3 gene deletion strain HB3, and controls pfhrp2 and pfhrp3 double deletion strain 3BD5 and pfhrp2 and pfhrp3 competent strain ITG. The commercial Standard Diagnostics, Inc. BIOLINE Malaria Ag P.f RDT (SD-RDT) and uRDT detected pfhrp2 positive strains down to 49 and 3.13 p/µL, respectively. The pfhrp2 deletion strains were detected down to 98 p/µL by both tests. The performance of the uRDT was variable depending on the protein, but overall showed a greater than 10-fold improvement over the SD-RDT. The uRDT also exhibited excellent specificity and showed the same cross-reactivity with HRP3 as the SD-RDT. Together, the results support the uRDT as a more sensitive HRP2 test that could be a potentially effective tool in elimination campaigns. Further clinical evaluations for this purpose are merited.

Persistent ICT Malaria P.f/P.v Panmalarial and HRP2 Antigen Reactivity after Treatment of Plasmodium falciparum Malaria Is Associated with Gametocytemia and Results in False-Positive Diagnoses of Plasmodium vivax in Convalescence

PubMed Central

Tjitra, Emiliana; Suprianto, Sri; McBroom, James; Currie, Bart J.; Anstey, Nicholas M.

2001-01-01

A problem with rapid Plasmodium falciparum-specific antigen histidine-rich protein 2 (HRP2) detection tests for malaria is the persistence of antigen in blood after the disappearance of asexual-stage parasitemia and clinical symptoms, resulting in false-positive (FP) test results following treatment. The ICT P.f/P.v immunochromatographic test detects both HRP2 and a panmalarial antigen (PMA) found in both P. falciparum and Plasmodium vivax. To examine posttreatment antigen persistence with this test and whether persistent sexual-stage forms (gametocytes) are a cause of FP tests after treatment, we compared serial antigen test results with microscopy results from patients symptomatic with P. falciparum malaria in Indonesia for 28 days following treatment with chloroquine (CQ; n = 66), sulfadoxine-pyrimethamine (SP; n = 36), and artesunate plus sulfadoxine-pyrimethamine (ART + SP; n = 15). Persistent FP antigenemia following SP treatment occurred in 29% (HRP2) and 42% (PMA) of the patients on day 7 and in 10% (HRP2) and 23% (PMA) on day 14. The high rates of persistent HRP2 and PMA antigenemia following CQ and SP treatment were strongly associated with the presence of gametocytemia, with the proportion with gametocytes on day 7 posttreatment being significantly greater in those with FP results than in those with true-negative PMA and HRP2 results. Gametocyte frequency on day 14 post-SP treatment was also greater in those with FP PMA results. Following SP treatment, PMA persisted longer than HRP2, giving an FP diagnosis of P. vivax in up to 16% of patients on day 14, with all FP P. vivax diagnoses having gametocytemia. In contrast, PMA was rapidly cleared following ART + SP treatment in association with rapid clearance of gametocytemia. Gametocytes appear to be an important cause of persistent posttreatment panmalarial antigenemia in areas of endemicity and may also contribute in part to persistent HRP2 antigenemia following treatment. PMID:11230422

Childhood Forearm Breaks Resulting from Mild Trauma May Indicate Bone Deficits

MedlinePlus

... a powerful new technology called high-resolution peripheral quantitative computed tomography (HRpQCT), which, unlike DXA, can assess ... persist throughout life. The investigators concluded that additional research is needed to determine if childhood bone weakness ...

Semi-Autonomous Rodent Habitat for Deep Space Exploration

NASA Technical Reports Server (NTRS)

Alwood, J. S.; Shirazi-Fard, Y.; Pletcher, D.; Globus, R.

2018-01-01

NASA has flown animals to space as part of trailblazing missions and to understand the biological responses to spaceflight. Mice traveled in the Lunar Module with the Apollo 17 astronauts and now mice are frequent research subjects in LEO on the ISS. The ISS rodent missions have focused on unravelling biological mechanisms, better understanding risks to astronaut health, and testing candidate countermeasures. A critical barrier for longer-duration animal missions is the need for humans-in-the-loop to perform animal husbandry and perform routine tasks during a mission. Using autonomous or telerobotic systems to alleviate some of these tasks would enable longer-duration missions to be performed at the Deep Space Gateway. Rodent missions performed using the Gateway as a platform could address a number of critical risks identified by the Human Research Program (HRP), as well as Space Biology Program questions identified by NRC Decadal Survey on Biological and Physical Sciences in Space, (2011). HRP risk areas of potentially greatest relevance that the Gateway rodent missions can address include those related to visual impairment (VIIP) and radiation risks to central nervous system, cardiovascular disease, as well as countermeasure testing. Space Biology focus areas addressed by the Gateway rodent missions include mechanisms and combinatorial effects of microgravity and radiation. The objectives of the work proposed here are to 1) develop capability for semi-autonomous rodent research in cis-lunar orbit, 2) conduct key experiments for testing countermeasures against low gravity and space radiation. The hardware and operations system developed will enable experiments at least one month in duration, which potentially could be extended to one year in duration. To gain novel insights into the health risks to crew of deep space travel (i.e., exposure to space radiation), results obtained from Gateway flight rodents can be compared to ground control groups and separate groups of mice exposed to simulated Galactic Cosmic Radiation (at the NASA Space Radiation Lab). Results can then be compared to identical experiments conducted on the ISS. Together results from Gateway, ground-based, and ISS rodent experiments will provide novel insight into the effects of space radiation.

New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA.

PubMed

Arakawa, Hidetoshi; Nakabayashi, Shigeo; Ohno, Ken-Ichi; Maeda, Masako

2012-04-01

Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10 -18 to 1.0×10 -15  mol/assay, with a detection limit of 1.0×10 -18  mol/assay. The coefficient of variation (CV, n =8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

Hooking horseradish peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization

NASA Astrophysics Data System (ADS)

Peng, Ye; Ling-Ling, Hu; Yu-Zhi, Du; Yong-Juan, Xu; Hua-Gang, Ni; Cong, Chen; Xiao-Lin, Lu; Xiao-Jun, Huang

2017-05-01

A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.

Molecular Engineering of Self-assembled Nanoreactors

DTIC Science & Technology

2014-08-15

substrate diffusion. We demonstrated spatial control of the GOx/HRP cascade organized by DNA origami structures. As shown in Figure 13, the...quantify the level of protein assembly on the DNA origami tiles - assembled enzymes exhibited higher surface landscapes than the underlying origami ... origami tiles with assembled Gox/HRP pairs with inter-enzyme distances ranging from 10 nm to 65 nm. GOx/HRP co-assembly yields were determined from AFM

HRP2 determines the efficiency and specificity of HIV-1 integration in LEDGF/p75 knockout cells but does not contribute to the antiviral activity of a potent LEDGF/p75-binding site integrase inhibitor.

PubMed

Wang, Hao; Jurado, Kellie A; Wu, Xiaolin; Shun, Ming-Chieh; Li, Xiang; Ferris, Andrea L; Smith, Steven J; Patel, Pratiq A; Fuchs, James R; Cherepanov, Peter; Kvaratskhelia, Mamuka; Hughes, Stephen H; Engelman, Alan

2012-12-01

The binding of integrase (IN) to lens epithelium-derived growth factor (LEDGF)/p75 in large part determines the efficiency and specificity of HIV-1 integration. However, a significant residual preference for integration into active genes persists in Psip1 (the gene that encodes for LEDGF/p75) knockout (KO) cells. One other cellular protein, HRP2, harbors both the PWWP and IN-binding domains that are important for LEDGF/p75 co-factor function. To assess the role of HRP2 in HIV-1 integration, cells generated from Hdgfrp2 (the gene that encodes for HRP2) and Psip1/Hdgfrp2 KO mice were infected alongside matched control cells. HRP2 depleted cells supported normal infection, while disruption of Hdgfrp2 in Psip1 KO cells yielded additional defects in the efficiency and specificity of integration. These deficits were largely restored by ectopic expression of either LEDGF/p75 or HRP2. The double-KO cells nevertheless supported residual integration into genes, indicating that IN and/or other host factors contribute to integration specificity in the absence of LEDGF/p75 and HRP2. Psip1 KO significantly increased the potency of an allosteric inhibitor that binds the LEDGF/p75 binding site on IN, a result that was not significantly altered by Hdgfrp2 disruption. These findings help to rule out the host factor-IN interactions as the primary antiviral targets of LEDGF/p75-binding site IN inhibitors.

Complexes of horseradish peroxidase with formate, acetate, and carbon monoxide.

PubMed

Carlsson, Gunilla H; Nicholls, Peter; Svistunenko, Dimitri; Berglund, Gunnar I; Hajdu, Janos

2005-01-18

Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the "hydrated" form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP-acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463-468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron-ligand distances of 2.3 and 1.8 A, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 A from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

Subchronic mild noise stress increases HRP permeability in rat small intestine in vitro.

PubMed

Bijlsma, P B; van Raaij, M T; Dobbe, C J; Timmerman, A; Kiliaan, A J; Taminiau, J A; Groot, J A

2001-05-01

Recently we reported an increased trans- and paracellular protein permeability in rat small intestine after acute cold restraint stress. In the present study, we applied randomized 95- or 105-dB white noise pulses during 45 min/h, 12 h/day, duration 8 days, as a milder, but more chronic stressor to male rats. At 8 days before the noise experiments, 50% of the animals were cannulated in the vena cava for blood sampling during the experimental period. The other 50% of the animals were sacrificed at Day 9, segments of ileum were mounted in Ussing chambers and perfused at 37 degrees C. Horseradish peroxidase (HRP) was added mucosally, serosal appearance was detected enzymatically and tissues were fixed for electron microscopy. In the animals exposed to 95-dB noise, plasma corticosterone levels were enhanced twofold compared to controls, and ileal HRP flux was enhanced twofold. Electron micrographs of tissue from stressed or control animals showed no detectable paracellular staining of HRP. Quantification of HRP-containing endosomes in enterocytes revealed a twofold increase in endosome number in the animals exposed to 95-db noise indicating that the increased HRP permeability was primarily due to increased endocytosis. In contrast to the animals exposed to 95-dB noise, rats exposed to 105-dB noise showed no increase in corticosterone levels and ileal HRP fluxes were not significantly different from controls. We conclude that mild subchronic noise stress may cause a decrease in intestinal barrier function by increased transcytosis of luminal antigens.

Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

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

Yun, Hong Shik; Hong, Eun-Hee; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791

2013-09-27

Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotypemore » of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.« less

Structure of poly(ethylene glycol)-modified horseradish peroxidase in organic solvents: infrared amide I spectral changes upon protein dehydration are largely caused by protein structural changes and not by water removal per se.

PubMed Central

Al-Azzam, Wasfi; Pastrana, Emil A; Ferrer, Yancy; Huang, Qing; Schweitzer-Stenner, Reinhard; Griebenow, Kai

2002-01-01

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure. PMID:12496131

Diagnostic potential of monoclonal antibodies developed against C-terminal polypeptide of P. falciparum Histidine Rich Protein2 (PfHRP2) in malaria infected patients from India.

PubMed

Verma, Reena; Chandy, Sara; Jayaprakash, N S; Manoharan, Anand; Vijayalakshmi, M A; Venkataraman, Krishnan

2017-09-01

Malaria, caused by Plasmodium falciparum has become a major health burden in most tropical and developing countries. P. falciparum Histidine Rich Protein2 (PfHRP2), which exhibits polymorphism, is being widely used as a diagnostic marker. Recently, we reported the development of monoclonal antibodies against conserved C-terminal 105 amino acids of PfHRP2 for malaria diagnosis. Now, in this study, the diagnostic performance of two anti-C-terminal PfHRP2 mAbs (b10c1 and Aa3c10) were evaluated with 100 blood samples from clinically identified malaria patients from seven different geographical centers in India. Sandwich ELISA, polymerase chain reaction (PCR) and statistical tools were used for the evaluation of the performance of the anti-C-terminal PfHRP2 mAb. These mAbs detected P. falciparum (mean OD value 1.525 ± 0.56) malaria with great accuracy with no cross reactivity with P. Plasmodium vivax (mean OD value 0.285 ± 0.051) and normal healthy control samples (mean OD value 0.185 ± 0.06) in Sandwich ELISA assay. The samples which were RDT negative for P. falciparum were also reactive in Sandwich ELISA with mean OD value of (1.303 ± 0.532). The amount of PfHRP2 antigen in the patients' blood sample was quantified and categorized into three distinct groups having the HRP2 antigen in high, intermediate and low amounts. The presence of Pfhrp2 gene was also confirmed by PCR analysis. The sensitivity and specificity of the mAb were found to be 95 and 96% respectively. These data strongly suggest that the anti-C-terminal PfHRP2 mAbs b10c1 and Aa3c10 have merits for improvising the existing malarial diagnostics.

HRP Integrated Research Plan Analysis

NASA Technical Reports Server (NTRS)

Elliott, Todd

2009-01-01

The charts, that are the totality of this document, presents tasks, duration of the tasks, the start and finish of the tasks, and subtasks. Also presented are PERT charts that display the beginning, external milestones, and end points for the tasks, and sub tasks.

Integrated Medical Model Overview

NASA Technical Reports Server (NTRS)

Myers, J.; Boley, L.; Foy, M.; Goodenow, D.; Griffin, D.; Keenan, A.; Kerstman, E.; Melton, S.; McGuire, K.; Saile, L.;

Behavioral Health and Performance (BHP) Standing Review Panel (SRP) Final Report

NASA Technical Reports Server (NTRS)

Suedfeld, Peter; Bootzin, Richard; Harvey, Allison; Leon, Gloria; Musson, David; Oltmanns, Thomas; Paulus, Martin

2010-01-01

The first meeting of the Behavioral Health and Performance (BHP) Standing Review Panel (SRP) was held in Houston, TX on November 1-3, 2009. Our task was to assess the Integrated Research Plan (IRP) related to the fields covered in the SRP charge (see section VIII). Having considered and discussed the extensive materials distributed prior to the meeting, and the highly informative briefings by the NASA BHP Human Research Program (HRP) personnel during the site visit, the SRP agreed that the IRP is comprehensive and was developed carefully. Question and answer periods with the presenters were particularly productive and helpful to the SRP. The presenters' willingness to share information and positive responses to the SRP's suggestions were greatly appreciated. Although the IRP and related documents are impressive, the SRP does have a number of recommendations regarding both the overall plan and its component parts.

NASA Human Research Wiki - An Online Collaboration Tool

NASA Technical Reports Server (NTRS)

Barr, Y. R.; Rasbury, J.; Johnson, J.; Barsten, K.; Saile, L.; Watkins, S. D.

2011-01-01

In preparation for exploration-class missions, the Exploration Medical Capability (ExMC) element of NASA's Human Research Program (HRP) has compiled a large evidence base, which previously was available only to persons within the NASA community. The evidence base is comprised of several types of data, for example: information on more than 80 medical conditions which could occur during space flight, derived from several sources (including data on incidence and potential outcomes of these medical conditions, as captured in the Integrated Medical Model's Clinical Finding Forms). In addition, approximately 35 gap reports are included in the evidence base, identifying current understanding of the medical challenges for exploration, as well as any gaps in knowledge and/or technology that would need to be addressed in order to provide adequate medical support for these novel missions. In an effort to make the ExMC information available to the general public and increase collaboration with subject matter experts within and outside of NASA, ExMC has developed an online collaboration tool, very similar to a wiki, titled the NASA Human Research Wiki. The platform chosen for this data sharing, and the potential collaboration it could generate, is a MediaWiki-based application that would house the evidence, allow "read only" access to all visitors to the website, and editorial access to credentialed subject matter experts who have been approved by the Wiki's editorial board. Although traditional wikis allow users to edit information in real time, the NASA Human Research Wiki includes a peer review process to ensure quality and validity of information. The wiki is also intended to be a pathfinder project for other HRP elements that may want to use this type of web-based tool. The wiki website will be released with a subset of the data described and will continue to be populated throughout the year.

Lessons learned from the use of HRP-2 based rapid diagnostic test in community-wide screening and treatment of asymptomatic carriers of Plasmodium falciparum in Burkina Faso.

PubMed

Tiono, Alfred B; Ouédraogo, Alphonse; Diarra, Amidou; Coulibaly, Sam; Soulama, Issiaka; Konaté, Amadou T; Barry, Aïssata; Mukhopadhyay, Amitava; Sirima, Sodiomon B; Hamed, Kamal

2014-01-27

Rapid diagnostic tests (RDTs) are immune chromatographic tests targeting antigens of one or more Plasmodium species and offer the potential to extend accurate malaria diagnosis in endemic areas. In this study, the performance of Plasmodium falciparum-specific histidine-rich protein-2 (PfHRP-2) RDT in the detection of asymptomatic carriers from a hyperendemic region of Burkina Faso was compared with microscopy to gain further insight on its relevance in community-based interventions. The performance of HRP-2 test was evaluated in terms of sensitivity, specificity, positive and negative predictive values, discordant values, likelihood ratios, accuracy, and precision using microscopy as the 'gold standard'. This analysis was carried out in a controlled, parallel, cluster-randomized (18 clusters; 1:1) study in children and adults. The effect of systematic treatment of P. falciparum asymptomatic carriers during three consecutive monthly community screening campaigns on the incidence of symptomatic malaria episodes over a 12-month period was compared with no treatment of asymptomatic carriers. Sensitivity of HRP-2 test in asymptomatic carriers was higher in campaign 1 (92.4%) when compared to campaign 2 (84.0%) and campaign 3 (77.8%). The sensitivity of HRP-2 test increased as parasite density increased across all the age groups. Highest sensitivity (≥97.0%) was recorded at parasite densities of 1,000-4,999/μl, except for children aged 10 to 14 years. The specificity of HRP-2 test was comparable across age groups and highest in campaign 3 (95.9%). The negative predictive values were high across the three campaigns (≥92.7%) while the positive predictive values ranged from 23.2 to 73.8%. False-positive and false-negative rates were high in campaign 1 and campaign 3, respectively. The performance of HRP-2 test in detecting asymptomatic carriers of P. falciparum varied by age and parasite density. Although the use of HRP-2 test is beneficial for the diagnosis of acute malaria, its low sensitivity in screening asymptomatic carriers may limit its utility in pre-elimination interventional settings. The use of a practical and more sensitive test such as loop-mediated isothermal amplification in combination with a cost effective HRP-2 test may be worth exploring in such settings.

Glyoxalase I is critical for human retinal capillary pericyte survival under hyperglycemic conditions.

PubMed

Miller, Antonia G; Smith, Dawn G; Bhat, Manjunatha; Nagaraj, Ram H

2006-04-28

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

Evaluation of lophine derivatives as L-012 (luminol analog)-dependent chemiluminescence enhancers for measuring horseradish peroxidase and H2O2.

PubMed

Ichibangase, T; Ohba, Y; Kishikawa, N; Nakashima, K; Kuroda, N

2014-03-01

8-Amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was recently synthesized as a new chemiluminescence (CL) probe; the light intensity and the sensitivity of L-012 are higher than those of other CL probes such as luminol. Previously, our group developed four lophine-based CL enhancers of the horseradish peroxidase (HRP)-catalyzed CL oxidation of luminol, namely 2-(4-hydroxyphenyl)-4,5-diphenylimidazole (HDI), 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole (HPI), 4-(4,5-diphenyl-1H-imidazol-2-yl)phenylboronic acid (DPA), and 4-[4,5-di(2-pyridyl)-1H-imidazol-2-yl]phenylboronic acid (DPPA), and showed that DPPA was suitable for the photographic detection of HRP. In this study, we replaced luminol with L-012 and evaluated these as L-012-dependent CL enhancers. In addition, to detect HRP and/or H2O2 with higher sensitivity, each detection condition for the L-012-HRP-H2O2 enhanced CL was optimized. All the derivatives enhanced the L-012-dependent CL as well as luminol CL; HPI generated the highest enhanced luminescence. Under optimized conditions for HRP detection, the detection limit of HRP was 0.08 fmol. By contrast, the detection limit of HRP with the enhanced L-012-dependent CL using 4-iodophenol, which is a common enhancer of luminol CL, was 1.1 fmol. With regard to H2O2 detection, the detection limits for enhanced CL with HPI and 4-iodophenol were 0.29 and 1.5 pmol, respectively. Therefore, it is demonstrated that HPI is the most superior L-012-dependent CL enhancer. Copyright © 2013 John Wiley & Sons, Ltd.

PopF1 and PopF2, Two Proteins Secreted by the Type III Protein Secretion System of Ralstonia solanacearum, Are Translocators Belonging to the HrpF/NopX Family†

PubMed Central

Meyer, Damien; Cunnac, Sébastien; Guéneron, Mareva; Declercq, Céline; Van Gijsegem, Frédérique; Lauber, Emmanuelle; Boucher, Christian; Arlat, Matthieu

2006-01-01

Ralstonia solanacearum GMI1000 is a gram-negative plant pathogen which contains an hrp gene cluster which codes for a type III protein secretion system (TTSS). We identified two novel Hrp-secreted proteins, called PopF1 and PopF2, which display similarity to one another and to putative TTSS translocators, HrpF and NopX, from Xanthomonas spp. and rhizobia, respectively. They also show similarities with TTSS translocators of the YopB family from animal-pathogenic bacteria. Both popF1 and popF2 belong to the HrpB regulon and are required for the interaction with plants, but PopF1 seems to play a more important role in virulence and hypersensitive response (HR) elicitation than PopF2 under our experimental conditions. PopF1 and PopF2 are not necessary for the secretion of effector proteins, but they are required for the translocation of AvrA avirulence protein into tobacco cells. We conclude that PopF1 and PopF2 are type III translocators belonging to the HrpF/NopX family. The hrpF gene of Xanthomonas campestris pv. campestris partially restored HR-inducing ability to popF1 popF2 mutants of R. solanacearum, suggesting that translocators of R. solanacearum and Xanthomonas are functionally conserved. Finally, R. solanacearum strain UW551, which does not belong to the same phylotype as GMI1000, also possesses two putative translocator proteins. However, although one of these proteins is clearly related to PopF1 and PopF2, the other seems to be different and related to NopX proteins, thus showing that translocators might be variable in R. solanacearum. PMID:16788199

Development of horseradish peroxidase-based cross-linked enzyme aggregates and their environmental exploitation for bioremediation purposes.

PubMed

Bilal, Muhammad; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2017-03-01

In the present study, horseradish peroxidase (HRP), in-house isolated crude cocktail enzyme, from Armoracia rusticana was cross-linked using a new type of cross-linking agent, i.e., ethylene glycol-bis [succinic acid N-hydroxysuccinimide, (EG-NHS)], which is mild in nature as compared to the glutaraldehyde (GA). The HRP-immobilized cross-linked enzyme aggregates (HRP-CLEAs) were developed using a wider range of EG-NHS and notably no adverse effect was observed. In a comparative evaluation, in the case of EG-NHS, a high-level stability in the residual activity was recorded, whereas a sharp decrease was observed in the case of glutaraldehyde. Following initial cross-linker evaluation, the HRP-CLEAs were tested to investigate their bio-catalytic efficacy for bioremediation purposes using a newly developed packed bed reactor system (PBRS). A maximal of 94.26% degradation of textile-based methyl orange dye was recorded within the shortest time frame, following 91.73% degradation of basic red 9, 84.35% degradation of indigo, 81.47% degradation of Rhodamin B, and 73.6% degradation of Rhodamine 6G, respectively, under the same working environment. Notably, the HRP-CLEAs retained almost 60% of its original activity after methyl orange dye degradation in seven consecutive cycles using PBRS. Furthermore, after HRP-CLEAs-mediated treatment in the PBRS, a significant toxicity reduction in the dye samples was recorded as compared to their pristine counterparts. In conclusion, the results suggest that the newly developed HRP-CLEAs have a great potential for industrial exploitation, to tackle numerous industrial dye-based emergent pollutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Determination of optimum process parameters for peroxidase-catalysed treatment of bisphenol A and application to the removal of bisphenol derivatives.

PubMed

Yamada, Kazunori; Ikeda, Naoya; Takano, Yoko; Kashiwada, Ayumi; Matsuda, Kiyomi; Hirata, Mitsuo

2010-03-01

Systematic investigations were carried out to determine the optimum process parameters such as the hydrogen peroxide (H2O2) concentration, concentration and molar mass of poly(ethylene glycol) (PEG) as an additive, pH value, temperature and enzyme dose for treatment of bisphenol A (BPA) with horseradish peroxidase (HRP). The HRP-catalysed treatment of BPA was effectively enhanced by adding PEG, and BPA was completely converted into phenoxy radicals by HRP dose of 0.10 U/cm3. The optimum conditions for HRP-catalysed treatment of BPA at 0.3 mM was determined to be 0.3 mM for H2O2 and 0.10 mg/cm3 for PEG with a molar mass of 1.0 x 10(4) in a pH 6.0 buffer at 30 degrees C. Different kinds of bisphenol derivatives were completely or effectively treated by HRP under the optimum conditions determined for treatment of BPA, although the HRP dose was further increased as necessary for some of them. The aggregation of water-insoluble oligomers generated by the enzymatic radicalization and radical coupling reaction was enhanced by decreasing the pH values to 4.0 with HCl after the enzymatic treatment, and BPA and bisphenol derivatives were removed from aqueous solutions by filtering out the oligomer precipitates.

Improved biodegradation of synthetic azo dye by horseradish peroxidase cross-linked on nano-composite support.

PubMed

Sun, Huaiyan; Jin, Xinyu; Long, Nengbing; Zhang, Ruifeng

2017-02-01

A ZnO nanowires/macroporous SiO 2 composite was used as support to immobilize horseradish peroxidase (HRP) by in-situ cross-linking method. Using diethylene glycol diglycidyl ether (DDE) as a long-chained cross-linker, it was adsorbed on the surface of ZnO nanowires before reaction with HRPs, the resulted composite was quite different from the traditional cross-linking enzyme aggregates (CLEAs) on both structure and catalytic performance. The immobilized HRP showed high activity in the decolorization of azo dyes. The effect of various conditions such as the loading amount of HRP, solution pH, temperature, contact time and concentration of dye were optimized on the decolorization. The decolorization percentage of Acid Blue 113 and Acid black 10 BX reached as high as 95.4% and 90.3%, respectively. The immobilized HRP gave the highest decolorization rate under dye concentration as 50mg/L and reaction time of 35min. The immobilized HRP exhibited much better resistance to temperature and pH inactivation than free HRP. The storage stability and reusability were greatly improved through the immobilization, from the decolorization of Acid blue 113 it was found that 80.4% of initial efficiency retained after incubation at 4°C for 60 days, and that 79.4% of decolorization efficiency retained after 12 cycles reuse. Copyright © 2016 Elsevier B.V. All rights reserved.

False-negative rapid diagnostic tests for malaria and deletion of the histidine-rich repeat region of the hrp2 gene.

PubMed

Koita, Ousmane A; Doumbo, Ogobara K; Ouattara, Amed; Tall, Lalla K; Konaré, Aoua; Diakité, Mahamadou; Diallo, Mouctar; Sagara, Issaka; Masinde, Godfred L; Doumbo, Safiatou N; Dolo, Amagana; Tounkara, Anatole; Traoré, Issa; Krogstad, Donald J

2012-02-01

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.

Accuracy of Two Malaria Rapid Diagnostic Tests (RDTS) for Initial Diagnosis and Treatment Monitoring in a High Transmission Setting in Uganda

PubMed Central

Mbabazi, Phoebe; Hopkins, Heidi; Osilo, Emmanuel; Kalungu, Michael; Byakika-Kibwika, Pauline; Kamya, Moses R.

2015-01-01

Malaria rapid diagnostic tests (RDTs) may improve fever management in areas without microscopy. We compared the accuracy of histidine-rich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (pLDH)-based RDTs, using expert microscopy as a gold standard, for initial diagnosis, treatment monitoring, and diagnosis of recurrent malaria in a cohort of children followed longitudinally in a high-transmission area in Uganda. For 305 initial fever episodes, sensitivity was 98% for HRP2 and 87% for pLDH, whereas specificity was 55% and 96%, respectively. The HRP2 gave 51% false-positive results on Day 28, whereas pLDH gave no false positives after Day 7. For 59 recurrent fever episodes during follow-up, sensitivity was 100% for HRP2 and 91% for pLDH, whereas specificity was 33% and 100%, respectively. The HRP2-based RDTs are useful for initial diagnosis of malaria caused by superior sensitivity; however, as a result of superior specificity, pLDH-based RDTs are more appropriate to monitor treatment and diagnose recurrent malaria. PMID:25624399

Risk as a Driver for Innovation

NASA Technical Reports Server (NTRS)

Davis, Jeff; Fogarty, Jennifer; Perchonok, Michele; Zapp, Neal; Ferebee, Melvin; Edwards, J. Michelle

2010-01-01

The Space Life Sciences directorate (SLSD) and Human Research Program (HRP) at NASA Johnson Space Center has implemented a system for managing human systems risks. These risks are defined as the health and performance risks posed to crew during and after spaceflight. Identification and evaluation of these risks has led to the identification of gaps in knowledge about the risks as well as gaps in technology needed to mitigate them. Traditional routes of closing technology gaps have, in some cases, proven to be too slow when a solution was required quickly. Therefore, certain gaps were used to drive the development of "challenges" for the scientific community. Partnering with open innovation service providers such as InnoCentive and Yet2.com, SLSD and HRP have decreased the amount of time from identification of a need to the evaluation of a solution. Although not all proposed solutions will result in a risk mitigation strategy or tool, the process has allowed faster evaluation of proposed solutions providing the researcher the ability to move to another possible solution if the first does not sufficiently address the problem. Moreover, this process engages the community outside of NASA and broadens the population from which to draw solutions. In the traditional grant funding structure, only those in the specific field will apply for the grant. However, using open innovation, solutions can come from individuals in many different fields. This can expand the general view of a field (way of thinking within a field) and the application of solutions form new fields while providing a pathway for the acquisition of novel solutions or refinements of current mitigations. Identification of the human systems risks has helped drive the development and evaluation of innovative solutions as well as engaging a broader scientific audience in working with NASA.

Vision Hampton Roads : strategy committee minutes & attendance.

DOT National Transportation Integrated Search

2010-02-22

February 20, 2009 : HRP Executive Committee Meeting, Looking Back, Looking Forward : March 20, 2009 : HRP Board Meeting, Accolades & Action, Doing Things Jointly with Comprehensive : Economic Development Strategies : April 17,...

Differences between the endocytosis of horseradish peroxidase and its conjugate with wheat germ agglutinin by cultured fibroblasts.

PubMed

Stieber, A; Gonatas, J O; Gonatas, N K

1984-04-01

A covalent conjugate of wheat germ agglutinin (WGA) with horseradish peroxidase (HRP) was used for a morphologic study of its adsorptive endocytosis by cultured human fibroblasts. Initial binding at 4 degrees C of the conjugate was observed over the entire plasma membrane, including "coated" and smooth pits. Endocytosis of HRP and the WGA-HRP conjugate was observed in lysosomes, but only the conjugate was seen in a cisterna of the Golgi apparatus (GERL), and in adjacent coated vesicles.

10 CFR 712.16 - DOE security review.

Code of Federal Regulations, 2010 CFR

2010-01-01

... of the HRP process, the HRP certifying official must be notified and the security concern evaluated... part. (c) Any mental/personality disorder or behavioral issues found in a personnel security file...

Association between HRP-2/pLDH rapid diagnostic test band positivity and malaria-related anemia at a peripheral health facility in Western Uganda.

PubMed

Boyce, Ross; Reyes, Raquel; Ntaro, Moses; Mulogo, Edgar; Matte, Michael; Boum, Yap; Siedner, Mark J

2015-12-01

The detection of severe malaria in resource-constrained settings is often difficult because of requirements for laboratory infrastructure and/or clinical expertise. The aim of this study, therefore, was to explore the utility of a multiple antigen (HRP-2/pLDH) rapid diagnostic test (RDT) as a low-cost, surrogate marker of patients at high risk for complications of severe malaria. We reviewed programmatic data at a peripheral health center in Western Uganda. Available demographic and clinical data on all individuals presenting to the center who underwent an RDT for suspected malaria infection were reviewed. We fit logistic regression models to identify correlates of two outcomes of interest: 1) severe malaria-related anemia, defined here as hemoglobin ≤7g/dL and 2) receipt of parenteral quinine. 1509 patients underwent malaria testing with an SD FK60 RDT during the observation period. A total of 637 (42%) RDTs were positive for at least one species of malaria, of which 326 (51%) exhibited a single HRP-2 band and 307 (48%) exhibited both HRP-2 and pLDH bands, while 4 exhibited only a single pLDH band. There was a trend towards more severe anemia in patients with a HRP-2/pLDH positive RDT compared to a HRP-2 only RDT (β = -0.99 g/dl, 95% CI -1.99 to 0.02, P = 0.055). A HRP-2/pLDH positive RDT was associated with an increased risk of severe malaria-related anemia compared to a negative RDT (adjusted odds ratio (AOR) 18.8, 95% CI 4.32 to 82.0, P < 0.001) and to a HRP-2 only RDT (AOR 2.46, 95% CI 0.75 to 8.04, P = 0.14). There was no significant association between RDT result and the administration of parenteral quinine. These results offer preliminary evidence that specific patterns of antigen positivity on RDTs could be utilized to identify patients at an increased risk for complications of severe malaria.

Modeling for Integrated Science Management and Resilient Systems Development

NASA Technical Reports Server (NTRS)

Shelhamer, M.; Mindock, J.; Lumpkins, S.

2014-01-01

Many physiological, environmental, and operational risks exist for crewmembers during spaceflight. An understanding of these risks from an integrated perspective is required to provide effective and efficient mitigations during future exploration missions that typically have stringent limitations on resources available, such as mass, power, and crew time. The Human Research Program (HRP) is in the early stages of developing collaborative modeling approaches for the purposes of managing its science portfolio in an integrated manner to support cross-disciplinary risk mitigation strategies and to enable resilient human and engineered systems in the spaceflight environment. In this talk, we will share ideas being explored from fields such as network science, complexity theory, and system-of-systems modeling. Initial work on tools to support these explorations will be discussed briefly, along with ideas for future efforts.

Case Report: A Case of Plasmodium falciparum hrp2 and hrp3 Gene Mutation in Bangladesh.

PubMed

Nima, Maisha Khair; Hougard, Thomas; Hossain, Mohammad Enayet; Kibria, Mohammad Golam; Mohon, Abu Naser; Johora, Fatema Tuj; Rahman, Rajibur; Haque, Rashidul; Alam, Mohammad Shafiul

2017-10-01

Several species of Plasmodium are responsible for causing malaria in humans. Proper diagnoses are crucial to case management, because severity and treatment varies between species. Diagnoses can be made using rapid diagnostic tests (RDTs), which detect Plasmodium proteins. Plasmodium falciparum causes the most virulent cases of malaria, and P. falciparum histidine-rich protein 2 (PfHRP2) is a common target of falciparum malaria RDTs. Here we report a case in which a falciparum malaria patient in Bangladesh tested negative on PfHRP2-based RDTs. The negative results can be attributed to a deletion of part of the pfhrp2 gene and frameshift mutations in both pfhrp2 and pfhrp3 gene. This finding may have implications for malaria diagnostics and case management in Bangladesh and other regions of South Asia.

The Enzymatic Oxidation of Graphene Oxide

PubMed Central

Kotchey, Gregg P.; Allen, Brett L.; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A.; Tyurina, Yulia Y.; Klein-Seetharaman, Judith; Kagan, Valerian E.; Star, Alexander

2011-01-01

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon – the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (~40 µM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, UV-Vis, EPR and FT-IR spectroscopy, TEM, AFM, SDS-PAGE, and GC-MS. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Due to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors. PMID:21344859

HrcU and HrpP are pathogenicity factors in the fire blight pathogen Erwinia amylovora required for the type III secretion of DspA/E.

PubMed

McNally, R Ryan; Zeng, Quan; Sundin, George W

2016-05-20

Many Gram-negative bacterial pathogens mediate host-microbe interactions via utilization of the type III secretion (T3S) system. The T3S system is a complex molecular machine consisting of more than 20 proteins. Collectively, these proteins translocate effectors across extracellular space and into the host cytoplasm. Successful translocation requires timely synthesis and allocation of both structural and secreted T3S proteins. Based on amino acid conservation in animal pathogenic bacteria, HrcU and HrpP were examined for their roles in regulation of T3S hierarchy. Both HrcU and HrpP were shown to be required for disease development in an immature pear infection model and respective mutants were unable to induce a hypersensitive response in tobacco. Using in vitro western blot analyses, both proteins were also shown to be required for the secretion of DspA/E, a type 3 effector and an important pathogenicity factor. Via yeast-two hybridization (Y2H), HrpP and HrcU were revealed to exhibit protein-protein binding. Finally, all HrcU and HrpP phenotypes identified were shown to be dependent on a conserved amino acid motif in the cytoplasmic tail of HrcU. Collectively, these data demonstrate roles for HrcU and HrpP in regulating T3S and represent the first attempt in understanding T3S heirarchy in E. amylovora.

H2O2 sensing using HRP modified catalyst-free ZnO nanorods synthesized by RF sputtering

NASA Astrophysics Data System (ADS)

Srivastava, Amit; Kumar, Naresh; Singh, Priti; Singh, Sunil Kumar

2017-06-01

Catalyst-free ( 00 l) oriented ZnO nanorods (NRs) -based biosensor for the H2O2 sensing has been reported. The (002) oriented ZnO NRs as confirmed by X-ray diffraction were successfully grown on indium tin oxide (ITO) coated glass substrate by radio frequency (RF) sputtering technique without using any catalyst. Horseradish peroxidase (HRP) enzyme was immobilized on ZnO NRs by physical adsorption technique to prepare the biosensor. In this HRP/ZnO NR/ITO bioelectrode, nafion solution was added to form a tight membrane on surface. The prepared bioelectrode has been used for biosensing measurements by electrochemical analyzer. The electrochemical studies reveal that the prepared HRP/ZnO NR/ITO biosensor is highly sensitive to the detection of H2O2 over a linear range of 0.250-10 μM. The ZnO NR-based biosensor showed lower value of detection limit (0.125 μM) and higher sensitivity (13.40 µA/µM cm2) towards H2O2. The observed value of higher sensitivity attributed to larger surface area of ZnO nanostructure for effective loading of HRP besides its high electron communication capability. In addition, the biosensor also shows lower value of enzyme's kinetic parameter (Michaelis-Menten constant, K m) of 0.262 μM which indicates enhanced enzyme affinity of HRP to H2O2. The reported biosensor may be useful for various applications in biosensing, clinical, food, and beverage industry.

Identification of a unique IgG Fc binding site in human intestinal epithelium.

PubMed

Kobayashi, K; Blaser, M J; Brown, W R

1989-10-15

In experiments to determine whether serum antibodies in patients with Crohn's disease could be used as probes for detecting potentially etiologic Ag in the patients' tissues, we found that peroxidase (HRP)-labeled IgG from healthy persons, as well as from the patients, bound to normal colonic and small intestinal epithelium, mostly or entirely to goblet cells. The binding was due to a reaction involving the Fc region of IgG because HRP-labeled Fc fragments of IgG bound, but HRP-Fab, HRP-IgA, and HRP-bovine albumin did not, and because binding of HRP-IgG was inhibited competitively by unlabeled IgG or Fc fragments but not by IgG Fab fragments or IgA. These immunohistochemical results were confirmed by ELISA with microtiter wells coated with a sonicated homogenate from human colonocytes. The epithelial IgG Fc binding site was characterized by SDS-PAGE as consisting of a high Mr (greater than 200,000 Da) and a 78,000-Da component. It bound all four subclasses of human IgG and bound aggregated as well as monomeric IgG. It is distinct from known human Fc-gamma R by lack of recognition by mAb to those receptors and differences in affinity for various subclasses of human and murine IgG. This unique IgG Fc binding site might be involved in immunologic defense of the gut, perhaps by mediating reactions between foreign Ag and the contents of goblet cells.

[Effects of Hippophae rhamnoides L. subsp. chinensis Rousi polysaccharide on alpha-glucosidase enzyme activity and level of blood glucose].

PubMed

Guo, Feng-Xia; Zeng, Yang; Li, Jin-Ping; Chen, Zhen-Ning; Ma, Ji-Xiong

2013-04-01

The enzyme-inhibitor model and the sugar tolerance mouse model were used to evaluate the relationship between the inhibition rate of enzyme activity and concentration of Hippophae rhamnoides L. subsp. chinensis Rousi polysaccharide (HRP). The inhibitory patterns of enzyme and dose-dependent effects of HRP's effect on blood glucose using acarbose tablets as control were also examined. The mechanism underlying hypoglycemic effects of HRP was discussed. The results showed: in the enzyme-inhibitor model, the inhibitory activity of different concentrations of HRP (9.80, 19.60, 39.20, 78.40, 156.80 and 312.50 mg x L(-1)) on alpha-glucosaminidase (AG) inhibitory activity were 6.62%, 18.02%, 33.26%, 48.23%, 62.11%, 76.31%, 90.12%, IC50 was 31.59 mg x L(-1). The inhibitory rate of 25.00 x 10(3) mg x L(-1) acarbose tablets was only 64.87%, and IC50 was 10.75 x 10(3) mg x L(-1). In the sugar tolerance mouse model, different doses of HRP (240, 480, 960 mg x kg(-1)) tended to decrease levels of blood glucose compared with control group (acarbose tablets 375 mg x kg(-1)) at 15, 30, 60 and 120 min. It's further confirmed that HRP is a kind of competitive inhibitor of AG activity. Its inhibition rate increases with the increase of concentration in normal mice, and it subsequently improves the sugar tolerance showing the effect of reducing blood sugar.

Synergistic Use of Gold Nanoparticles (AuNPs) and “Capillary Enzyme-Linked Immunosorbent Assay (ELISA)” for High Sensitivity and Fast Assays

PubMed Central

Kim, Wan-Joong; Cho, Hyo Young; Jeong, Bongjin; Byun, Sangwon; Huh, JaeDoo; Kim, Young Jun

2017-01-01

Using gold nanoparticles (AuNPs) on “capillary enzyme-linked immunosorbent assay (ELISA)”, we produced highly sensitive and rapid assays, which are the major attributes for point-of-care applications. First, in order to understand the size effect of AuNPs, AuNPs of varying diameters (5 nm, 10 nm, 15 nm, 20 nm, 30 nm, and 50 nm) conjugated with Horseradish Peroxidase (HRP)-labeled anti-C reactive protein (antiCRP) (AuNP•antiCRP-HRP) were used for well-plate ELISA. AuNP of 10 nm produced the largest optical density, enabling detection of 0.1 ng/mL of CRP with only 30 s of incubation, in contrast to 10 ng/mL for the ELISA run in the absence of AuNP. Then, AuNP of 10 nm conjugated with antiCRP-HRP (AuNP•antiCRP-HRP) was used for “capillary ELISA” to detect as low as 0.1 ng/mL of CRP. Also, kinetic study on both 96-well plates and in a capillary tube using antiCRP-HRP or AuNP•antiCRP-HRP showed a synergistic effect between AuNP and the capillary system, in which the fastest assay was observed from the “AuNP capillary ELISA”, with its maximum absorbance reaching 2.5 min, while the slowest was the typical well-plate ELISA with its maximum absorbance reaching in 13.5 min. PMID:29278402

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

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

Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791; Baek, Jeong-Hwa

2014-07-11

Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates themore » radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells.« less

Medical System Concept of Operations for Mars Exploration Missions

NASA Technical Reports Server (NTRS)

Urbina, Michelle; Rubin, D.; Hailey, M.; Reyes, D.; Antonsen, Eric

2017-01-01

Future exploration missions will be the first time humanity travels beyond Low Earth Orbit (LEO) since the Apollo program, taking us to cis-lunar space, interplanetary space, and Mars. These long-duration missions will cover vast distances, severely constraining opportunities for emergency evacuation to Earth and cargo resupply opportunities. Communication delays and blackouts between the crew and Mission Control will eliminate reliable, real-time telemedicine consultations. As a result, compared to current LEO operations onboard the International Space Station, exploration mission medical care requires an integrated medical system that provides additional in-situ capabilities and a significant increase in crew autonomy. The Medical System Concept of Operations for Mars Exploration Missions illustrates how a future NASA Mars program could ensure appropriate medical care for the crew of this highly autonomous mission. This Concept of Operations document, when complete, will document all mission phases through a series of mission use case scenarios that illustrate required medical capabilities, enabling the NASA Human Research Program (HRP) Exploration Medical Capability (ExMC) Element to plan, design, and prototype an integrated medical system to support human exploration to Mars.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP.

PubMed

Czulak, J; Guerreiro, A; Metran, K; Canfarotta, F; Goddard, A; Cowan, R H; Trochimczuk, A W; Piletsky, S

2016-06-07

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

PubMed Central

Capone, Simona; Ćorajević, Lejla; Bonifert, Günther; Murth, Patrick; Maresch, Daniel; Altmann, Friedrich; Herwig, Christoph; Spadiut, Oliver

2015-01-01

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins. PMID:26404235

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay.

PubMed

Lakshmipriya, Thangavel; Gopinath, Subash C B; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors.

Biotin-Streptavidin Competition Mediates Sensitive Detection of Biomolecules in Enzyme Linked Immunosorbent Assay

PubMed Central

Lakshmipriya, Thangavel; Gopinath, Subash C. B.; Tang, Thean-Hock

2016-01-01

Enzyme Linked Immunosorbent Assay (ELISA) is the gold standard assay for detecting and identifying biomolecules using antibodies as the probe. Improving ELISA is crucial for detecting disease-causing agents and facilitating diagnosis at the early stages of disease. Biotinylated antibody and streptavidin-conjugated horse radish peroxide (streptavidin-HRP) often are used with ELISA to enhance the detection of various kinds of targets. In the present study, we used a competition-based strategy in which we pre-mixed free biotin with streptavidin-HRP to generate high-performance system, as free biotin occupies some of the biotin binding sites on streptavidin, thereby providing more chances for streptavidin-HRP to bind with biotinylated antibody. ESAT-6, which is a protein secreted early during tuberculosis infection, was used as the model target. We found that 8 fM of free biotin mixed with streptavidin-HRP anchored the higher detection level of ESAT-6 by four-fold compared with detection without free biotin (only streptavidin-HRP), and the limit of detection of the new method was 250 pM. These results suggest that biotin-streptavidin competition can be used to improve the diagnosis of analytes in other types of sensors. PMID:26954237

A novel approach to construct a horseradish peroxidase|hydrophilic ionic liquids|Au nanoparticles dotted titanate nanotubes biosensor for amperometric sensing of hydrogen peroxide.

PubMed

Liu, Xiaoqiang; Feng, Heqing; Zhao, Ruoxia; Wang, Yanbing; Liu, Xiuhua

2012-01-15

The direct electrochemistry of horseradish peroxidase (HRP) on a novel sensing platform modified glassy carbon electrode (GCE) has been achieved. This sensing platform consists of Nafion, hydrophilic room-temperature ionic liquid (RTIL) and Au nanoparticles dotted titanate nanotubes (GNPs-TNTs). The composite of RTIL and GNPs-TNTs was immobilized on the electrode surface through the gelation of a small amount of HRP aqueous solution. The composite was characterized by transmission electron microscopy (TEM), powder X-ray diffraction (XRD) and infrared spectroscopy (IR). UV-Vis and IR spectroscopy demonstrated that HRP in the composite could retain its native secondary structure and biochemical activity. The HRP-immobilized electrode was investigated by cyclic voltammetry and chronoamperometry. The results from both techniques showed that the direct electron transfer between the nanocomposite modified electrodes and heme in HRP could be realized. The biosensor responded to H(2)O(2) in the linear range from 5×10(-6) to 1×10(-3) mol L(-1) with a detection limit of 2.1×10(-6) mol L(-1) (based on the S/N=3). Copyright © 2011 Elsevier B.V. All rights reserved.

Quantitative detection of PfHRP2 in saliva of malaria patients in the Philippines

PubMed Central

2012-01-01

Background Malaria is a global health priority with a heavy burden of fatality and morbidity. Improvements in field diagnostics are needed to support the agenda for malaria elimination. Saliva has shown significant potential for use in non-invasive diagnostics, but the development of off-the-shelf saliva diagnostic kits requires best practices for sample preparation and quantitative insight on the availability of biomarkers and the dynamics of immunoassay in saliva. This pilot study measured the levels of the PfHRP2 in patient saliva to inform the development of salivary diagnostic tests for malaria. Methods Matched samples of blood and saliva were collected between January and May, 2011 from eight patients at Palawan Baptist Hospital in Roxas, Palawan, Philippines. Parasite density was determined from thick-film blood smears. Concentrations of PfHRP2 in saliva of malaria-positive patients were measured using a custom chemiluminescent ELISA in microtitre plates. Sixteen negative-control patients were enrolled at UCLA. A substantive difference between this protocol and previous related studies was that saliva samples were stabilized with protease inhibitors. Results Of the eight patients with microscopically confirmed P. falciparum malaria, seven tested positive for PfHRP2 in the blood using rapid diagnostic test kits, and all tested positive for PfHRP2 in saliva. All negative-control samples tested negative for salivary PfHRP2. On a binary-decision basis, the ELISA agreed with microscopy with 100 % sensitivity and 100 % specificity. Salivary levels of PfHRP2 ranged from 17 to 1,167 pg/mL in the malaria-positive group. Conclusion Saliva is a promising diagnostic fluid for malaria when protein degradation and matrix effects are mitigated. Systematic quantitation of other malaria biomarkers in saliva would identify those with the best clinical relevance and suitability for off-the-shelf diagnostic kits. PMID:22631858

Integrated Medical Model Project - Overview and Summary of Historical Application

NASA Technical Reports Server (NTRS)

Myers, J.; Boley, L.; Butler, D.; Foy, M.; Goodenow, D.; Griffin, D.; Keenan, A.; Kerstman, E.; Melton, S.; McGuire, K.;

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

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

Liu, Guodong; Timchalk, Chuck; Lin, Yuehe

2006-07-01

A magnetic beads-based bioelectrochemical magnetic immunosensor was developed for the fast and sensitive determination of the trichloropyridinol (TCP) biomarker in environmental samples. After liquid phrase competitive immunoreaction among a limited amount of TCP antibody coated-magnetic beads (Ab-MBs), TCP analyte, and horseradish peroxidase (HRP) labeled TCP (HRP-TCP), a magnet/glassy carbon (MGC) electrode was used to collect a TCP-Abs-MBs and a HRP-TCP-Ab-MBs immunocomplex assembly. The activity of HRP tracers bound to the beads was monitored with highly sensitive square wave voltammetry (SWV) by accumulating an electroactive enzymatic product to the MGC electrode surface under constant potential (0.5 V) during enzymatic reaction inmore » the presence of 3’,3’,5’,5’-tetramethylbenzidine (TMB)-H2O2 substrate solution. The electrochemical characteristics of substrate and product were investigated, and the parameters of the immunoassay were optimized.« less

Purification and partial characterization of PfHRP-II protein of Plasmodium falciparum.

PubMed

Ghimire, Prakash; Samantaray, J C; Mirdha, B R; Patra, A K; Panda, A K

2003-12-01

The human malarial parasite Plasmodium falciparum secretes various intra-and extra-cellular proteins during its asexual life cycle in human RBC. Histidine rich protein-II (HRP-II) is one of the most prominent proteins, found to be secreted by P. falciparum throughout the asexual cycle with the peak during mature schizont stage of the parasite development in human IRBC. The high histidine content (35% of the total amino acids in protein) of this protein suggested the potential to bind divalent metal ions. We have demonstrated by metal chelate chromatography, an extraordinary capacity of HRP-II to bind nickel ions (Ni++) and employed this characteristic to purify the extra-cellular HRP-II protein secreted by P. falciparum from culture supernatant. The identity of the purified protein was verified by the relative molecular weight on SDS-PAGE, by reacting with polyclonal antibodies directed against it using Western blot technique.

2013 Pharmacology Risk SRP Status Review Comments to Chief Scientist. The Risk of Clinically Relevant Unpredicted Effects of Medication

NASA Technical Reports Server (NTRS)

2014-01-01

On December 5, 2013, the Pharmacology Risk SRP, participants from the JSC, HQ, the NSBRI, and NRESS participated in a WebEx/teleconference. The purpose of the call (as stated in the Statement of Task) was to allow the SRP members to: 1. Receive an update by the HRP Chief Scientist or Deputy Chief Scientist on the status of NASA's current and future exploration plans and the impact these will have on the HRP. 2. Receive an update on any changes within the HRP since the 2012 SRP meeting. 3. Receive an update by the Element or Project Scientist(s) on progress since the 2012 SRP meeting. 4. Participate in a discussion with the HRP Chief Scientist, Deputy Chief Scientist, and the Element regarding possible topics to be addressed at the next SRP meeting.

A green approach to the synthesis of novel ``Desert rose stone''-like nanobiocatalytic system with excellent enzyme activity and stability

NASA Astrophysics Data System (ADS)

Wang, Min; Bao, Wen-Jing; Wang, Jiong; Wang, Kang; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

2014-10-01

3D hierarchical layer double hydroxides (LDHs) have attracted extensive interest due to their unique electronic and catalytic properties. Unfortunately, the existing preparation methods require high temperature or toxic organic compounds, which limits the applications of the 3D hierarchical LDHs in biocatalysis and biomedicine. Herein, we present a green strategy to synthesize ``Desert Rose Stone''-like Mg-Al-CO3 LDH nanoflowers in situ deposited on aluminum substrates via a coprecipitation method using atmospheric carbon dioxide. Using this method, we construct a novel ``Desert Rose Stone''-like nanobiocatalytic system by using HRP as the model enzyme. Compared with the free HRP, the HRP/Mg-Al-LDH nanobiocatalytic system exhibits higher catalytic activity and stability. A smaller apparent Michaelis-Menten constant (0.16 mM) of this system suggests that the encapsulated HRP shows higher affinity towards H2O2.

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing.

PubMed

Su, Huilan; Yuan, Ruo; Chai, Yaqin; Mao, Li; Zhuo, Ying

2011-07-15

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H₂O₂. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL. Copyright © 2011 Elsevier B.V. All rights reserved.

Improved peroxide biosensor based on Horseradish Peroxidase/Carbon Nanotube on a thiol-modified gold electrode.

PubMed

Kafi, A K M; Naqshabandi, M; Yusoff, Mashitah M; Crossley, Maxwell J

2018-06-01

A new 3-dimensional (3D) network of crosslinked Horseradish Peroxidase/Carbon Nanotube (HRP/CNT) on a thiol-modified Au surface has been described in order to build up the effective electrical wiring of the enzyme units with the electrode. The synthesized 3D HRP/CNT network has been characterized with cyclic voltammetry and amperometry which results the establishment of direct electron transfer between the redox active unit of HRP and the Au surface. Electrochemical measurements reveal that the high biological activity and stability is exhibited by the immobilized HRP and a quasi-reversible redox peak of the redox centre of HRP was observed at about -0.355 and -0.275V vs. Ag/AgCl. The electron transfer rate constant, K S and electron transfer co-efficient α were found as 0.57s -1 and 0.42, respectively. Excellent electrocatalytic activity for the reduction of H 2 O 2 was exhibited by the developed biosensor. The proposed biosensor modified with HRP/CNT 3D network displays a broader linear range and a lower detection limit for H 2 O 2 determination. The linear range is from 1.0×10 -7 to 1.2×10 -4 M with a detection limit of 2.2.0×10 -8 M at 3σ. The Michaelies-Menten constant Kapp M value is estimated to be 0.19mM. Moreover, this biosensor exhibits very high sensitivity, good reproducibility and long-time stability. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of the neuroprotective drugs somatostatin and brimonidine on retinal cell models of diabetic retinopathy.

PubMed

Beltramo, Elena; Lopatina, Tatiana; Mazzeo, Aurora; Arroba, Ana I; Valverde, Angela M; Hernández, Cristina; Simó, Rafael; Porta, Massimo

2016-12-01

Diabetic retinopathy is considered a microvascular disease, but recent evidence has underlined early involvement of the neuroretina with interactions between microvascular and neural alterations. Topical administration of somatostatin (SST), a neuroprotective molecule with antiangiogenic properties, prevents diabetes-induced retinal neurodegeneration in animals. The α 2 -adrenergic receptor agonist brimonidine (BRM) decreases vitreoretinal vascular endothelial growth factor and inhibits blood-retinal barrier breakdown in diabetic rats. However, SST and BRM effects on microvascular cells have not yet been studied. We investigated the behaviour of these drugs on the crosstalk between microvasculature and neuroretina. Expression of SST receptors 1-5 in human retinal pericytes (HRP) was checked. We subsequently evaluated the effects of diabetic-like conditions (high glucose and/or hypoxia) with/without SST/BRM on HRP survival. Endothelial cells (EC) and photoreceptors were maintained in the above conditions and their conditioned media (CM) used to culture HRP. Vice versa, HRP-CM was used on EC and photoreceptors. Survival parameters were assessed. HRP express the SST receptor 1 (SSTR1). Glucose fluctuations mimicking those occurring in diabetic subjects are more damaging for pericytes and photoreceptors than stable high glucose and hypoxic conditions. SST/BRM added to HRP in diabetic-like conditions decrease EC apoptosis. However, neither SST nor BRM changed the response of pericytes and neuroretina-vascular crosstalk under diabetic-like conditions. Retinal pericytes express SSTR1, indicating that they can be a target for SST. Exposure to SST/BRM had no adverse effects, direct or mediated by the neuroretina, suggesting that these molecules could be safely evaluated for the treatment of ocular diseases.

The enzymatic oxidation of graphene oxide.

PubMed

Kotchey, Gregg P; Allen, Brett L; Vedala, Harindra; Yanamala, Naveena; Kapralov, Alexander A; Tyurina, Yulia Y; Klein-Seetharaman, Judith; Kagan, Valerian E; Star, Alexander

2011-03-22

Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon--the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (∼40 μM), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, ultraviolet-visible, electron paramagnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gas chromatography-mass spectrometry. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Owing to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors.

An enhancer-like region regulates hrp3 promoter stage-specific gene expression in the human malaria parasite Plasmodium falciparum

PubMed Central

López-Estraño, Carlos; Gopalakrishnan, Anusha M.; Semblat, Jean-Philippe; Fergus, M. Ross; Mazier, Dominique; Haldar, Kasturi

2008-01-01

The asexual blood stage of Plasmodium falciparum is comprised of morphologically distinct ring, trophozoite and schizont stages. Each of these developmental stages possesses a distinct pattern of gene expression. Regulation of P. falciparum gene expression is thought to occur, at least in part, at the promoter level. Previously, we have found that although the RNA of the P. falciparum hrp3 gene is only seen in ring-stage parasites, deletion of a specific sequensce in the 5’ end of the promoter region decreased ring-stage expression of hrp3 and enabled detection of its transcripts in trophozoite-stage parasites. In order to investigate this stage specific regulation of gene expression, we employed a series of nested deletions of the 1.7-kb hrp3 promoter. Firefly luciferase gene was used as a reporter to evaluate the role of promoter sequences in gene regulation. Using this approach, we identified a ring-stage specific regulatory region on the hrp3 promoter located between -1.7-kb and -1.1-kb from the ATG initiation codon. Small 100–150 bp truncations on this enhancer-like region failed to uncover discrete regulatory sequences, suggesting the multipartite nature of this element. The data presented in this study demonstrates that stage specific promoter activity of the hrp3 gene in P. falciparum blood stage parasites is supported, at least in-part, by a small promoter region that can function in the absence of a larger chromosomal context. PMID:17570541

Biofunctionalization of carbon nanostructures through enzyme immobilization in colloidal silica

NASA Astrophysics Data System (ADS)

Goulet, Evan M.

Multi-walled carbon nanotubes (MWNT) and carbon nanopipettes (CNP) provide interesting high aspect ratio scaffolds on which to base functionally gradient materials. In this dissertation, we present a general method for the production of an enzymatically active composite material based on MWNTs. Polyethyleneimine (PEI) was applied to purified MWNTs, generating a positive electrostatic potential on the MWNTs. This positive potential was used to apply negatively charged colloidal silica particle in the presence of a high concentration of enzyme. The silica coating continued to grow via localized condensation of silica particles driven by the buffered saline conditions, immobilizing the enzyme within the coating. The mesoporous nanostructure was characterized via transmission electron microscopy. Optical spectroscopy experiments on the material employed as an active suspension showed that the immobilized enzymes horseradish peroxidase (HRP) and tyrosinase (TV) retained their activity upon incorporation into the material. Using HRP as a model enzyme, it was determined that the MWNT-HRP-Silica material showed similar pH and temperature dependencies in activity to those of free HRP in solution. An examination of the Michaelis-Menten kinetics showed that the material had a slightly higher value of KM than did free HRP. The MWNT-HRP-Silica material was also employed as an active filter membrane, which allowed us to explore the reusable nature of the material. We were able to show the denaturation of the filter due to the loss of Ca2+ cations at low pH and then restore the activity by soaking the filter membrane in 1 mM CaCl2. The MWNT-HRP-Silica material was used to modify a carbon microelectrode and produce a functioning electrochemical sensor for H2O2 . Utilizing cyclic voltammetry, the sensor was shown to have a linear response in limiting current versus concentration of H2O2 of 4.26 pA/microM. We also determined a lower detection limit of 0.67 microM H2O2. CNPs were investigated as functional microelectrodes. Colloidal silica was applied to the CNP with HRP, but it was difficult to prove functionality. One irregularly coated CNP showed a clear response to H2O2, but we were not able to reproduce the response in other samples. This work indicated the CNPs have promise as functional microelectrodes.

Formation of target-specific binding sites in enzymes: solid-phase molecular imprinting of HRP

NASA Astrophysics Data System (ADS)

Czulak, J.; Guerreiro, A.; Metran, K.; Canfarotta, F.; Goddard, A.; Cowan, R. H.; Trochimczuk, A. W.; Piletsky, S.

2016-05-01

Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates.Here we introduce a new concept for synthesising molecularly imprinted nanoparticles by using proteins as macro-functional monomers. For a proof-of-concept, a model enzyme (HRP) was cross-linked using glutaraldehyde in the presence of glass beads (solid-phase) bearing immobilized templates such as vancomycin and ampicillin. The cross-linking process links together proteins and protein chains, which in the presence of templates leads to the formation of permanent target-specific recognition sites without adverse effects on the enzymatic activity. Unlike complex protein engineering approaches commonly employed to generate affinity proteins, the method proposed can be used to produce protein-based ligands in a short time period using native protein molecules. These affinity materials are potentially useful tools especially for assays since they combine the catalytic properties of enzymes (for signaling) and molecular recognition properties of antibodies. We demonstrate this concept in an ELISA-format assay where HRP imprinted with vancomycin and ampicillin replaced traditional enzyme-antibody conjugates for selective detection of templates at micromolar concentrations. This approach can potentially provide a fast alternative to raising antibodies for targets that do not require high assay sensitivities; it can also find uses as a biochemical research tool, as a possible replacement for immunoperoxidase-conjugates. Electronic supplementary information (ESI) available: Additional circular dichroism data and nanoparticle tracking analysis trace. See DOI: 10.1039/c6nr02009g

A fluorescence approach to the unfolding thermodynamics of horseradish peroxidase based on heme degradation by hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ke, Zhigang; Ma, Shanshan; Li, Lamei; Huang, Qing

2016-07-01

Horseradish peroxidase (HRP) is a classical heme-containing protein which has been applied in many fields. The prosthetic group heme in HRP, especially in unfolded state, can react with hydrogen peroxide (H2O2) to produce a fluorescent product with the maximum emission wavelength at 450 nm. Utilizing this emission band as a fluorescence probe, the unfolding process of HRP in urea can be assessed quantitatively, and the calculated thermodynamic parameters are consistent with those determined by circular dichroism (CD) at 222 nm and steady-state tryptophan (Trp) fluorescence methods.

Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

PubMed

Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

2003-10-01

Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

A three-enzyme cascade reaction through positional assembly of enzymes in a polymersome nanoreactor.

PubMed

van Dongen, Stijn F M; Nallani, Madhavan; Cornelissen, Jeroen J L M; Nolte, Roeland J M; van Hest, Jan C M

2009-01-01

Porous polymersomes based on block copolymers of isocyanopeptides and styrene have been used to anchor enzymes at three different locations, namely, in their lumen (glucose oxidase, GOx), in their bilayer membrane (Candida antarctica lipase B, CalB) and on their surface (horseradish peroxidase, HRP). The surface coupling was achieved by click chemistry between acetylene-functionalised anchors on the surface of the polymersomes and azido functions of HRP, which were introduced by using a direct diazo transfer reaction to lysine residues of the enzyme. To determine the encapsulation and conjugation efficiency of the enzymes, they were decorated with metal-ion labels and analysed by mass spectrometry. This revealed an almost quantitative immobilisation efficiency of HRP on the surface of the polymersomes and a more than statistical incorporation efficiency for CalB in the membrane and for GOx in the aqueous compartment. The enzyme-decorated polymersomes were studied as nanoreactors in which glucose acetate was converted by CalB to glucose, which was oxidised by GOx to gluconolactone in a second step. The hydrogen peroxide produced was used by HRP to oxidise 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) to ABTS(.+). Kinetic analysis revealed that the reaction step catalysed by HRP is the fastest in the cascade reaction.

The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae.

PubMed

Schmidt, Maria Augusta; Balsanelli, Eduardo; Faoro, Hellison; Cruz, Leonardo M; Wassem, Roseli; de Baura, Valter A; Weiss, Vinícius; Yates, Marshall G; Madeira, Humberto M F; Pereira-Ferrari, Lilian; Fungaro, Maria H P; de Paula, Francine M; Pereira, Luiz F P; Vieira, Luiz G E; Olivares, Fábio L; Pedrosa, Fábio O; de Souza, Emanuel M; Monteiro, Rose A

2012-06-06

Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.

Paper Microzone Plates as Analytical Tools for Studying Enzyme Stability: A Case Study on the Stabilization of Horseradish Peroxidase Using Trehalose and SU-8 Epoxy Novolac Resin.

PubMed

Ganaja, Kirsten A; Chaplan, Cory A; Zhang, Jingyi; Martinez, Nathaniel W; Martinez, Andres W

2017-05-16

Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents.

Electrochemical immunosensor for interferon-γ based on disposable ITO detector and HRP-antibody-conjugated nano gold as signal tag.

PubMed

Zhang, Yaru; Zhang, Bin; Ye, Xiaoli; Yan, Yuqi; Huang, Langhuan; Jiang, Zhenyou; Tan, Shaozao; Cai, Xiang

2016-02-01

Tuberculosis is the most frequent cause of infection-related death worldwide. A new disposable electrochemical immunosensor with low cost and simple fabrication was proposed to detect interferon-γ (IFN-γ). Diallyldimethylammonium chloride (PDDA) and Au nanoparticle (AuNP) composite were used to provide an efficient biointerface, horseradish peroxidase (HRP)-labeled antibody-conjugated AuNP (HRP-Ab2-AuNP) bioconjugates were used as a novel signal tag. The large amounts of HRP on the signal tag can catalyze the oxidation of Hydroquinone (HQ) by H2O2, which can induce an amplified reductive current. The catalytic reduction current was related to the amount of HRP immobilized on the surface, which itself was related to the concentration of IFN-γ. Under optimized conditions, the proposed immunosensor showed a high sensitivity and a linear range of 0.1-10,000pg/mL with a detection limit of 0.048pg/mL. The assay results of clinical serum samples obtained by the immunosensor were in acceptable agreement with the reference values. Therefore, the immunosensor possessed excellent clinical value in early diagnosis and control of tuberculosis. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

Carbon Felt-Based Bioelectrocatalytic Flow-Through Detectors: 2,6-Dichlorophenol Indophenol and Peroxidase Coadsorbed Carbon-Felt for Flow-Amperometric Determination of Hydrogen Peroxide

PubMed Central

Wang, Yue; Hasebe, Yasushi

2014-01-01

2,6-dichlorophenol indophenol (DCIP) and horseradish peroxidase (HRP) were coadsorbed on a porous carbon felt (CF) from their mixed aqueous solution under ultrasound irradiation for 5 min. The resulting DCIP and HRP-coadsorbed CF (DCIP/HRP-CF) showed an excellent bioelectrocatalytic activity for the reduction of H2O2. The coadsorption of DCIP together with HRP was essential to obtain larger bioelectrocatalytic current to H2O2. The DCIP/HRP-CF was successfully used as a working electrode unit of a bioelectrocatalytic flow-through detector for highly sensitive and continuous amperometric determination of H2O2. Under the optimized operational conditions (i.e., applied potential, +0.2 V versus Ag/AgCl; carrier pH 5.0, and carrier flow rate, 1.9 mL/min), the cathodic peak current of H2O2 linearly increased over the concentration range from 0.1 to 30 μM (the sensitivity, 0.88 μA/μM (slope of linear part); the limit of detection, 0.1 μM (S/N = 3) current noise level, 30 nA) with a sample through-put of ca. 40–90 samples/h. PMID:28788505

Advanced Technology Applications for Combat Casualty Care

NASA Technical Reports Server (NTRS)

Watkins, Sharmila; Baumann, David; Wu, Jimmy

2010-01-01

Exploration Medical Capability (ExMC) is an element of NASA s Human Research Program (HRP). ExMC s goal is to address the risk of the "Inability to Adequately Recognize or Treat an Ill or Injured Crewmember." This poster highlights the approach ExMC has taken to address this goal and our current areas of interest. The Space Medicine Exploration Medical Condition List (SMEMCL) was created to identify medical conditions of concern during exploration missions. The list was derived from space flight medical incidents, the shuttle medical checklist, the International Space Station medical checklist, and expert opinion. The conditions on the list were prioritized according to mission type by a panel comprised of flight surgeons, physician astronauts, engineers, and scientists. From the prioritized list, the ExMC element determined the capabilities needed to address the medical conditions of concern. Where such capabilities were not currently available, a gap was identified. The element s research plan outlines these gaps and the tasks identified to achieve the desired capabilities for exploration missions.

Life Sciences Data Archives (LSDA) in the Post-Shuttle Era

NASA Technical Reports Server (NTRS)

Fitts, Mary A.; Johnson-Throop, Kathy; Havelka, Jacque; Thomas, Diedre

2010-01-01

Now, more than ever before, NASA is realizing the value and importance of their intellectual assets. Principles of knowledge management-the systematic use and reuse of information, experience, and expertise to achieve a specific goal-are being applied throughout the agency. LSDA is also applying these solutions, which rely on a combination of content and collaboration technologies, to enable research teams to create, capture, share, and harness knowledge to do the things they do well, even better. In the early days of spaceflight, space life sciences data were collected and stored in numerous databases, formats, media-types and geographical locations. These data were largely unknown/unavailable to the research community. The Biomedical Informatics and Health Care Systems Branch of the Space Life Sciences Directorate at JSC and the Data Archive Project at ARC, with funding from the Human Research Program through the Exploration Medical Capability Element, are fulfilling these requirements through the systematic population of the Life Sciences Data Archive. This project constitutes a formal system for the acquisition, archival and distribution of data for HRP-related experiments and investigations. The general goal of the archive is to acquire, preserve, and distribute these data and be responsive to inquiries for the science communities. Information about experiments and data, as well as non-attributable human data and data from other species' are available on our public Web site http://lsda.jsc.nasa.gov. The Web site also includes a repository for biospecimens, and a utilization process. NASA has undertaken an initiative to develop a Shuttle Data Archive repository. The Shuttle program is nearing its end in 2010 and it is critical that the medical and research data related to the Shuttle program be captured, retained, and usable for research, lessons learned, and future mission planning. Communities of practice are groups of people who share a concern or a passion for something they do, and learn how to do it better as they interact regularly. LSDA works with the HRP community of practice to ensure that we are preserving the relevant research and data they need in the LSDA repository. An evidence-based approach to risk management is required in space life sciences. Evidence changes over time. LSDA has a pilot project with Collexis, a new type of Web-based search engine. Collexis differentiates itself from full-text search engines by making use of thesauri for information retrieval. The high-quality search is based on semantics that have been defined in a life sciences ontology. Additionally, Collexis' matching technology is unique, allowing discovery of partially matching dicuments. Users do not have to construct a complicated (Boolean) search query, but can simply enter a free text search without the risk of getting "no results". Collexis may address these issues by virtue of its retrieval and discovery capabilities across multiple repositories.

EEG-Informed fMRI Reveals a Disturbed Gamma-Band–Specific Network in Subjects at High Risk for Psychosis

PubMed Central

Leicht, Gregor; Vauth, Sebastian; Polomac, Nenad; Andreou, Christina; Rauh, Jonas; Mußmann, Marius; Karow, Anne; Mulert, Christoph

2016-01-01

Objectives. Abnormalities of oscillatory gamma activity are supposed to reflect a core pathophysiological mechanism underlying cognitive disturbances in schizophrenia. The auditory evoked gamma-band response (aeGBR) is known to be reduced across all stages of the disease. The present study aimed to elucidate alterations of an aeGBR-specific network mediated by gamma oscillations in the high-risk state of psychosis (HRP) by means of functional magnetic resonance imaging (fMRI) informed by electroencephalography (EEG). Methods. EEG and fMRI were simultaneously recorded from 27 HRP individuals and 26 healthy controls (HC) during performance of a cognitively demanding auditory reaction task. We used single trial coupling of the aeGBR with the corresponding blood oxygen level depending response (EEG-informed fMRI). Results. A gamma-band–specific network was significantly lower active in HRP subjects compared with HC (random effects analysis, P < .01, Bonferroni-corrected for multiple comparisons) accompanied by a worse task performance. This network involved the bilateral auditory cortices, the thalamus and frontal brain regions including the anterior cingulate cortex, as well as the bilateral dorsolateral prefrontal cortex. Conclusions. For the first time we report a reduced activation of an aeGBR-specific network in HRP subjects brought forward by EEG-informed fMRI. Because the HRP reflects the clinical risk for conversion to psychotic disorders including schizophrenia and the aeGBR has repeatedly been shown to be altered in patients with schizophrenia the results of our study point towards a potential applicability of aeGBR disturbances as a marker for the prediction of transition of HRP subjects to schizophrenia. PMID:26163477

Study on the reaction mechanism and the static injection chemiluminescence method for detection of acetaminophen.

PubMed

Wu, Yongjun; Zhang, Huili; Yu, Songcheng; Yu, Fei; Li, Yanqiang; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B

2013-01-01

Acetaminophen, also called paracetamol, is found in Tylenol, Excedrin and other products as over-the-counter medicines. In this study, acetaminophen as a luminol signal enhancer was used in the chemiluminescence (CL) substrate solution of horseradish peroxidase (HRP) for the first time. The use of acetaminophen in the luminol-HRP-H2O2 system affected not only the intensity of the obtained signal, but also its kinetics. It was shown that acetaminophen was to be a potent enhancer of the luminol-HRP-H2O2 system. A putative enhancement mechanism for the luminol-H2O2-HRP-acetaminophen system is presented. The resonance of the nucleophilic amide group and the benzene ring of acetaminophen structure have a great effect on O-H bond dissociation energy of the phenol group and therefore on phenoxyl radical stabilization. These radicals act as mediators between HRP and luminol in an electron transfer reaction that generates luminol radicals and subsequently light emission, in which the intensity of CL is enhanced in the presence of acetaminophen. In addition, a simple method was developed to detect acetaminophen by static injection CL based on the enhanced CL system of luminol-H2O2-HRP by acetaminophen. Experimental conditions, such as pH and concentrations of substrates, have been examined and optimized. The proposed method exhibited good performance, the linear range was from 0.30 to 7.5 mM, the relative standard deviation was 1.86% (n = 10), limit of detection was 0.16 mM and recovery was 99 ± 4%. Copyright © 2013 John Wiley & Sons, Ltd.

Rapid diagnostic tests for malaria at sites of varying transmission intensity in Uganda.

PubMed

Hopkins, Heidi; Bebell, Lisa; Kambale, Wilson; Dokomajilar, Christian; Rosenthal, Philip J; Dorsey, Grant

2008-02-15

In Africa, fever is often treated presumptively as malaria, resulting in misdiagnosis and the overuse of antimalarial drugs. Rapid diagnostic tests (RDTs) for malaria may allow improved fever management. We compared RDTs based on histidine-rich protein 2 (HRP2) and RDTs based on Plasmodium lactate dehydrogenase (pLDH) with expert microscopy and PCR-corrected microscopy for 7000 patients at sites of varying malaria transmission intensity across Uganda. When all sites were considered, the sensitivity of the HRP2-based test was 97% when compared with microscopy and 98% when corrected by PCR; the sensitivity of the pLDH-based test was 88% when compared with microscopy and 77% when corrected by PCR. The specificity of the HRP2-based test was 71% when compared with microscopy and 88% when corrected by PCR; the specificity of the pLDH-based test was 92% when compared with microscopy and >98% when corrected by PCR. Based on Plasmodium falciparum PCR-corrected microscopy, the positive predictive value (PPV) of the HRP2-based test was high (93%) at all but the site with the lowest transmission rate; the pLDH-based test and expert microscopy offered excellent PPVs (98%) for all sites. The negative predictive value (NPV) of the HRP2-based test was consistently high (>97%); in contrast, the NPV for the pLDH-based test dropped significantly (from 98% to 66%) as transmission intensity increased, and the NPV for expert microscopy decreased significantly (99% to 54%) because of increasing failure to detect subpatent parasitemia. Based on the high PPV and NPV, HRP2-based RDTs are likely to be the best diagnostic choice for areas with medium-to-high malaria transmission rates in Africa.

Reduction of Sodium Arsenite-Mediated Adverse Effects in Mice using Dietary Supplementation of Water Hyacinth (Eichornia crassipes) Root Powder.

PubMed

Sarker, Rim Sabrina Jahan; Ahsan, Nazmul; Hossain, Khaled; Ghosh, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Akhand, Anwarul Azim

2012-07-01

In this study, we evaluated the protective effects of water Hyacinth Root Powder (HRP) on arsenic-mediated toxic effects in mice. Swiss albino mice, used in this study, were divided into four different groups (for each group n=5). The control group was supplied with normal feed and water, Arsenic group (As-group) was supplied with normal feed plus arsenic (sodium arsenite)-containing water, and arsenic+hyacinth group (As+Hy group) was supplied with feed supplemented with HRP plus arsenic water. The remaining Hy-group was supplied with feed supplemented with HRP plus normal water. Oral administration of arsenic reduced the normal growth of the mice as evidenced by weight loss. Interestingly, tip of the tails of these mice developed wound that caused gradual reduction of the tail length. Supplementation of HRP in feed significantly prevented mice growth retardation and tail wounding in As+Hy group mice. However, the growth pattern in Hy-group mice was observed to be almost similar to that of the control group indicating that HRP itself has no toxic or negative effect in mice. Ingested arsenic also distorted the shape of the blood cells and elevated the serum enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and serum glutamic pyruvic transaminase (SGPT). Importantly, elevation of these enzymes and distortion of blood cell shape were partially reduced in mice belong to As+Hy group, indicating HRP-mediated reduction of arsenic toxicity. Therefore, the preventive effect of hyacinth root on arsenic-poisoned mice suggested the future application of hyacinth to reduce arsenic toxicity in animal and human.

Reduction of Sodium Arsenite-Mediated Adverse Effects in Mice using Dietary Supplementation of Water Hyacinth (Eichornia crassipes) Root Powder

PubMed Central

Sarker, Rim Sabrina Jahan; Ahsan, Nazmul; Hossain, Khaled; Ghosh, Paritosh Kumar; Ahsan, Chowdhury Rafiqul; Akhand, Anwarul Azim

2012-01-01

Background In this study, we evaluated the protective effects of water Hyacinth Root Powder (HRP) on arsenic-mediated toxic effects in mice. Methods Swiss albino mice, used in this study, were divided into four different groups (for each group n=5). The control group was supplied with normal feed and water, Arsenic group (As-group) was supplied with normal feed plus arsenic (sodium arsenite)-containing water, and arsenic+hyacinth group (As+Hy group) was supplied with feed supplemented with HRP plus arsenic water. The remaining Hy-group was supplied with feed supplemented with HRP plus normal water. Results Oral administration of arsenic reduced the normal growth of the mice as evidenced by weight loss. Interestingly, tip of the tails of these mice developed wound that caused gradual reduction of the tail length. Supplementation of HRP in feed significantly prevented mice growth retardation and tail wounding in As+Hy group mice. However, the growth pattern in Hy-group mice was observed to be almost similar to that of the control group indicating that HRP itself has no toxic or negative effect in mice. Ingested arsenic also distorted the shape of the blood cells and elevated the serum enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and serum glutamic pyruvic transaminase (SGPT). Importantly, elevation of these enzymes and distortion of blood cell shape were partially reduced in mice belong to As+Hy group, indicating HRP-mediated reduction of arsenic toxicity. Conclusion Therefore, the preventive effect of hyacinth root on arsenic-poisoned mice suggested the future application of hyacinth to reduce arsenic toxicity in animal and human. PMID:23407303

The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase.

PubMed

Campomanes, Pablo; Rothlisberger, Ursula; Alfonso-Prieto, Mercedes; Rovira, Carme

2015-09-02

Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H2O2). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H2O2 at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, ∼ 6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H2O2, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the "wet" active site of HRP have a substantial impact on the reaction barrier, compared to the "dry" active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H2O2 disproportionation.

Interplay between chromatin modulators and histone acetylation regulates the formation of accessible chromatin in the upstream regulatory region of fission yeast fbp1.

PubMed

Adachi, Akira; Senmatsu, Satoshi; Asada, Ryuta; Abe, Takuya; Hoffman, Charles S; Ohta, Kunihiro; Hirota, Kouji

2018-05-03

Numerous noncoding RNA transcripts are detected in eukaryotic cells. Noncoding RNAs transcribed across gene promoters are involved in the regulation of mRNA transcription via chromatin modulation. This function of noncoding RNA transcription was first demonstrated for the fission yeast fbp1 gene, where a cascade of noncoding RNA transcription events induces chromatin remodeling to facilitate transcription factor binding. We recently demonstrated that the noncoding RNAs from the fbp1 upstream region facilitate binding of the transcription activator Atf1 and thereby promote histone acetylation. Histone acetylation by histone acetyl transferases (HATs) and ATP-dependent chromatin remodelers (ADCRs) are implicated in chromatin remodeling, but the interplay between HATs and ADCRs in this process has not been fully elucidated. Here, we examine the roles played by two distinct ADCRs, Snf22 and Hrp3, and by the HAT Gcn5 in the transcriptional activation of fbp1. Snf22 and Hrp3 redundantly promote disassembly of chromatin in the fbp1 upstream region. Gcn5 critically contributes to nucleosome eviction in the absence of either Snf22 or Hrp3, presumably by recruiting Hrp3 in snf22∆ cells and Snf22 in hrp3∆ cells. Conversely, Gcn5-dependent histone H3 acetylation is impaired in snf22∆/hrp3∆ cells, suggesting that both redundant ADCRs induce recruitment of Gcn5 to the chromatin array in the fbp1 upstream region. These results reveal a previously unappreciated interplay between ADCRs and histone acetylation in which histone acetylation facilitates recruitment of ADCRs, while ADCRs are required for histone acetylation.

Performance of pfHRP2 versus pLDH antigen rapid diagnostic tests for the detection of Plasmodium falciparum: a systematic review and meta-analysis.

PubMed

Li, Bo; Sun, Zhiqiang; Li, Xiaohan; Li, Xiaoxi; Wang, Han; Chen, Weijiao; Chen, Peng; Qiao, Mengran; Mao, Yuanli

2017-04-01

There have been many inconsistent reports about the performance of histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH) antigens as rapid diagnostic tests (RDTs) for the diagnosis of past Plasmodium falciparum infections. This meta-analysis was performed to determine the performance of pfHRP2 versus pLDH antigen RDTs in the detection of P. falciparum . After a systematic review of related studies, Meta-DiSc 1.4 software was used to calculate the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Forest plots and summary receiver operating characteristic curve (SROC) analysis were used to summarize the overall test performance. Fourteen studies which met the inclusion criteria were included in the meta-analysis. The summary performances for pfHRP2- and pLDH-based tests in the diagnosis of P. falciparum infections were as follows: pooled sensitivity, 96.3% (95.8-96.7%) vs. 82.6% (81.7-83.5%); specificity, 86.1% (85.3-86.8%) vs. 95.9% (95.4-96.3%); diagnostic odds ratio (DOR), 243.31 (97.679-606.08) vs. 230.59 (114.98-462.42); and area under ROCs, 0.9822 versus 0.9849 (all p < 0.001). The two RDTs performed satisfactorily for the diagnosis of P. falciparum , but the pLDH tests had higher specificity, whereas the pfHRP2 tests had better sensitivity. The pfHRP2 tests had slightly greater accuracy compared to the pLDH tests. A combination of both antigens might be a more reliable approach for the diagnosis of malaria.

Phospholipid-templated silica nanocapsules as efficient polyenzymatic biocatalysts.

PubMed

Phuoc, Lai Truong; Laveille, Paco; Chamouleau, Françoise; Renard, Gilbert; Drone, Jullien; Coq, Bernard; Fajula, François; Galarneau, Anne

2010-09-28

Solid polyenzymatic biocatalysts have been designed by combining two immobilized enzymes, the first one allowing the in situ generation of H(2)O(2) from air and the second one performing an oxidation reaction. The in situ H(2)O(2) generation system is based on the reaction of glucose with air using a glucose oxidase (GOx). The optimization of the encapsulation of GOx into phospholipids-templated silica capsules (NPS) was performed. A bienzymatic system made of GOx and horseradish peroxidase (HRP) was studied. Optimal conditions for the activity of the GOx/HRP bienzymatic system have been determined for both homogeneous and heterogeneous conditions. The encapsulation in NPS materials increases the stability of both enzymes. The performance of the encapsulated bienzymatic GOx/HRP system in the model reaction of 4-aminoantipyridine with phenol is similar when the enzymes are immobilized separately in two NPS or coencapsulated in the same NPS. An excess of peroxidase compared to GOx ([HRP]/[GOx] = 5-10) is necessary to obtain the optimal activity. To show the potentiality of bienzymatic systems in real applications, HRP has been replaced by hemoglobin, which is known for its ability to oxidize polycyclic aromatic hydrocarbons (PAH) pollutants through a pseudoperoxidase pathway. A larger excess of Hb compared to GOx ([Hb]/[GOx] = 1000) was necessary to obtain the maximum PAH removal, as Hb is not a real peroxidase as HRP but a hemoprotein with some pseudoperoxidase activity. In opposite to real enzymes, the immobilization of Hb by adsorption in mesoporous silica is preferable as its encapsulation. Therefore, the bienzymatic system made of GOx encapsulated in NPS and Hb adsorbed in mesoporous silica has been used for the removal of 11 PAH from water. This heterogeneous bienzymatic system allows 64% of PAH removal from water using simple air as oxidant.

The NASA Human Research Wiki - An Online Collaboration Tool

NASA Technical Reports Server (NTRS)

Barr, Yael; Rasbury, Jack; Johnson, Jordan; Barstend, Kristina; Saile, Lynn; Watkins, Sharmi

2012-01-01

The Exploration Medical Capability (ExMC) element is one of six elements of the Human Research Program (HRP). ExMC is charged with decreasing the risk of: "Inability to adequately recognize or treat an ill or injured crew member" for exploration-class missions In preparation for exploration-class missions, ExMC has compiled a large evidence base, previously available only to persons within the NASA community. ExMC has developed the "NASA Human Research Wiki" in an effort to make the ExMC information available to the general public and increase collaboration within and outside of NASA. The ExMC evidence base is comprised of several types of data, including: (1)Information on more than 80 medical conditions which could occur during space flight (a)Derived from several sources (b)Including data on incidence and potential outcomes, as captured in the Integrated Medical Model s (IMM) Clinical Finding Forms (CliFFs). (2)Approximately 25 gap reports (a)Identify any "gaps" in knowledge and/or technology that would need to be addressed in order to provide adequate medical support for these novel missions.

Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology.

PubMed

Wang, Baojun; Kitney, Richard I; Joly, Nicolas; Buck, Martin

2011-10-18

Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ(54)-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts. © 2011 Macmillan Publishers Limited. All rights reserved.

Overview of the Contributing Factor Map and How it can Support your Research

NASA Technical Reports Server (NTRS)

Mindock, Jennifer A.

2014-01-01

The Contributing Factor Map (CFM) is a visual representation of a taxonomy of factors influencing human health and performance in space. This presentation will give an overview of its development and its structure. It will describe various uses of the CFM that can support researchers working within the Human Research Program (HRP) Architecture of Evidence-Risk-Gap-Task-Deliverable. For example, during the Risk phase, the CFM can be used as a "menu" to help formulate a qualitative model of the factors contributing to specific consequences of concern. It provides a reference set of factors from across the operational, vehicle design, and human domains that otherwise might not be considered if approaching a risk from a specific domain perspective. Using the CFM as a reference can increase awareness of potential cross-disciplinary collaborations for overall risk mitigation. The CFM can also be used as a framework for identifying gaps in knowledge about a risk. This identification can support the subsequent development of gaps and tasks comprising the research plan aimed at risk mitigation. Examples of these types of applications of the CFM will be discussed and information on the support available to researchers in using it will be provided.

Improved activity and thermo-stability of the horse radish peroxidase with graphene quantum dots and its application in fluorometric detection of hydrogen peroxide

NASA Astrophysics Data System (ADS)

Xiaoyan, Zhou; Yuanyuan, Jiang; Zaijun, Li; Zhiguo, Gu; Guangli, Wang

2016-08-01

Graphene quantum dots (GQDs) have received extensive concern in many fields such as optical probe, bioimaging and biosensor. However, few reports refer on the influence of GQDs on enzyme performance. The paper reports two kinds of graphene quantum dots (termed as GO-GQDs and N,S-GQDs) that were prepared by cutting of graphene oxide and pyrolysis of citric acid and L-cysteine, and their use for the horse radish peroxidase (HRP) modification. The study reveals that GO-GQDs and N,S-GQDs exhibit an opposite effect on the HRP performance. Only HRP modified with GO-GQDs offers an enhanced activity (more than 1.9 times of pristine enzyme) and thermo-stability. This is because GO-GQDs offer a larger conjugate rigid plane and fewer hydrophilic groups compared to N,S-GQDs. The characteristics can make GO-GQDs induce a proper conformational change in the HRP for the catalytic performance, improving the enzyme activity and thermo-stability. The HRP modified with green luminescent GO-GQDs was also employed as a biocatalyst for sensing of H2O2 by a fluorometric sensor. The colorless tetramethylbenzidine (TMB) is oxidized into blue oxidized TMB in the presence of H2O2 by the assistance of HRP/GO-GQDs, leading to an obvious fluorescence quenching. The fluorescence intensity linearly decreases with the increase of H2O2 concentration in the range from 2 × 10 - 9 to 2 × 10 - 4 M with the detection limit of 6.8 × 10 - 10 M. The analytical method provides the advantage of sensitivity, stability and accuracy compared with present H2O2 sensors based on the pristine HRP. It has been successfully applied in the determination of H2O2 in real water samples. The study also opens a new avenue for modification of enzyme activity and stability that offers great promise in applications such as biological catalysis, biosensing and enzyme engineering.

Accuracy of a Plasmodium falciparum specific histidine-rich protein 2 rapid diagnostic test in the context of the presence of non-malaria fevers, prior anti-malarial use and seasonal malaria transmission.

PubMed

Kiemde, Francois; Bonko, Massa Dit Achille; Tahita, Marc Christian; Lompo, Palpouguini; Rouamba, Toussaint; Tinto, Halidou; van Hensbroek, Michael Boele; Mens, Petra F; Schallig, Henk D F H

2017-07-20

It remains challenging to distinguish malaria from other fever causing infections, as a positive rapid diagnostic test does not always signify a true active malaria infection. This study was designed to determine the influence of other causes of fever, prior anti-malarial treatment, and a possible seasonality of the performance of a PfHRP2 RDT for the diagnosis of malaria in children under-5 years of age living in a malaria endemic area. A prospective etiology study was conducted in 2015 among febrile children under 5 years of age in Burkina Faso. In order to assess the influence of other febrile illnesses, prior treatment and seasonality on the performance of a PfHRP2 RDT in diagnosing malaria, the RDT results were compared with the gold standard (expert microscopic diagnosis of Plasmodium falciparum) and test results were analysed by assuming that prior anti-malarial use and bacterial/viral infection status would have been known prior to testing. To assess bacterial and viral infection status blood, urine and stool samples were analysed. In total 683 blood samples were analysed with microscopy and RDT-PfHRP2. Plasmodium falciparum malaria was diagnosed in 49.8% (340/683) by microscopy compared to 69.5% (475/683) by RDT-PfHRP2. The RDT-PfHRP2 reported 29.7% (141/475) false positive results and 1.8% (6/340) false negative cases. The RDT-PfHRP2 had a high sensitivity (98.2%) and negative predictive value (97.1%), but a low specificity (58.9%) and positive predictive value (70.3%). Almost 50% of the alternative cause of fever were diagnosed by laboratory testing in the RDT false positive malaria group. The use of a malaria RDT-PfHRP2 in a malaria endemic area may cause misdiagnosis of the actual cause of fever due to false positive test results. The development of a practical diagnostic tool to screen for other causes of fever in malaria endemic areas is required to save lives.

Microfluidic biosensing systems. Part I. Development and optimisation of enzymatic chemiluminescent micro-biosensors based on silicon microchips.

PubMed

Davidsson, Richard; Genin, Frédéric; Bengtsson, Martin; Laurell, Thomas; Emnéus, Jenny

2004-10-01

Chemiluminescent (CL) enzyme-based flow-through microchip biosensors (micro-biosensors) for detection of glucose and ethanol were developed for the purpose of monitoring real-time production and release of glucose and ethanol from microchip immobilised yeast cells. Part I of this study focuses on the development and optimisation of the micro-biosensors in a microfluidic sequential injection analysis (microSIA) system. Glucose oxidase (GOX) or alcohol oxidase (AOX) was co-immobilised with horseradish peroxidase (HRP) on porous silicon flow through microchips. The hydrogen peroxide produced from oxidation of the corresponding analyte (glucose or ethanol) took part in the chemiluminescent (CL) oxidation of luminol catalysed by HRP enhanced by addition of p-iodophenol (PIP). All steps in the microSIA system, including control of syringe pump, multiposition valve (MPV) and data readout, were computer controlled. The influence of flow rate and luminol- and PIP concentration were investigated using a 2(3)-factor experiment using the GOX-HRP sensor. It was found that all estimated single factors and the highest order of interaction were significant. The optimum was found at 250 microM luminol and 150 microM PIP at a flow rate of 18 microl min(-1), the latter as a compromise between signal intensity and analysis time. Using the optimised system settings one sample was processed within 5 min. Two different immobilisation chemistries were investigated for both micro-biosensors based on 3-aminopropyltriethoxsilane (APTS)- or polyethylenimine (PEI) functionalisation followed by glutaraldehyde (GA) activation. GOX-HRP micro-biosensors responded linear in a log-log format within the range 10-1000 microM glucose. Both had an operational stability of at least 8 days, but the PEI-GOX-HRP sensor was more sensitive. The AOX-HRP micro-biosensors responded linear (log-log) in the range between 1 and 10 mM ethanol, but the PEI-AOX-HRP sensor was in general more sensitive. Both sensors had an operational stability of at least 8 h, but with a half-life of 2-3 days.

Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

PubMed

Danielson, Alex P; Bailey-Van Kuren, Dylan; Lucius, Melissa E; Makaroff, Katherine; Williams, Cameron; Page, Richard C; Berberich, Jason A; Konkolewicz, Dominik

2016-02-01

Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae

PubMed Central

2012-01-01

Background Herbaspirillum rubrisubalbicans was first identified as a bacterial plant pathogen, causing the mottled stripe disease in sugarcane. H. rubrisubalbicans can also associate with various plants of economic interest in a non pathogenic manner. Results A 21 kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization. Conclusions Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice. PMID:22672506

Two New Preyssler-Type Polyoxometalate-Based Coordination Polymers and Their Application in Horseradish Peroxidase Immobilization.

PubMed

Du, Jing; Cao, Mei-Da; Feng, Shu-Li; Su, Fang; Sang, Xiao-Jing; Zhang, Lan-Cui; You, Wan-Sheng; Yang, Mei; Zhu, Zai-Ming

2017-10-17

Enzyme immobilization is of increasing importance for biocatalysis, for which good supports are critical. Herein, two new Preyssler-type polyoxometalate (POM)-based coordination polymers, namely, {[Cu(H 2 biim) 2 ][{Cu(H 2 biim) 2 (μ-H 2 O)} 2 Cu(H 2 biim)(H 2 O) 2 ]H[({Cu(H 2 biim)(H 2 O) 2 } 0.5 ) 2 ((μ-C 3 HN 2 Cl 2 ){Cu(H 2 biim)} 2 ){Z(H 2 O)P 5 W 30 O 110 }]⋅x H 2 O} n (1: Z=Na, x=9; 2: Z=Ag, x=10; H 2 biim=2,2'-biimidazole) were designed and synthesized. Compounds 1 and 2 exhibit the same skeletons, which contain multiple Cu II complex fragments and penta-supported {ZP 5 W 30 } (Z=Na, Ag) clusters. They were first employed to immobilize horseradish peroxidase (HRP). Results show that compounds 1 and 2 are good supports for HRP immobilization, and exhibit higher enzyme loading, lower loading times, and excellent reusability. The immobilized HRP (HRP/1 or HRP/2) was further applied to detect H 2 O 2 , and good sensitivity, wide linear range, low detection limit, and fast response were achieved. This work shows that POM-based hybrid materials are a new kind of promising support for enzyme immobilization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative analysis of the Hrp pathogenicity island of Rubus- and Spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element.

PubMed

Mann, Rachel A; Blom, Jochen; Bühlmann, Andreas; Plummer, Kim M; Beer, Steven V; Luck, Joanne E; Goesmann, Alexander; Frey, Jürg E; Rodoni, Brendan C; Duffy, Brion; Smits, Theo H M

2012-08-01

The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation. Copyright © 2012 Elsevier B.V. All rights reserved.

Performances of malaria P.f/Pan rapid test device Acon® (Pf HRP2/pan aldolase) and malaria Pf rapid test device Acon® (Pf HRP2) for the diagnosis of malaria in adults and children living in Gabon, Central Africa.

PubMed

Bouyou Akotet, Marielle Karine; Mawili-Mboumba, Denise Patricia; Madoungou, Blondel; Kombila, Maryvonne

2013-09-01

The Malaria Pf Rapid Test Device Acon® (Acon Labs) and the pan HRP2/aldolase RDT, Malaria P.f/Pan Rapid Test Device Acon® (Acon Labs), performances were evaluated for malaria species diagnosis in 592 febrile patients living in Gabon using microscopy as gold standard. Sensitivities were equal or above 96.0% for Plasmodium falciparum detection, of 62.5% for non-P. falciparum malaria species detection and higher in younger children (100%). Negative predictive values were greater than 97.0%. Acon®HRP2 had a higher specificity (96.6%) and lower false-positive (FP) rate (9.3%) compared to Acon®Pf/Pan, which had a specificity of 87.3% and a FP rate of 27.1% (P < 0.01). Overall, 32.5% of all Acon® Pf/Pan tests resulted in a "faint band" with only 2 resulted from samples with a parasitemia below 100 p/μL. The accuracy of Acon®HRP2 RDT for the diagnosis of P. falciparum infection is confirmed. However, the high FP rate observed with Acon®Pf/Pan is a limitation for its use. Copyright © 2013 Elsevier Inc. All rights reserved.

Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

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

Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.

2007-07-01

A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´-more » tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.« less

Catalase-like activity of horseradish peroxidase: relationship to enzyme inactivation by H2O2.

PubMed Central

Hernández-Ruiz, J; Arnao, M B; Hiner, A N; García-Cánovas, F; Acosta, M

2001-01-01

H2O2 is the usual oxidizing substrate of horseradish peroxidase C (HRP-C). In the absence in the reaction medium of a one-electron donor substrate, H2O2 is able to act as both oxidizing and reducing substrate. However, under these conditions the enzyme also undergoes a progressive loss of activity. There are several pathways that maintain the activity of the enzyme by recovering the ferric form, one of which is the decomposition of H2O2 to molecular oxygen in a similar way to the action of catalase. This production of oxygen has been kinetically characterized with a Clark-type electrode coupled to an oxygraph. HRP-C exhibits a weak catalase-like activity, the initial reaction rate of which is hyperbolically dependent on the H2O2 concentration, with values for K(2) (affinity of the first intermediate, compound I, for H2O2) and k(3) (apparent rate constant controlling catalase activity) of 4.0 +/- 0.6 mM and 1.78 +/- 0.12 s(-1) respectively. Oxygen production by HRP-C is favoured at pH values greater than approx. 6.5; under similar conditions HRP-C is also much less sensitive to inactivation during incubations with H2O2. We therefore suggest that this pathway is a major protective mechanism of HRP-C against such inactivation. PMID:11171085

Activation of Hydrogen Peroxide in Horseradish Peroxidase Occurs within ∼200 μs Observed by a New Freeze-Quench Device

PubMed Central

Tanaka, Motomasa; Matsuura, Koji; Yoshioka, Shiro; Takahashi, Satoshi; Ishimori, Koichiro; Hori, Hiroshi; Morishima, Isao

2003-01-01

To observe the formation process of compound I in horseradish peroxidase (HRP), we developed a new freeze-quench device with ∼200 μs of the mixing-to-freezing time interval and observed the reaction between HRP and hydrogen peroxide (H2O2). The developed device consists of a submillisecond solution mixer and rotating copper or silver plates cooled at 77 K; it freezes the small droplets of mixed solution on the surface of the rotating plates. The ultraviolet-visible spectra of the sample quenched at ∼1 ms after the mixing of HRP and H2O2 suggest the formation of compound I. The electron paramagnetic resonance spectra of the same reaction quenched at ∼200 μs show a convex peak at g = 2.00, which is identified as compound I due to its microwave power and temperature dependencies. The absence of ferric signals in the electron paramagnetic resonance spectra of the quenched sample indicates that compound I is formed within ∼200 μs after mixing HRP and H2O2. We conclude that the activation of H2O2 in HRP at ambient temperature completes within ∼200 μs. The developed device can be generally applied to investigate the electronic structures of short-lived intermediates of metalloenzymes. PMID:12609902

Gel-based immunotest for simultaneous detection of 2,4,6-trichlorophenol and ochratoxin A in red wine.

PubMed

Beloglazova, N V; Goryacheva, I Yu; Rusanova, T Yu; Yurasov, N A; Galve, R; Marco, M-P; De Saeger, S

2010-07-05

A new rapid method which allows simultaneous one step detection of two analytes of different nature (2,4,6,-trichlorophenol (TCP) and ochratoxin A (OTA)) in red wine was developed. It was based on a column test with three separate immunolayers: two test layers and one control layer. Each layer consisted of sepharose gel with immobilized anti-OTA (OTA test layer), anti-TCP (TCP test layer) or anti-HRP (control layer) antibodies. Analytes bind to the antibodies in the corresponding test layer while sample flows through the column. Then a mixture of OTA-HRP and TCP-HRP in appropriate dilutions was used, followed by the application of chromogenic substrate. Colour development of the test layer occurred when the corresponding analyte was absent in the sample. HRP-conjugates bound to anti-HRP antibody in the control layer independently of presence or absence of analytes and a blue colour developed in the control layer. Cut-off values for both analytes were 2 microg L(-1). The described method was applied to the simultaneous detection of TCP and OTA in wine samples. To screen the analytes in red wine samples, clean-up columns were used for sample pre-treatment in combination with the test column. Results were confirmed by chromatographic methods. Copyright 2010 Elsevier B.V. All rights reserved.

Genetic variation of pfhrp2 in Plasmodium falciparum isolates from Yemen and the performance of HRP2-based malaria rapid diagnostic test.

PubMed

Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Al-Delaimy, Ahmed K; Nasr, Nabil A; Dawaki, Salwa; Abdulsalam, Awatif M; Ithoi, Init; Lau, Yee Ling; Fong, Mun Yik; Surin, Johari

2015-07-22

The genetic variation in the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene that may compromise the use of pfhrp2-based rapid diagnostic tests (RDTs) for the diagnosis of malaria was assessed in P. falciparum isolates from Yemen. This study was conducted in Hodeidah and Al-Mahwit governorates, Yemen. A total of 622 individuals with fever were examined for malaria by CareStart malaria HRP2-RDT and Giemsa-stained thin and thick blood films. The Pfhrp2 gene was amplified and sequenced from 180 isolates, and subjected to amino acid repeat types analysis. A total of 188 (30.2%) participants were found positive for P. falciparum by the RDT. Overall, 12 different amino acid repeat types were identified in Yemeni isolates. Six repeat types were detected in all the isolates (100%) namely types 1, 2, 6, 7, 10 and 12 while types 9 and 11 were not detected in any of the isolates. Moreover, the sensitivity and specificity of the used PfHRP2-based RDTs were high (90.5% and 96.1%, respectively). The present study provides data on the genetic variation within the pfhrp2 gene, and its potential impact on the PfHRP2-based RDTs commonly used in Yemen. CareStart Malaria HRP2-based RDT showed high sensitivity and specificity in endemic areas of Yemen.

Lifetime Surveillance of Astronaut Health (LSAH) / Life Sciences Data Archive (LSDA) Data Request Helpdesk

NASA Technical Reports Server (NTRS)

Young, Millennia; Van Baalen, Mary

2016-01-01

This session is intended to provide to HRP IWS attendees instant feedback on archived astronaut data, including such topics as content of archives, access, request processing, and data format. Members of the LSAH and LSDA teams will be available at a 'help desk' during the poster sessions to answer questions from researchers.

Behavioral Health and Performance Laboratory Standard Measures (BHP-SM)

NASA Technical Reports Server (NTRS)

Williams, Thomas J.; Cromwell, Ronita

2017-01-01

The Spaceflight Standard Measures is a NASA Johnson Space Center Human Research Project (HRP) project that proposes to collect a set of core measurements, representative of many of the human spaceflight risks, from astronauts before, during and after long-duration International Space Station (ISS) missions. The term "standard measures" is defined as a set of core measurements, including physiological, biochemical, psychosocial, cognitive, and functional, that are reliable, valid, and accepted in terrestrial science, are associated with a specific and measurable outcome known to occur as a consequence of spaceflight, that will be collected in a standardized fashion from all (or most) crewmembers. While such measures might be used to define standards of health and performance or readiness for flight, the prime intent in their collection is to allow longitudinal analysis of multiple parameters in order to answer a variety of operational, occupational, and research-based questions. These questions are generally at a high level, and the approach for this project is to populate the standard measures database with the smallest set of data necessary to indicate further detailed research is required. Also included as standard measures are parameters that are not outcome-based in and of-themselves, but provide ancillary information that supports interpretation of the outcome measures, e.g., nutritional assessment, vehicle environmental parameters, crew debriefs, etc. The project's main aim is to ensure that an optimized minimal set of measures is consistently captured from all ISS crewmembers until the end of Station in order to characterize the human in space. -This allows the HRP to identify, establish, and evaluate a common set of measures for use in spaceflight and analog research to: develop baselines, systematically characterize risk likelihood and consequences, and assess effectiveness of countermeasures that work for behavioral health and performance risk factors. -By standardizing the battery of measures on all crewmembers, it will allow the HRP to evaluate countermeasures that work for one physiological system and ensure another system is not negatively affected. -These measures, named "Standard Measures," will serve as a data repository and be available to other studies under data sharing agreements.

Space Human Factors Engineering Gap Analysis Project Final Report

NASA Technical Reports Server (NTRS)

Hudy, Cynthia; Woolford, Barbara

2006-01-01

Humans perform critical functions throughout each phase of every space mission, beginning with the mission concept and continuing to post-mission analysis (Life Sciences Division, 1996). Space missions present humans with many challenges - the microgravity environment, relative isolation, and inherent dangers of the mission all present unique issues. As mission duration and distance from Earth increases, in-flight crew autonomy will increase along with increased complexity. As efforts for exploring the moon and Mars advance, there is a need for space human factors research and technology development to play a significant role in both on-orbit human-system interaction, as well as the development of mission requirements and needs before and after the mission. As part of the Space Human Factors Engineering (SHFE) Project within the Human Research Program (HRP), a six-month Gap Analysis Project (GAP) was funded to identify any human factors research gaps or knowledge needs. The overall aim of the project was to review the current state of human factors topic areas and requirements to determine what data, processes, or tools are needed to aid in the planning and development of future exploration missions, and also to prioritize proposals for future research and technology development.

2013 Advanced Environmental Health/Advanced Food Technology Standing Review Panel Final Report

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

The 2013 Advanced Environmental Health/Advanced Food Technology (AEH/AFT) Standing Review Panel (from here on referred to as the SRP) participated in a WebEx/teleconference with members of the Space Human Factors and Habitability (SHFH) Element, representatives from the Human Research Program (HRP), and NASA Headquarters on November 22, 2013 (list of participants is in Section IX of this report). The SRP reviewed the updated research plans for the Risk of Adverse Health Effects Due to Alterations in Host-Microorganism Interactions (Host Microbe Risk) and the Risk of Performance Decrement and Crew Illness Due to an Inadequate Food System (Food Risk). The SRP also received a status update on the Risk of Adverse Health Effects of Exposure to Dust and Volatiles during Exploration of Celestial Bodies (Dust Risk). Overall, the SRP was impressed with the strong research plans presented by the scientists and staff associated with the SHFH Element. The SRP also thought that the updated research plans were thorough, well organized, and presented in a comprehensive manner. The SRP agrees with the changes made to the Host Microbe Risk and Food Risk portfolios and thinks that the targets for Gap closure are appropriate.

2015 Occupant Protection Standing Review Panel

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2015-01-01

The 2015 Occupant Protection (OP) Risk Standing Review Panel (from here on referred to as the SRP) participated in a WebEx/teleconference with members of the Space Human Factors and Habitability (SHFH) Element, representatives from the Human Research Program (HRP), NASA Headquarters, and NASA Research and Education Support Services on November 3, 2015 (list of participants is in Section VII of this report). The SRP reviewed the updated research plans for the Risk of Injury from Dynamic Loads (OP Risk). The SRP agrees that the Gaps are relevant and appropriate to mitigate the injury risk. All the appropriate and relevant Tasks have been identified to fill the Gaps. Depending upon the findings, additional tasks may need to be identified or modified. Excellent progress has been made since the 2014 SRP meeting. Publications in peer-reviewed journals validate the scientific merit of the research findings. As detailed in this report, the SRP has specific comments, guidance, and information in the following areas: human finite element modeling, human vs. surrogate dynamic responses, chest injury risk curves, matched pair testing of Test device for Human Occupant Restraint (THOR) and Hybrid III, and disc herniation risk analysis.

Rapidly responsive silk fibroin hydrogels as an artificial matrix for the programmed tumor cells death.

PubMed

Ribeiro, Viviana P; Silva-Correia, Joana; Gonçalves, Cristiana; Pina, Sandra; Radhouani, Hajer; Montonen, Toni; Hyttinen, Jari; Roy, Anirban; Oliveira, Ana L; Reis, Rui L; Oliveira, Joaquim M

2018-01-01

Timely and spatially-regulated injectable hydrogels, able to suppress growing tumors in response to conformational transitions of proteins, are of great interest in cancer research and treatment. Herein, we report rapidly responsive silk fibroin (SF) hydrogels formed by a horseradish peroxidase (HRP) crosslinking reaction at physiological conditions, and demonstrate their use as an artificial biomimetic three-dimensional (3D) matrix. The proposed SF hydrogels presented a viscoelastic nature of injectable hydrogels and spontaneous conformational changes from random coil to β-sheet conformation under physiological conditions. A human neuronal glioblastoma (U251) cell line was used for screening cell encapsulation and in vitro evaluation within the SF hydrogels. The transparent random coil SF hydrogels promoted cell viability and proliferation up to 10 days of culturing, while the crystalline SF hydrogels converted into β-sheet structure induced the formation of TUNEL-positive apoptotic cells. Therefore, this work provides a powerful tool for the investigation of the microenvironment on the programed tumor cells death, by using rapidly responsive SF hydrogels as 3D in vitro tumor models.

Human Resource Planning: An Introduction. Report 312.

ERIC Educational Resources Information Center

Reilly, Peter

This report is designed to give readers an introduction to the principles of human resource planning (HRP) and the areas in which it can be used, including those facing today's managers. Chapter 1 outlines why some organizations no longer plan, describes the background of change and uncertainty that discouraged them, and defines HRP. Chapter 2…

A new gene in A. rubens: A sea star Ig kappa gene.

PubMed

Vincent, Nadine; Osteras, Magne; Otten, Patricia; Leclerc, Michel

2014-12-01

The sea star Asterias rubens reacts specifically to the antigen:HRP (horse-radish peroxydase) and produces an antibody anti-HRP. We previously identified a candidate Ig kappa gene corresponding to this manuscript. We show now the gene referred to as: "sea star Ig kappa gene in its specificity".

Nanobody based immunoassay for human soluble epoxide hydrolase detection using polyHRP for signal enhancement—the rediscovery of polyHRP

USDA-ARS?s Scientific Manuscript database

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain and multiple cardiovascular related diseases. A variable domain of a heavy chain only antibody (termed sdAb, nanobody or VHH) possesses advantages of small size, high ...

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicola M2.

PubMed

Álvarez-Mejía, César; Rodríguez-Ríos, Dalia; Hernández-Guzmán, Gustavo; López-Ramírez, Varinia; Valenzuela-Soto, Humberto; Marsch, Rodolfo

2015-01-01

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection.

Characterization of the hrpZ gene from Pseudomonas syringae pv. maculicolaM2

PubMed Central

Álvarez-Mejía, César; Rodríguez-Ríos, Dalia; Hernández-Guzmán, Gustavo; López-Ramírez, Varinia; Valenzuela-Soto, Humberto; Marsch, Rodolfo

2015-01-01

Pseudomonas syringae pv. maculicola is a natural pathogen of members of the Brassicaceae plant family. Using a transposon-based mutagenesis strategy in Pseudomonas syringaepv. maculicola M2 (PsmM2), we conducted a genetic screen to identify mutants that were capable of growing in M9 medium supplemented with a crude extract from the leaves of Arabidopsis thaliana. A mutant containing a transposon insertion in the hrpZ gene (PsmMut8) was unable to infect adult plants from Arabidopsis thaliana or Brassica oleracea, suggesting a loss of pathogenicity. The promotorless cat reporter present in the gene trap was expressed if PsmMut8 was grown in minimal medium (M9) supplemented with the leaf extract but not if grown in normal rich medium (KB). We conducted phylogenetic analysis using hrpAZB genes, showing the classical 5-clade distribution, and nucleotide diversity analysis, showing the putative position for selective pressure in this operon. Our results indicate that the hrpAZB operon from Pseudomonas syringaepv. maculicola M2 is necessary for its pathogenicity and that its diversity would be under host-mediated diversifying selection. PMID:26413080

Enzymatic oxidation of rutin by horseradish peroxidase: kinetic mechanism and identification of a dimeric product by LC-Orbitrap mass spectrometry.

PubMed

Savic, Sasa; Vojinovic, Katarina; Milenkovic, Sanja; Smelcerovic, Andrija; Lamshoeft, Marc; Petronijevic, Zivomir

2013-12-15

Flavonoid oxidation is important issue in food processing and quality. The kinetic mechanism of enzymatic oxidation of rutin by horseradish peroxidase (HRP) was studied. Rutin oxidation reaction was followed by recording of spectral changes over the time at 360 nm. The studied oxidation is mostly enzymatic and less part non-enzymatic. The reaction with HRP has a higher rate compared with the reaction without of HRP, whereby is part of non-enzymatic reaction about 10% of the total reaction. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation, and it was found that investigated reactions of rutin oxidation by HRP take place in a ping-pong kinetic mechanism. High resolution HPLC-MS analysis of the mixture of oxidized products of rutin revealed the presence of rutin dimer. Because of widely distribution of rutin as well as presence of peroxidases and hydrogen peroxide in fresh foods identification of this enzymatic modification product can be beneficial for foods quality and safety. Copyright © 2013 Elsevier Ltd. All rights reserved.

Accelerated Innovation Pilot

NASA Technical Reports Server (NTRS)

Davis, Jeffrey

2012-01-01

Opportunities: I. Engage NASA team (examples) a) Research and technology calls . provide suggestions to AES, HRP, OCT. b) Use NASA@Work to solicit other ideas; (possibly before R+D calls). II. Stimulate collaboration (examples) a) NHHPC. b) Wharton Mack Center for Technological Innovation (Feb 2013). c) International ] DLR ] :envihab (July 2013). d) Accelerated research models . NSF, Myelin Repair Foundation. III. Engage public Prizes (open platform: InnoCentive, yet2.com, NTL; Rice Business Plan, etc.) IV. Use same methods to engage STEM.

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer.

PubMed

Rahman, Md Aminur; Park, Deog-Soo; Shim, Yoon-Bo

2004-07-15

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2':5',2"-terthiophene-3'-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H(2)O(2) in a choline solution at +0.6V. The other one modified with ChO/HRP utilized the reduction process of H(2)O(2) in a choline solution at -0.2V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0 x 10(-6) to 8.0 x 10(-5) M and the other based on ChO/CPME from 1.0 x 10(-6) to 5.0 x 10(-5) M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0 x 10(-7) and 4.0 x 10(-7) M, respectively. The response time of sensors was less than 5s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.

Identification of Optimal Epitopes for Plasmodium falciparum Rapid Diagnostic Tests That Target Histidine-Rich Proteins 2 and 3

PubMed Central

Lee, Nelson; Gatton, Michelle L.; Pelecanos, Anita; Bubb, Martin; Gonzalez, Iveth; Bell, David; Cheng, Qin

2012-01-01

Rapid diagnostic tests (RDTs) represent important tools to diagnose malaria infection. To improve understanding of the variable performance of RDTs that detect the major target in Plasmodium falciparum, namely, histidine-rich protein 2 (HRP2), and to inform the design of better tests, we undertook detailed mapping of the epitopes recognized by eight HRP-specific monoclonal antibodies (MAbs). To investigate the geographic skewing of this polymorphic protein, we analyzed the distribution of these epitopes in parasites from geographically diverse areas. To identify an ideal amino acid motif for a MAb to target in HRP2 and in the related protein HRP3, we used a purpose-designed script to perform bioinformatic analysis of 448 distinct gene sequences from pfhrp2 and from 99 sequences from the closely related gene pfhrp3. The frequency and distribution of these motifs were also compared to the MAb epitopes. Heat stability testing of MAbs immobilized on nitrocellulose membranes was also performed. Results of these experiments enabled the identification of MAbs with the most desirable characteristics for inclusion in RDTs, including copy number and coverage of target epitopes, geographic skewing, heat stability, and match with the most abundant amino acid motifs identified. This study therefore informs the selection of MAbs to include in malaria RDTs as well as in the generation of improved MAbs that should improve the performance of HRP-detecting malaria RDTs. PMID:22259210

Novel monoclonal antibodies against Plasmodium falciparum histidine-rich protein 2: development and application in rapid diagnostic tests of malaria in hyperendemic regions of China and Myanmar.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Li, Wenmei; Xie, Mingquan; Luo, Xiaochun; Liu, Hui

2015-05-12

Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT). The recombinant protein was expressed in soluble form in E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 × 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar. The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200-2000, and > 2000 parasites/μL was 87.5, 98.7, and 100%, respectively. These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities.

Evaluation of a rapid diagnostic test (CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test) for the diagnosis of malaria in a reference setting

PubMed Central

2010-01-01

Background Malaria Rapid Diagnostic Tests (RDTs) are widely used for diagnosing malaria. The present retrospective study evaluated the CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test targeting the Plasmodium falciparum specific antigen histidine-rich protein (HRP-2) and the pan-Plasmodium antigen lactate dehydrogenase (pLDH) in a reference setting. Methods The CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test was evaluated on a collection of samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included were P. falciparum (n = 320), Plasmodium vivax (n = 76), Plasmodium ovale (n = 76), Plasmodium malariae (n = 23) and Plasmodium negative samples (n = 95). Results Overall sensitivity for the detection of P. falciparum was 88.8%, increasing to 94.3% and 99.3% at parasite densities above 100 and 1,000/μl respectively. For P. vivax, P. ovale and P. malariae, overall sensitivities were 77.6%, 18.4% and 30.4% respectively. For P. vivax sensitivity reached 90.2% for parasite densities above 500/μl. Incorrect species identification occurred in 11/495 samples (2.2%), including 8/320 (2.5%) P. falciparum samples which generated only the pan-pLDH line. For P. falciparum samples, 205/284 (72.2%) HRP-2 test lines had strong or medium line intensities, while for all species the pan-pLDH lines were less intense, especially in the case of P. ovale. Agreement between observers was excellent (kappa values > 0.81 for positive and negative readings) and test results were reproducible. The test was easy to perform with good clearing of the background. Conclusion The CareStart™ Malaria HRP-2/pLDH (Pf/pan) Combo Test performed well for the detection of P. falciparum and P. vivax, but sensitivities for P. ovale and P. malariae were poor. PMID:20565816

Evaluation of a rapid diagnostic test (CareStart Malaria HRP-2/pLDH (Pf/pan) Combo Test) for the diagnosis of malaria in a reference setting.

PubMed

Maltha, Jessica; Gillet, Philippe; Bottieau, Emmanuel; Cnops, Lieselotte; van Esbroeck, Marjan; Jacobs, Jan

2010-06-18

Malaria Rapid Diagnostic Tests (RDTs) are widely used for diagnosing malaria. The present retrospective study evaluated the CareStart Malaria HRP-2/pLDH (Pf/pan) Combo Test targeting the Plasmodium falciparum specific antigen histidine-rich protein (HRP-2) and the pan-Plasmodium antigen lactate dehydrogenase (pLDH) in a reference setting. The CareStart Malaria HRP-2/pLDH (Pf/pan) Combo Test was evaluated on a collection of samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included were P. falciparum (n = 320), Plasmodium vivax (n = 76), Plasmodium ovale (n = 76), Plasmodium malariae (n = 23) and Plasmodium negative samples (n = 95). Overall sensitivity for the detection of P. falciparum was 88.8%, increasing to 94.3% and 99.3% at parasite densities above 100 and 1,000/microl respectively. For P. vivax, P. ovale and P. malariae, overall sensitivities were 77.6%, 18.4% and 30.4% respectively. For P. vivax sensitivity reached 90.2% for parasite densities above 500/microl. Incorrect species identification occurred in 11/495 samples (2.2%), including 8/320 (2.5%) P. falciparum samples which generated only the pan-pLDH line. For P. falciparum samples, 205/284 (72.2%) HRP-2 test lines had strong or medium line intensities, while for all species the pan-pLDH lines were less intense, especially in the case of P. ovale. Agreement between observers was excellent (kappa values > 0.81 for positive and negative readings) and test results were reproducible. The test was easy to perform with good clearing of the background. The CareStart Malaria HRP-2/pLDH (Pf/pan) Combo Test performed well for the detection of P. falciparum and P. vivax, but sensitivities for P. ovale and P. malariae were poor.

Evaluation of the Palutop+4 malaria rapid diagnostic test in a non-endemic setting.

PubMed

van Dijk, David P J; Gillet, Philippe; Vlieghe, Erika; Cnops, Lieselotte; van Esbroeck, Marjan; Jacobs, Jan

2009-12-12

Palutop+4 (All. Diag, Strasbourg, France), a four-band malaria rapid diagnostic test (malaria RDT) targeting the histidine-rich protein 2 (HRP-2), Plasmodium vivax-specific parasite lactate dehydrogenase (Pv-pLDH) and pan Plasmodium-specific pLDH (pan-pLDH) was evaluated in a non-endemic setting on stored whole blood samples from international travellers suspected of malaria. Microscopy corrected by PCR was the reference method. Samples include those infected by Plasmodium falciparum (n = 323), Plasmodium vivax (n = 97), Plasmodium ovale (n = 73) and Plasmodium malariae (n = 25) and 95 malaria negative samples. The sensitivities for the diagnosis of P. falciparum, P. vivax, P. malariae and P. ovale were 85.1%, 66.0%, 32.0% and 5.5%. Sensitivities increased at higher parasite densities and reached 90.0% for P. falciparum >100/microl and 83.8% for P. vivax > 500/microl. Fourteen P. falciparum samples reacted with the Pv-pLDH line, one P. vivax sample with the HRP-2 line, and respectively two and four P. ovale and P. malariae samples reacted with the HRP-2 line. Two negative samples gave a signal with the HRP-2 line. Faint and weak line intensities were observed for 129/289 (44.6%) HRP-2 lines in P. falciparum samples, for 50/64 (78.1%) Pv-pLDH lines in P. vivax samples and for 9/13 (69.2%) pan-pLDH lines in P. ovale and P. malariae samples combined. Inter-observer reliabilities for positive and negative readings were excellent for the HRP-2 and Pv-pLDH lines (overall agreement > 92.0% and kappa-values for each pair of readers >or= 0.88), and good for the pan-pLDH line (85.5% overall agreement and kappa-values >or= 0.74). Palutop+4 performed moderately for the detection of P. falciparum and P. vivax, but sensitivities were lower than those of three-band malaria RDTs.

Use of a three-band HRP2/pLDH combination rapid diagnostic test increases diagnostic specificity for falciparum malaria in Ugandan children.

PubMed

Hawkes, Michael; Conroy, Andrea L; Opoka, Robert O; Namasopo, Sophie; Liles, W Conrad; John, Chandy C; Kain, Kevin C

2014-02-01

Rapid diagnostic tests (RDTs) for malaria provide a practical alternative to light microscopy for malaria diagnosis in resource-limited settings. Three-band RDTs incorporating two parasite antigens may have enhanced diagnostic specificity, relative to two-band RDTs with a single parasite antigen (typically histidine-rich protein 2 [HRP2]). Phase 1: 2,000 children, two months to five years of age, admitted to a referral hospital in Jinja, Uganda, with acute febrile illness were enrolled. A WHO highly rated three-band RDT was compared to light microscopy of thick peripheral blood films read by local expert microscopists.Phase 2: the three-band RDT was used as a screening tool for inclusion of patients in a clinical trial, and subjects with three positive RDT bands were tested by microscopy using blood samples drawn in parallel. Discordant results were adjudicated by PCR. Phase 1: 1,648 children had both a RDT and peripheral blood smear performed. The specificity of a RDT with all three bands positive was 82% (95% CI: 79-85%) compared to 62% (95% CI: 59-66%) for HRP2 alone. The sensitivity was 88% (95% CI: 85-89%) and 94% (95% CI: 92-95%) for three-band positive RDT and HRP2 antigen, respectively. 119 patients (7.2%) had a positive HRP2 band, but negative parasite lactate dehydrogenase (pLHD) band and negative peripheral smear, and 72 (61%) of these had received pre-treatment with anti-malarials, suggesting a false positive HRP2 result (p = 0.002).Phase 2: the positive predictive value (PPV) of the three-band RDT was 94% (95% CI 89%-97%) using microscopy as the reference standard. However, microscopy-discordant results were shown to be positive for P. falciparum by PCR in all cases, suggesting that the PPV was in fact higher. The pLDH antigen on three-band RDTs, used in combination with HRP2, provides added diagnostic specificity for malaria parasitaemia and may be useful to distinguish acute infection from recently treated infection. In situations where diagnostic specificity is desirable (e.g., for selection of malaria-infected participants in clinical trials), a three-band RDT should be considered in a sub-Saharan African setting.

Vision restoration therapy (VRT) efficacy as assessed by comparative perimetric analysis and subjective questionnaires.

PubMed

Sabel, Bernhard A; Kenkel, Sigrid; Kasten, Erich

2004-01-01

We wished to evaluate the efficacy of vision restoration therapy (VRT) in patients with post-chiasmatic brain damage using different functional perimetric tests. These were compared with measures of subjective vision and reaction time. An open trial was conducted with hemianopia/scotoma (n=16) patients. Before and after 6 months of VRT results of high resolution (HRP) and Tuebingen automated perimetry (TAP) were evaluated and compared to performance in a Scanning Laser Ophthalmoscope (SLO) as previously reported. Whereas TAP and HRP used above-threshold or near-threshold individual target stimuli on grey background, the SLO used a psychophysical task of detection of three black targets (reverse stimulus) on bright red, patterned background. Subjective testimonials of activities of daily living (ADL) were probed with questionnaires and interviews. Before VRT, the visual field border as assessed by SLO was located significantly closer to the vertical midline than the HRP and TAP border (border mismatch). After VRT the SLO border was still unchanged whereas HRP measurements revealed significant border shifts due to improved stimulus detection (p<0.0001) and improved reaction time (p<0.005) . Fewer misses were also observed in both eyes with TAP (p<0.01) which was primarily due to a significant shift of the absolute borders. Thus, VRT potentiated the mismatch between the SLO borders and the HRP/TAP borders. Fixation performance and the blind spot position remained unchanged after VRT. ADL ratings in the questionnaire improved significantly after VRT which was confirmed by independent patient testimonials. We replicated earlier findings that VRT improves stimulus detection in HRP and TAP perimetry which were accompanied by subjective, visual improvements. These changes are not caused by fixation or eye movement artifacts. Because the SLO border was located significantly closer to the vertical midline before VRT ("border mismatch") and, in contrast to HRP and TAP, did not change after VRT, we interpret this border mismatch to indicate that the SLO task was too difficult to perform and thus insensitive to VRT effects. Significant reaction time improvements indicate that plasticity of temporal processing might play an important role in vision restoration after brain damage. A further description of the precise psychophysical nature of the restored areas of residual vision is now warranted.

Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

PubMed Central

2011-01-01

Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and as a deterrent effect of HrpNEa treatment in WT Arabidopsis rather than the atpp2-a1/E/142 mutant suggest that AtPP2-A1 plays a role in plant resistance to the insect, particularly at the phloem-feeding stage. The accompanied change of aphid population in leaf colonies suggests that the function of AtPP2-A1 is related to colonization of the plant. PMID:21226963

Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae.

PubMed

Zhang, Chunling; Shi, Haojie; Chen, Lei; Wang, Xiaomeng; Lü, Beibei; Zhang, Shuping; Liang, Yuan; Liu, Ruoxue; Qian, Jun; Sun, Weiwei; You, Zhenzhen; Dong, Hansong

2011-01-13

Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. The repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and as a deterrent effect of HrpNEa treatment in WT Arabidopsis rather than the atpp2-a1/E/142 mutant suggest that AtPP2-A1 plays a role in plant resistance to the insect, particularly at the phloem-feeding stage. The accompanied change of aphid population in leaf colonies suggests that the function of AtPP2-A1 is related to colonization of the plant.

Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in São Tomé.

PubMed

Lee, Pei-Wen; Ji, Dar-Der; Liu, Chia-Tai; Rampao, Herodes S; do Rosario, Virgilio E; Lin, I-Feng; Shaio, Men-Fang

2012-12-06

A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up.

Application of loop-mediated isothermal amplification for malaria diagnosis during a follow-up study in São Tomé

PubMed Central

2012-01-01

Background A reliable and simple test for the detection of malaria parasite is crucial in providing effective treatment and therapeutic follow-up, especially in malaria elimination programmes. A comparison of four methods, including nested polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were used for the malaria diagnosis and treatment follow-up in São Tomé and Príncipe, during a successful pre-elimination campaign. Method During the period September to November 2009, blood samples from 128 children (five to 14 years old) with temperature ≥38°C (tympanic) in the District of Agua Grande were examined using four different methods, i.e., histidine-rich protein 2 (HRP-2) based rapid diagnostic tests (HRP-2-RDTs), optical microscopy, nested PCR, and LAMP. First-line treatment with artesunate-amodiaquine was given for uncomplicated malaria and intravenous quinine was given for complicated malaria. Children with persistent positivity for malaria by microscopy, or either by nested PCR, or by LAMP on day 7 were given second-line treatment with artemether-lumefantrine. Treatment follow-up was made weekly, for up to four weeks. Results On day 0, positive results for HRP-2-RDTs, microscopy, nested PCR, and LAMP, were 68(53%), 47(37%), 64(50%), and 65(51%), respectively. When nested PCR was used as a reference standard, only LAMP was comparable; both HRP-2-RDTs and microscopy had moderate sensitivity; HRP-2-RDTs had poor positive predictive value (PPV) and a moderate negative predictive value (NPV) for the treatment follow-up. Seventy-one children with uncomplicated malaria and eight children with complicated falciparum malaria were diagnosed based on at least one positive result from the four tests as well as clinical criteria. Twelve of the 79 children receiving first-line treatment had positive results by nested PCR on day 7 (nested PCR-corrected day 7 cure rate was 85%). After the second-line treatment, nested PCR/LAMP-corrected day 28 cure rate was 83% for these 12 children. Conclusions HRP-2-RDTs have similar sensitivity as microscopy but less specificity. However, as compared to nested PCR, the poor sensitivity of HRP-2-RDTs indicates that low parasitaemia may not be detected after treatment, as well as the low specificity of HRP-2-RDTs indicates it cannot be applied for treatment follow-up. LAMP has similar sensitivity and specificity to nested PCR. With high PPV and NPV, LAMP is simpler and faster as compared to nested PCR with the advantage of detecting low parasitaemia becoming a potential point-of-care test for treatment follow-up. PMID:23217163

Technology Development to Support Human Health and Performance in Exploration Beyond Low Earth Orbit

NASA Technical Reports Server (NTRS)

Kundrot, C.E.; Steinberg, S. L.; Charles, J. B.

2011-01-01

In the course of defining the level of risks and mitigating the risks for exploration missions beyond low Earth orbit, NASA s Human Research Program (HRP) has identified the need for technology development in several areas. Long duration missions increase the risk of serious medical conditions due to limited options for return to Earth; no resupply; highly limited mass, power, volume; and communication delays. New space flight compatible medical capabilities required include: diagnostic imaging, oxygen concentrator, ventilator, laboratory analysis (saliva, blood, urine), kidney stone diagnosis & treatment, IV solution preparation and delivery. Maintenance of behavioral health in such an isolated, confined and extreme environment requires new sensory stimulation (e.g., virtual reality) technology. Unobtrusive monitoring of behavioral health and treatment methods are also required. Prolonged exposure to weightlessness deconditions bone, muscle, and the cardiovascular system. Novel exercise equipment or artificial gravity are necessary to prevent deconditioning. Monitoring of the degree of deconditioning is required to ensure that countermeasures are effective. New technologies are required in all the habitable volumes (e.g., suit, capsule, habitat, exploration vehicle, lander) to provide an adequate food system, and to meet human environmental standards for air, water, and surface contamination. Communication delays require the crew to be more autonomous. Onboard decision support tools that assist crew with real-time detection and diagnosis of vehicle and habitat operational anomalies will enable greater autonomy. Multi-use shield systems are required to provide shielding from solar particle events. The HRP is pursuing the development of these technologies in laboratories, flight analog environments and the ISS so that the human health and performance risks will be acceptable with the available resources.

Over-expression of the Pseudomonas syringae harpin-encoding gene hrpZm confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean.

PubMed

Du, Qian; Yang, Xiangdong; Zhang, Jinhua; Zhong, Xiaofang; Kim, Kyung Seok; Yang, Jing; Xing, Guojie; Li, Xiaoyu; Jiang, Zhaoyuan; Li, Qiyun; Dong, Yingshan; Pan, Hongyu

2018-06-01

Phytophthora root and stem rot (PRR) caused by Phytophthora sojae is one of the most devastating diseases reducing soybean (Glycine max) production all over the world. Harpin proteins in many plant pathogenic bacteria were confirmed to enhance disease and insect resistance in crop plants. Here, a harpin protein-encoding gene hrpZpsta from the P. syringae pv. tabaci strain Psta218 was codon-optimized (renamed hrpZm) and introduced into soybean cultivars Williams 82 and Shennong 9 by Agrobacterium-mediated transformation. Three independent transgenic lines over-expressing hrpZm were obtained and exhibited stable and enhanced tolerance to P. sojae infection in T 2 -T 4 generations compared to the non-transformed (NT) and empty vector (EV)-transformed plants. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression of salicylic acid-dependent genes PR1, PR12, and PAL, jasmonic acid-dependent gene PPO, and hypersensitive response (HR)-related genes GmNPR1 and RAR was significantly up-regulated after P. sojae inoculation. Moreover, the activities of defense-related enzymes such as phenylalanine ammonia lyase (PAL), polyphenoloxidase (PPO), peroxidase, and superoxide dismutase also increased significantly in the transgenic lines compared to the NT and EV-transformed plants after inoculation. Our results suggest that over-expression of the hrpZm gene significantly enhances PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways, thus providing an alternative approach for development of soybean varieties with improved tolerance against the soil-borne pathogen PRR.

Evaluation of a PfHRP2 and a pLDH-based Rapid Diagnostic Test for the Diagnosis of Severe Malaria in 2 Populations of African Children

PubMed Central

Hendriksen, Ilse C. E.; Mtove, George; Pedro, Alínia José; Gomes, Ermelinda; Silamut, Kamolrat; Lee, Sue J.; Mwambuli, Abraham; Gesase, Samwel; Reyburn, Hugh; Day, Nicholas P. J.; White, Nicholas J.; von Seidlein, Lorenz

2011-01-01

Background. Rapid diagnostic tests (RDTs) now play an important role in the diagnosis of falciparum malaria in many countries where the disease is endemic. Although these tests have been extensively evaluated in uncomplicated falciparum malaria, reliable data on their performance for diagnosing potentially lethal severe malaria is lacking. Methods. We compared a Plasmodium falciparum histidine-rich-protein2 (PfHRP2)–based RDT and a Plasmodium lactate dehydrogenase (pLDH)–based RDT with routine microscopy of a peripheral blood slide and expert microscopy as a reference standard for the diagnosis of severe malaria in 1898 children who presented with severe febrile illness at 2 centers in Mozambique and Tanzania. Results. The overall sensitivity, specificity, positive predictive value, and negative predictive values of the PfHRP2-based test were 94.0%, 70.9%, 85.4%, and 86.8%, respectively, and for the pLDH-based test, the values were 88.0%, 88.3%, 93.2%, and 80.3%, respectively. At parasite counts <1000 parasites/μL (n = 173), sensitivity of the pLDH-based test was low (45.7%), compared with that of the PfHRP2-based test (69.9%). Both RDTs performed better than did the routine slide reading in a clinical laboratory as assessed in 1 of the centers. Conclusion. The evaluated PfHRP2-based RDT is an acceptable alternative to routine microscopy for diagnosing severe malaria in African children and performed better than did the evaluated pLDH-based RDT. PMID:21467015

Deterioration of kidney function by the (pro)renin receptor blocker handle region peptide in aliskiren-treated diabetic transgenic (mRen2)27 rats.

PubMed

te Riet, Luuk; van den Heuvel, Mieke; Peutz-Kootstra, Carine J; van Esch, Joep H M; van Veghel, Richard; Garrelds, Ingrid M; Musterd-Bhaggoe, Usha; Bouhuizen, Angelique M; Leijten, Frank P J; Danser, A H Jan; Batenburg, Wendy W

2014-05-15

Dual renin-angiotensin system (RAS) blockade in diabetic nephropathy is no longer feasible because of the profit/side effect imbalance. (Pro)renin receptor [(P)RR] blockade with handle region peptide (HRP) has been reported to exert beneficial effects in various diabetic models in a RAS-independent manner. To what degree (P)RR blockade adds benefits on top of RAS blockade is still unknown. In the present study, we treated diabetic TGR(mREN2)27 rats, a well-established nephropathy model with high prorenin levels [allowing continuous (P)RR stimulation in vivo], with HRP on top of renin inhibition with aliskiren. Aliskiren alone lowered blood pressure and exerted renoprotective effects, as evidenced by reduced glomerulosclerosis, diuresis, proteinuria, albuminuria, and urinary aldosterone levels as well as diminished renal (P)RR and ANG II type 1 receptor expression. It also suppressed plasma and tissue RAS activity and suppressed cardiac atrial natriuretic peptide and brain natriuretic peptide expression. HRP, when given on top of aliskiren, did not alter the effects of renin inhibition on blood pressure, RAS activity, or aldosterone. However, it counteracted the beneficial effects of aliskiren in the kidney, induced hyperkalemia, and increased plasma plasminogen activator-inhibitor 1, renal cyclooxygenase-2, and cardiac collagen content. All these effects have been linked to (P)RR stimulation, suggesting that HRP might, in fact, act as a partial agonist. Therefore, the use of HRP on top of RAS blockade in diabetic nephropathy is not advisable. Copyright © 2014 the American Physiological Society.

ApoHRP-based assay to measure intracellular regulatory heme.

PubMed

Atamna, Hani; Brahmbhatt, Marmik; Atamna, Wafa; Shanower, Gregory A; Dhahbi, Joseph M

2015-02-01

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-β (Aβ) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

Nucleotide sequences of two genomic DNAs encoding peroxidase of Arabidopsis thaliana.

PubMed

Intapruk, C; Higashimura, N; Yamamoto, K; Okada, N; Shinmyo, A; Takano, M

1991-02-15

The peroxidase (EC 1.11.1.7)-encoding gene of Arabidopsis thaliana was screened from a genomic library using a cDNA encoding a neutral isozyme of horseradish, Armoracia rusticana, peroxidase (HRP) as a probe, and two positive clones were isolated. From the comparison with the sequences of the HRP-encoding genes, we concluded that two clones contained peroxidase-encoding genes, and they were named prxCa and prxEa. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, GT and AG, at the 5' and 3' ends, respectively. The lengths of each putative exon of the prxEa gene were the same as those of the HRP-basic-isozyme-encoding gene, prxC3, and coded for 349 amino acids (aa) with a sequence homology of 89% to that encoded by prxC3. The prxCa gene was very close to the HRP-neutral-isozyme-encoding gene, prxC1b, and coded for 354 aa with 91% homology to that encoded by prxC1b. The aa sequence homology was 64% between the two peroxidases encoded by prxCa and prxEa.

Colorimetric Aptasensor Based on Enzyme for the Detection of Vibrio parahemolyticus.

PubMed

Wu, Shijia; Wang, Yinqiu; Duan, Nuo; Ma, Haile; Wang, Zhouping

2015-09-09

A simple colorimetric aptasensor system has been developed to detect Vibrio parahemolyticus. Magnetic nanoparticles (MNPs) are synthesized and conjugated with specific aptamers against target and used as capture probes. In addition, this method employs gold nanoparticles (AuNPs) as carriers of horseradish peroxidase (HRP) and aptamers, which served as signal probes. In the presence of target, a "sandwich-type" complex of AuNPs-HRP-aptamer-target-aptamer-MNPs is formed through specific recognition of aptamers and corresponding target. As a result, HRP molecules confined at the surface of the "sandwich" complexes catalyze the enzyme substrate, 3,3',5,5'-tetramethylbenzidine (TMB) and H2O2 and generate an optical signal. Under optimal conditions, the signals are linearly dependent on V. parahemolyticus concentrations from 10 to 10(6) colony-forming units (cfu)/mL in a logarithmic plot, with a limit of detection of 10 cfu/mL. Owing to AuNPs, a large amount of HRP could be loaded, resulting in an amplified signal, and the sensitivity would be improved. This strategy has the potential of being extended to the construction of simple monitor systems for a variety of biomolecules related to food safety.

Horseradish Peroxidase-Encapsulated Hollow Silica Nanospheres for Intracellular Sensing of Reactive Oxygen Species

NASA Astrophysics Data System (ADS)

Chen, Hsin-Yi; Wu, Si-Han; Chen, Chien-Tsu; Chen, Yi-Ping; Chang, Feng-Peng; Chien, Fan-Ching; Mou, Chung-Yuan

2018-04-01

Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of κ-casein in bovine milk samples.

PubMed

Li, Y S; Zhou, Y; Meng, X Y; Zhang, Y Y; Liu, J Q; Zhang, Y; Wang, N N; Hu, P; Lu, S Y; Ren, H L; Liu, Z S

2014-11-15

A dual labeled probe was synthesized by coating gold nanoparticles (AuNPs) with anti-κ-CN monoclonal antibody (McAb) and horseradish peroxidase (HRP) enzyme on their surface. The McAb was used as detector and HRP was used as label for signal amplification catalytically oxidize the substrate. AuNPs were used as bridges between the McAb and HRP. Based on the probe, an immunoassay was developed for ultrasensitive detection of κ-CN in bovine milk samples. The assay has a linear response range within 4.2-560 ng mL(-1). The limit of detection (LOD) was 4.2 ng mL(-1) which was 10 times lower than that of traditional McAb-HRP based ELISA. The recoveries of κ-CN from three brand bovine milk samples were from 95.8% to 111.0% that had a good correlation (R(2)=0.998) with those obtained by official standard Kjeldahl method. For higher sensitivity and as simple as the traditional ELISA, the developed immunoassay could provide an alternative approach for ultrasensitive detection of κ-CN in bovine milk sample. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of Plasmodium Lactate Dehydrogenase and Histidine-Rich Protein 2 Clearance Patterns via Rapid On-Bead Detection from a Single Dried Blood Spot

PubMed Central

Markwalter, Christine F.; Gibson, Lauren E.; Mudenda, Lwiindi; Kimmel, Danielle W.; Mbambara, Saidon; Thuma, Philip E.; Wright, David W.

2018-01-01

Abstract. A rapid, on-bead enzyme-linked immunosorbent assay for Plasmodium lactate dehydrogenase (pLDH) and Plasmodium falciparum histidine-rich protein 2 (HRP2) was adapted for use with dried blood spot (DBS) samples. This assay detected both biomarkers from a single DBS sample with only 45 minutes of total incubation time and detection limits of 600 ± 500 pM (pLDH) and 69 ± 30 pM (HRP2), corresponding to 150 and 24 parasites/μL, respectively. This sensitive and reproducible on-bead detection method was used to quantify pLDH and HRP2 in patient DBS samples from rural Zambia collected at multiple time points after treatment. Biomarker clearance patterns relative to parasite clearance were determined; pLDH clearance followed closely with parasite clearance, whereas most patients maintained detectable levels of HRP2 for 35–52 days after treatment. Furthermore, weak-to-moderate correlations between biomarker concentration and parasite densities were found for both biomarkers. This work demonstrates the utility of the developed assay for epidemiological study and surveillance of malaria. PMID:29557342

Armored Enzyme-Nanohybrids and Their Catalytic Function Under Challenging Conditions.

PubMed

Zore, Omkar V; Kasi, Rajeswari M; Kumar, Challa V

2017-01-01

Synthesis and characterization of highly stable and functional bienzyme-polymer triads assembled on layered graphene oxide (GO) are described here. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were used as model enzymes and polyacrylic acid (PAA) as model polymer to armor the enzymes. PAA-armored GOx and HRP covalent conjugates were further protected from denaturation by adsorption onto GO nanosheets. Structure and morphology of this enzyme-polymer-nanosheet hybrid biocatalyst (GOx-HRP-PAA/GO) were confirmed by agarose gel electrophoresis, zeta potential, circular dichroism, and transmission electron microscopy. The armored biocatalysts retained full enzymatic activities under challenging conditions of pH (2.5-7.4), warm temperatures (65°C), and presence of chemical denaturants, 4mM sodium dodecyl sulfate, while GOx/HRP physical mixtures without the armor had very little activity under the same conditions. Therefore, this novel combination of two orthogonal approaches, enzyme conjugation with PAA and subsequent physical adsorption onto GO nanosheets, resulted in super stable hybrid biocatalysts that function under harsh conditions. Therefore, this general and powerful approach may be used to design environmentally friendly, green, biocompatible, and biodegradable biocatalysts for energy production in biofuel cell or biobattery applications. © 2017 Elsevier Inc. All rights reserved.

Life Sciences Data Archive (LSDA) in the Post-Shuttle Era

NASA Technical Reports Server (NTRS)

Fitts, Mary A.; Johnson-Throop, Kathy; Havelka, Jacque; Thomas, Diedre

2009-01-01

Now, more than ever before, NASA is realizing the value and importance of their intellectual assets. Principles of knowledge management, the systematic use and reuse of information/experience/expertise to achieve a specific goal, are being applied throughout the agency. LSDA is also applying these solutions, which rely on a combination of content and collaboration technologies, to enable research teams to create, capture, share, and harness knowledge to do the things they do well, even better. In the early days of spaceflight, space life sciences data were been collected and stored in numerous databases, formats, media-types and geographical locations. These data were largely unknown/unavailable to the research community. The Biomedical Informatics and Health Care Systems Branch of the Space Life Sciences Directorate at JSC and the Data Archive Project at ARC, with funding from the Human Research Program through the Exploration Medical Capability Element, are fulfilling these requirements through the systematic population of the Life Sciences Data Archive. This project constitutes a formal system for the acquisition, archival and distribution of data for HRP-related experiments and investigations. The general goal of the archive is to acquire, preserve, and distribute these data and be responsive to inquiries from the science communities.

From Process to Product: Your Risk Process at Work

NASA Technical Reports Server (NTRS)

Kundrot, Craig E.; Fogarty, Jenifer; Charles, John; Buquo, Lynn; Sibonga, Jean; Alexander, David; Horn, Wayne G.; Edwards, J. Michelle

2010-01-01

The Space Life Sciences Directorate (SLSD) and Human Research Program (HRP) at the NASA/Johnson Space Center work together to address and manage the human health and performance risks associated with human space flight. This includes all human system requirements before, during, and after space flight, providing for research, and managing the risk of adverse long-term health outcomes for the crew. We previously described the framework and processes developed for identifying and managing these human system risks. The focus of this panel is to demonstrate how the implementation of the framework and associated processes has provided guidance in the management and communication of human system risks. The risks of early onset osteoporosis, CO2 exposure, and intracranial hypertension in particular have all benefitted from the processes developed for human system risk management. Moreover, we are continuing to develop capabilities, particularly in the area of information architecture, which will also be described. We are working to create a system whereby all risks and associated actions can be tracked and related to one another electronically. Such a system will enhance the management and communication capabilities for the human system risks, thereby increasing the benefit to researchers and flight surgeons.

A novel membrane-based process to isolate peroxidase from horseradish roots: optimization of operating parameters.

PubMed

Liu, Jianguo; Yang, Bo; Chen, Changzhen

2013-02-01

The optimization of operating parameters for the isolation of peroxidase from horseradish (Armoracia rusticana) roots with ultrafiltration (UF) technology was systemically studied. The effects of UF operating conditions on the transmission of proteins were quantified using the parameter scanning UF. These conditions included solution pH, ionic strength, stirring speed and permeate flux. Under optimized conditions, the purity of horseradish peroxidase (HRP) obtained was greater than 84 % after a two-stage UF process and the recovery of HRP from the feedstock was close to 90 %. The resulting peroxidase product was then analysed by isoelectric focusing, SDS-PAGE and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. The effects of calcium ion on HRP specific activities were also experimentally determined.

Horseradish-Peroxidase-Catalyzed Tyrosine Click Reaction.

PubMed

Sato, Shinichi; Nakamura, Kosuke; Nakamura, Hiroyuki

2017-03-02

The efficiency of protein chemical modification on tyrosine residues with N-methylluminol derivatives was drastically improved by using horseradish peroxidase (HRP). In the previous method, based on the use of hemin and H 2 O 2 , oxidative side reactions such as cysteine oxidation were problematic for functionalization of proteins selectively on tyrosine residues. Oxidative activation of N-methylluminol derivatives with a minimum amount of H 2 O 2 prevented the occurrence of oxidative side reactions under HRP-catalyzed conditions. As probes for HRP-catalyzed protein modification, N-methylluminol derivatives showed much higher efficiency than tyramide without inducing oligomerization of probe molecules. Tyrosine modification also proceeded in the presence of β-nicotinamide adenine dinucleotide (NADH, H 2 O 2 -free conditions). © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer

NASA Astrophysics Data System (ADS)

Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

2015-02-01

Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal ``off'' state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an ``open'' configuration of the hairpin probe and a CRET signal ``on'' state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing. Electronic supplementary information (ESI) available: Sequences of RNA and DNA used in this study, relationship of the proposed three modes, CRET mechanism of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein system, calculation of the surface coverage of hairpin probe I-1 on MGO, control experiment, comparison between different modes for microRNA detection, and advantages of the proposed strategy. See DOI: 10.1039/c4nr06603k

A gold standard method for the evaluation of antibody-based materials functionality: Approach to forced degradation studies.

PubMed

Coussot, Gaëlle; Le Postollec, Aurélie; Faye, Clément; Dobrijevic, Michel

2018-04-15

The scope of this paper is to present a gold standard method to evaluate functional activity of antibody (Ab)-based materials during the different phases of their development, after their exposure to forced degradations or even during routine quality control. Ab-based materials play a central role in the development of diagnostic devices, for example, for screening or therapeutic target characterization, in formulation development, and in novel micro(nano)technology approaches to develop immunosensors useful for the analysis of trace substances in pharmaceutical and food industries, clinical and environmental fields. A very important aspect in diagnostic device development is the construction of its biofunctional surfaces. These Ab surfaces require biocompatibility, homogeneity, stability, specificity and functionality. Thus, this work describes the validation and applications of a unique ligand binding assay to directly perform the quantitative measurement of functional Ab binding sites immobilized on the solid surfaces. The method called Antibody Anti-HorseRadish Peroxidase (A2HRP) method, uses a covalently coated anti-HRP antibody (anti-HRP Ab) and does not need for a secondary Ab during the detection step. The A2HRP method was validated and gave reliable results over a wide range of absorbance values. Analyzed validation criteria were fulfilled as requested by the food and drug administration (FDA) and European Medicines Agency (EMA) guidance for the validation of bioanalytical methods with 1) an accuracy mean value within +15% of the nominal value; 2) the within-assay precision less than 7.1%, and 3) the inter-day variability under 12.1%. With the A2HRP method, it is then possible to quantify from 0.04 × 10 12 to 2.98 × 10 12 functional Ab binding sites immobilized on the solid surfaces. A2HRP method was validated according to FDA and EMA guidance, allowing the creation of a gold standard method to evaluate Ab surfaces for their resistance under laboratory constraints. Stability testing was described through forced degradation studies after exposure of Ab-surfaces to storage, pH and aqueous-organic solvent mixture stresses. Copyright © 2018 Elsevier B.V. All rights reserved.

A Large Proportion of P. falciparum Isolates in the Amazon Region of Peru Lack pfhrp2 and pfhrp3: Implications for Malaria Rapid Diagnostic Tests

PubMed Central

Gamboa, Dionicia; Ho, Mei-Fong; Bendezu, Jorge; Torres, Katherine; Chiodini, Peter L.; Barnwell, John W.; Incardona, Sandra; Perkins, Mark; Bell, David; McCarthy, James; Cheng, Qin

2010-01-01

Background Malaria rapid diagnostic tests (RDTs) offer significant potential to improve the diagnosis of malaria, and are playing an increasing role in malaria case management, control and elimination. Peru, along with other South American countries, is moving to introduce malaria RDTs as components of malaria control programmes supported by the Global Fund for AIDS, TB and malaria. The selection of the most suitable malaria RDTs is critical to the success of the programmes. Methods Eight of nine microscopy positive P. falciparum samples collected in Iquitos, Peru tested negative or weak positive using HRP2-detecting RDTs. These samples were tested for the presence of pfhrp2 and pfhrp3 and their flanking genes by PCR, as well as the presence of HRP proteins by ELISA. To investigate for geographic extent of HRP-deleted parasites and their temporal occurrence a retrospective study was undertaken on 148 microscopy positive P. falciparum samples collected in different areas of the Amazon region of Peru. Findings Eight of the nine isolates lacked the pfhrp2 and/or pfhrp3 genes and one or both flanking genes, and the absence of HRP was confirmed by ELISA. The retrospective study showed that 61 (41%) and 103 (70%) of the 148 samples lacked the pfhrp2 or pfhrp3 genes respectively, with 32 (21.6%) samples lacking both hrp genes. Conclusions This is the first documentation of P. falciparum field isolates lacking pfhrp2 and/or pfhrp3. The high frequency and wide distribution of different parasites lacking pfhrp2 and/or pfhrp3 in widely dispersed areas in the Peruvian Amazon implies that malaria RDTs targeting HRP2 will fail to detect a high proportion of P. falciparum in malaria-endemic areas of Peru and should not be used. RDTs detecting parasite LDH or aldolase and quality microscopy should be use for malaria diagnosis in this region. There is an urgent need for investigation of the abundance and geographic distribution of these parasites in Peru and neighbouring countries. PMID:20111602

2013 Immune Risk Standing Review Panel Evidence Review for: The Risk of Crew Adverse Health Event Due to Altered Immune Response

NASA Technical Reports Server (NTRS)

Steinberg, Susan

2014-01-01

The 2013 Immune Risk Standing Review Panel (from here on referred to as the SRP) met for a site visit in Houston, TX on February 3-4, 2014. The SRP reviewed the new Evidence Report for the Risk of Crew Adverse Health Event Due to Altered Immune Response (from here on referred to as the 2013 Immune Evidence Report), as well as the Research Plan for this Risk that is in the current version of the Human Research Program’s (HRP) Integrated Research Plan (IRP).

Sexual health-related information delivery - are patient information leaflets still relevant?

PubMed

Varma, Rick; Chung, Charles; Townsend, Amanda; Power, Melissa

2016-06-01

Background Patient information leaflets (PILs) are widely utilised within publically funded sexual health clinics to deliver sexual health-related information (SHRI); however, their continued value to clients in the era of social media is unclear. This study aimed to evaluate clients' opinions on three newly developed PILs and examine client views on other forms of SHRI delivery. An anonymous self-administered questionnaire was completed by clients attending the Western Sydney Sexual Health Centre (WSSHC) in 2012. High-risk population (HRP) vs non-high-risk population (non-HRP) views on PILs vs alternative methods of SHRI delivery were analysed by using Mann-Whitney U, Wilcoxon, McNemar and χ(2) tests. Over half (210/315; (67%)) of the consecutive clients from a culturally diverse population completed the survey. Sex workers (SW) and young people (YP) were significantly likely to have a high school education than non-HRP (P<0.039 and P<0.032). Overall, PILs, a clinic website and the Sexual Health Information Link (SHIL), a state-wide website and telephone line, were ranked significantly higher as a means of SHRI delivery on a Likert scale than newer technologies including Facebook (P<0.001), email (P<0.001), mobile phone applications (P<0.001), TVs in waiting rooms (P<0.001) and business cards (P<0.001). There was no significant difference in opinion between HRP and non-HRP. This study provides evidence for the ongoing use of PILs to deliver SHRI to clinic attendees, in conjunction with other forms of SHRI delivery such as websites and SHIL. Novel methods may require additional consumer engagement and a greater understanding of specific population's needs.

Sol-gel derived silica/chitosan/Fe3O4 nanocomposite for direct electrochemistry and hydrogen peroxide biosensing

NASA Astrophysics Data System (ADS)

Satvekar, R. K.; Rohiwal, S. S.; Tiwari, A. P.; Raut, A. V.; Tiwale, B. M.; Pawar, S. H.

2015-01-01

A novel strategy to fabricate hydrogen peroxide third generation biosensor has been developed from sol-gel of silica/chitosan (SC) organic-inorganic hybrid material assimilated with iron oxide magnetic nanoparticles (Fe3O4). The large surface area of Fe3O4 and porous morphology of the SC composite facilitates a high loading of horseradish peroxidase (HRP). Moreover, the entrapped enzyme preserves its conformation and biofunctionality. The fabrication of hydrogen peroxide biosensor has been carried out by drop casting of the SC/F/HRP nanocomposite on glassy carbon electrode (GCE) for study of direct electrochemistry. The x-ray diffraction (XRD) pattern and transmission electron microscopy (TEM) confirms the phase purity and particle size of as-synthesized Fe3O4 nanoparticles, respectively. The nanocomposite was characterized by UV-vis spectroscopy, fluorescence spectroscopy and Fourier transform infrared (FTIR) for the characteristic structure and conformation of enzyme. The surface topographies of the nanocomposite thin films were investigated by scanning electron microscopy (SEM). Dynamic light scattering (DLS) was used to determine the particle size distribution. The electrostatic interactions of the SC composite with Fe3O4 nanoparticles were studied by the zeta potential measurement. Electrochemical impedance spectroscopy (EIS) of the SC/F/HRP/GCE electrode displays Fe3O4 nanoparticles as an excellent candidate for electron transfer. The SC/F/HRP/GCE exhibited a pair of well-defined quasi reversible cyclic voltammetry peaks due to the redox couple of HRP-heme Fe (III)/Fe (II) in pH 7.0 potassium phosphate buffer. The biosensor was employed to detect H2O2 with linear range of 5 μM to 40 μM and detection limit of 5 μM. The sensor displays excellent selectivity, sensitivity, good reproducibility and long term stability.

Trabecular Bone Score (TBS)—A Novel Method to Evaluate Bone Microarchitectural Texture in Patients With Primary Hyperparathyroidism

PubMed Central

Boutroy, Stephanie; Zhang, Chiyuan; McMahon, Donald Jay; Zhou, Bin; Wang, Ji; Udesky, Julia; Cremers, Serge; Sarquis, Marta; Guo, Xiang-Dong Edward; Hans, Didier

2013-01-01

Context: In the milder form of primary hyperparathyroidism (PHPT), cancellous bone, represented by areal bone mineral density at the lumbar spine by dual-energy x-ray absorptiometry (DXA), is preserved. This finding is in contrast to high-resolution peripheral quantitative computed tomography (HRpQCT) results of abnormal trabecular microstructure and epidemiological evidence for increased overall fracture risk in PHPT. Because DXA does not directly measure trabecular bone and HRpQCT is not widely available, we used trabecular bone score (TBS), a novel gray-level textural analysis applied to spine DXA images, to estimate indirectly trabecular microarchitecture. Objective: The purpose of this study was to assess TBS from spine DXA images in relation to HRpQCT indices and bone stiffness in radius and tibia in PHPT. Design and Setting: This was a cross-sectional study conducted in a referral center. Patients: Participants were 22 postmenopausal women with PHPT. Main Outcome Measures: Outcomes measured were areal bone mineral density by DXA, TBS indices derived from DXA images, HRpQCT standard measures, and bone stiffness assessed by finite element analysis at distal radius and tibia. Results: TBS in PHPT was low at 1.24, representing abnormal trabecular microstructure (normal ≥1.35). TBS was correlated with whole bone stiffness and all HRpQCT indices, except for trabecular thickness and trabecular stiffness at the radius. At the tibia, correlations were observed between TBS and volumetric densities, cortical thickness, trabecular bone volume, and whole bone stiffness. TBS correlated with all indices of trabecular microarchitecture, except trabecular thickness, after adjustment for body weight. Conclusion: TBS, a measurement technology readily available by DXA, shows promise in the clinical assessment of trabecular microstructure in PHPT. PMID:23526463

Multi-input and -output logic circuits based on bioelectrocatalysis with horseradish peroxidase and glucose oxidase immobilized in multi-responsive copolymer films on electrodes.

PubMed

Yu, Xue; Lian, Wenjing; Zhang, Jiannan; Liu, Hongyun

2016-06-15

Herein, poly(N-isopropylacrylamide-co-N,N'-dimethylaminoethylmethacrylate) copolymer films were polymerized on electrode surface with a simple one-step method, and the enzyme horseradish peroxidase (HRP) was embedded in the films simultaneously, which were designated as P(NiPAAm-co-DMEM)-HRP. The films exhibited a reversible structure change with the external stimuli, such as pH, CO2, temperature and SO4(2-), causing the cyclic voltammetric (CV) response of electroactive K3Fe(CN)6 at the film electrodes to display the corresponding multi-stimuli sensitive ON-OFF behavior. Based on the switchable CV property of the system and the electrochemical reduction of H2O2 catalyzed by HRP in the films and mediated by Fe(CN)6(3-) in solution, a 5-input/3-output logic gate was established. To further increase the complexity of the logic system, another enzyme glucose oxidase (GOD) was added into the films, designated as P(NiPAAm-co-DMEM)-HRP-GOD. In the presence of oxygen, the oxidation of glucose in the solution was catalyzed by GOD in the films, and the produced H2O2 in situ was recognized and electrocatalytically reduced by HRP and mediated by Fe(CN)6(3-). Based on the bienzyme films, a cascaded or concatenated 4-input/3-output logic gate system was proposed. The present work combined the multi-responsive interface with bioelectrocatalysis to construct cascaded logic circuits, which might open a new avenue to develop biocomputing elements with more sophisticated functions and design novel glucose biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Monosynaptic inputs from the nucleus tractus solitarii to the laryngeal motoneurons in the nucleus ambiguus of the rat.

PubMed

Hayakawa, T; Takanaga, A; Maeda, S; Ito, H; Seki, M

2000-11-01

The cricothyroid (CT) and the posterior cricoarytenoid (PCA) muscles in the larynx are activated by the laryngeal motoneurons located within the nucleus ambiguus; these motoneurons receive the laryngeal sensory information from the nucleus tractus solitarii (NTS) during respiration and swallowing. We investigated whether the neurons in the NTS projected directly to the laryngeal motoneurons, and what is the synaptic organization of their nerve terminals on the laryngeal motoneurons using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) was injected into the CT muscle or the PCA muscle, the anterogradely WGA-HRP-labeled terminals from the NTS were found to directly contact the retrogradely CT-HRP-labeled dendrites and soma of both the CT and the PCA motoneurons. The labeled NTS terminals comprised about 4% of the axosomatic terminals in a section through the CT motoneurons, and about 9% on both the small (PCA-A) and the large (PCA-B) PCA motoneurons. The number of labeled axosomatic terminals containing round vesicles and making asymmetric synaptic contacts (Gray's type I) was almost equal to that of the labeled terminals containing pleomorphic vesicles and making symmetric synaptic contacts (Gray's type II) on the CT motoneurons. The labeled axosomatic terminals were mostly Gray's type II on the PCA-A motoneurons, while the majority of them were Gray's type I on the PCA-B motoneurons. These results indicate that the laryngeal CT and PCA motoneurons receive a few direct excitatory and inhibitory inputs from the neurons in the NTS.

Synaptology of the direct projections from the nucleus of the solitary tract to pharyngeal motoneurons in the nucleus ambiguus of the rat.

PubMed

Hayakawa, T; Zheng, J Q; Seki, M; Yajima, Y

1998-04-13

During the pharyngeal phase of the swallowing reflex, the nucleus of the solitary tract (NTS) receives peripheral inputs from the pharynx by means of the glossopharyngeal ganglion and is the location of premotor neurons for the pharyngeal (PH) motoneurons. The semicompact formation of the nucleus ambiguus (AmS) is composed of small and medium-sized neurons that do not project to the pharynx, and large PH motoneurons. We investigated whether the neurons in the NTS projected directly to the PH motoneurons or to the other kinds of neurons in the AmS by using the electron microscope. When wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) was injected into the NTS after cholera toxin subunit B-conjugated HRP (CT-HRP) injections into the pharyngeal muscles of male Sprague-Dawley rats, many nerve terminals anterogradely labeled with WGA-HRP were found to contact PH motoneurons retrogradely labeled with CT-HRP. Most of the labeled axodendritic terminals (63%) contained pleomorphic vesicles with symmetric synaptic contacts (Gray's type II), and the remaining ones contained round vesicles with asymmetric synaptic contacts (Gray's type I). About 14% of the axosomatic terminals on PH motoneuron in a sectional plane were anterogradely labeled, and about 70% of the labeled axosomatic terminals were Gray's type II. Observations of serial ultrathin sections revealed that both the small and the medium-sized neurons received only a few labeled axosomatic terminals that were exclusively Gray's type I. These results indicate that the NTS neurons may send mainly inhibitory as well as a few excitatory inputs directly to the PH motoneurons in the AmS.

Quantitative Analysis of Immunohistochemistry in Melanoma Tumors

PubMed Central

Lilyquist, Jenna; White, Kirsten Anne Meyer; Lee, Rebecca J.; Philips, Genevieve K.; Hughes, Christopher R.; Torres, Salina M.

2017-01-01

Abstract Identification of positive staining is often qualitative and subjective. This is particularly troublesome in pigmented melanoma lesions, because melanin is difficult to distinguish from the brown stain resulting from immunohistochemistry (IHC) using horse radish peroxidase developed with 3,3′-Diaminobenzidine (HRP-DAB). We sought to identify and quantify positive staining, particularly in melanoma lesions. We visualized G-protein coupled estrogen receptor (GPER) expression developed with HRP-DAB and counterstained with Azure B (stains melanin) in melanoma tissue sections (n = 3). Matched sections (n = 3), along with 22 unmatched sections, were stained only with Azure B as a control. Breast tissue (n = 1) was used as a positive HRP-DAB control. Images of the stained tissues were generated using a Nuance Spectral Imaging Camera. Analysis of the images was performed using the Nuance Spectral Imaging software and SlideBook. Data was analyzed using a Kruskal–Wallis one way analysis of variance (ANOVA). We showed that a pigmented melanoma tissue doubly stained with anti-GPER HRP-DAB and Azure B can be unmixed using spectra derived from a matched, Azure B-only section, and an anti-GPER HRP-DAB control. We unmixed each of the melanoma lesions using each of the Azure B spectra, evaluated the mean intensity of positive staining, and examined the distribution of the mean intensities (P = .73; Kruskal–Wallis). These results suggest that this method does not require a matched Azure B-only stained control tissue for every melanoma lesion, allowing precious tissues to be conserved for other studies. Importantly, this quantification method reduces the subjectivity of protein expression analysis, and provides a valuable tool for accurate evaluation, particularly for pigmented tissues. PMID:28403073

UNMYELINATED FIBERS OF THE ANTERIOR ETHMOIDAL NERVE IN THE RAT CO-LOCALIZE WITH NEURONS IN THE MEDULLARY DORSAL HORN AND VENTROLATERAL MEDULLA ACTIVATED BY NASAL STIMULATION

PubMed Central

Hollandsworth, Michael P.; DiNovo, Karyn M.; McCulloch, Paul F.

2009-01-01

The anterior ethmoidal nerve (AEN) innervates the nasal passages and external nares, and serves as the afferent limb of the nasopharyngeal and diving responses. However, although 65% of the AEN is composed of unmyelinated fibers, it has not been determined whether this afferent signal is carried by unmyelinated or myelinated fibers. We used the transganglionic tracers WGA-HRP, IB4-HRP, and CTB-HRP to trace the central projections of the AEN of the rat. Interpretation of the labeling patterns suggests that AEN unmyelinated fibers project primarily to the ventral tip of the ipsilateral medullary dorsal horn (MDH) at the level of the area postrema. Other unmyelinated projections were to the ventral paratrigeminal nucleus and ventrolateral medulla, specifically the Bötzinger and RVLM/C1 regions. Myelinated AEN fibers projected to the ventral paratrigeminal and mesencephalic trigeminal nuclei. Stimulating the nasal passages of urethane-anesthetized rats with ammonia vapors produced the nasopharyngeal response that included apnea, bradycardia and an increase in arterial blood pressure. Central projections of the AEN co-localized with neurons within both MDH and RVLM/C1 that were activated by nasal stimulation. Within the ventral MDH the density of AEN terminal projections positively correlated with the rostral-caudal location of activated neurons, especially at and just caudal to the obex. We conclude that unmyelinated AEN terminal projections are involved in the activation of neurons in the MDH and ventrolateral medulla that participate in the nasopharyngeal response in the rat. We also found that IB4-HRP was a much less robust tracer than WGA-HRP. PMID:19732757

Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

PubMed

Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

2008-01-23

Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

Three-dimensionally ordered macroporous (3DOM) gold-nanoparticle-doped titanium dioxide (GTD) photonic crystals modified electrodes for hydrogen peroxide biosensor.

PubMed

Li, Jianlin; Han, Tao; Wei, Nannan; Du, Jiangyan; Zhao, Xiangwei

2009-12-15

Gold nanoparticles have been introduced into the wall framework of titanium dioxide photonic crystals by the colloidal crystal template technique. The three-dimensionally ordered macroporous gold-nanoparticle-doped titanium dioxide (3DOM GTD) film was modified on the indium-tin oxide (ITO) electrode surface and used for the hydrogen peroxide biosensor. The direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on this film have been investigated. The 3DOM GTD film could provide a good microenvironment for retaining the biological bioactivity, large internal area, and superior conductivity. The HRP/3DOM GTD/ITO electrode exhibited two couples of redox peaks corresponding to the HRP intercalated in the mesopores and adsorbed on the external surface of the film with the formal potential of -0.19 and -0.52V in 0.1M PBS (pH 7.4), respectively. The HRP intercalated in the mesopores showed a surface-controlled process with a single proton transfer. The direct electron transfer between the adsorbed HRP and the electrode is achieved without the aid of an electron mediator. The H(2)O(2) biosensor displayed a rapid eletrocatalytic response (less than 3s), a wide linear range from 0.5 microM to 1.4mM with a detection limit of 0.2 microM, high sensitivity (179.9 microAmM(-1)), good stability and reproducibility. Compared with the free-Au doped titanium dioxide photonic crystals modified electrode, the GTD modified electrode could greatly enhance the response current signal, linear detection range and higher sensitivity. The 3DOM GTD provided a new matrix for protein immobilization and direct transfer study and opened a way for low conductivity electrode biosensor.

Development of a highly sensitive chemiluminescence enzyme immunoassay using enhanced luminol as substrate.

PubMed

Tao, Xiaoqi; Wang, Wenjun; Wang, Zhanhui; Cao, Xingyuan; Zhu, Jinghui; Niu, Lanlan; Wu, Xiaoping; Jiang, Haiyang; Shen, Jianzhong

2014-06-01

In this study, a high sensitivity chemiluminescence enzyme immunoassay (CLEIA) based on novel enhancers was developed. Under optimal conditions, we developed an enhanced chemiluminescence reaction (ECR) catalyzed by horseradish peroxidase (HRP-C) in the presence of 3-(10'-phenothiazinyl) propane-1-sulfonate (SPTZ) and 4-morpholinopyridine (MORP) as enhancers. The limit of detection of the newly prepared chemiluminescent cocktail for HRP was 0.33 pg/well, which is lower than that of commercial Super Signal substrate. The results showed that this novel chemiluminescent cocktail can significantly increase the light output of HRP-catalyzed ECR, which can be translated into a corresponding improvement in sensitivity. Similar improvements were observed in CLEIA for the determination of chloramphenicol in milk. In addition, the ECR of N-azoles as secondary enhancer was also presented. Copyright © 2013 John Wiley & Sons, Ltd.

The nervus terminalis ganglion in Anguilla rostrata: an immunocytochemical and HRP histochemical analysis.

PubMed

Grober, M S; Bass, A H; Burd, G; Marchaterre, M A; Segil, N; Scholz, K; Hodgson, T

1987-12-08

Immunocytochemistry and retrograde horseradish peroxidase (HRP) transport were used to study the ganglion of the nervus terminalis in the American eel, Anguilla rostrata. Luteinizing hormone releasing hormone (LHRH) like immunoreactivity was found in large, ganglion-like cells located ventromedially at the junction of the telencephalon and olfactory bulb and in fibers within the retina and olfactory epithelium. HRP transport from the retina demonstrated direct connections with both the ipsi- and contralateral populations of these ganglion-like cells. Given the well-documented role of both olfaction and vision during migratory and reproductive phases of the life cycle of eels, the robust nature of a nervus terminalis system in these fish may present a unique opportunity to study the behavioral correlates of structure-function organization in a discrete population of ganglion-like cells.

Monodispersed silk fibroin microdroplets for protein stabilization

NASA Astrophysics Data System (ADS)

Liu, Qiang; Jiang, Nan; Liu, Dewen; Ying, Guoliang; Shi, Qiusheng; Yetisen, Ali K.; Liu, Haifeng; Fan, Yubo

2018-04-01

Low stability of globular protein droplets in emulsion significantly limits their applications in drug encapsulation, long-term storage, and controlled drug release. Here, a microfluidic flow-focusing device was utilized to synthesize horseradish peroxidase (HRP)-loaded silk fibroin microdroplets. The two immiscible streams of microfluidic flow-focusing were regenerated by silk fibroin solution and a mixture of 95 wt. % sunflower oil and 5 wt. % span 80 as the dispersed and continuous phases, respectively. In this study, the water-in-oil silk fibroin microdroplets were homogeneously produced by leveraging the discrete and periodic breakup of microdroplets and regulating the flow rates. Moreover, the result showed that the stability of encapsulated HRP in microdroplets was 25% higher than that of HRP after 6 weeks incubation. Thus, the microfluidic flow-focusing is a promising technique to form monodisperse microdroplets and maximize the stability of protein droplets.

Reviewing the Relationship between Human Resource Practices and Psychological Contract and Their Impact on Employee Attitude and Behaviours: A Conceptual Model

ERIC Educational Resources Information Center

Aggarwal, Upasana; Bhargava, Shivganesh

2009-01-01

Purpose: The purpose of this paper is to review and synthesise literature on the role of human resource practices (HRP) in shaping employee psychological contract (PC). Based on this review, a conceptual framework for examining the relationship between HRP and PC and their impact on employee attitudes as well as behaviour has been put forward for…

Amorphous carbon nanoparticle used as novel resonance energy transfer acceptor for chemiluminescent immunoassay of transferrin.

PubMed

Gao, Hongfei; Wang, Wenwen; Wang, Zhenxing; Han, Jing; Fu, Zhifeng

2014-03-28

Amorphous carbon nanoparticles (ACNPs) showing highly efficient quenching of chemiluminescence (CL) were prepared from candle soot with a very simple protocol. The prepared ACNP was employed as the novel energy acceptor for a chemiluminescence resonance energy transfer (CRET)-based immunoassay. In this work, ACNP was linked with transferrin (TRF), and horseradish peroxidase (HRP) was conjugated to TRF antibody (HRP-anti-TRF). The immunoreaction rendered the distance between the ACNP acceptor and the HRP-catalyzed CL emitter to be short enough for CRET occurring. In the presence of TRF, this antigen competed with ACNP-TRF for HRP-anti-TRF, thus led to the decreased occurrence of CRET. A linear range of 20-400 ng mL(-1) and a limit of detection of 20 ng mL(-1) were obtained in this immunoassay. The proposed method was successfully applied for detection of TRF levels in human sera, and the results were in good agreement with ELISA method. Moreover, the ACNPs show higher energy transfer efficiency than other conventional nano-scaled energy acceptors such as graphene oxide in CRET assay. It is anticipated that this approach can be developed for determination of other analytes with low cost, simple manipulation and high specificity. Copyright © 2014 Elsevier Ltd. All rights reserved.

Peroxidase-catalyzed stabilization of 2,4-dichlorophenol in alkali-extracted soils.

PubMed

Palomo, Mónica; Bhandari, Alok

2011-01-01

Horseradish peroxidase- (HRP) mediated stabilization of phenolic contaminants is a topic of interest due to its potential for remediation of contaminated soils. This study evaluated the sorption of 2,4-dichlorophenol (DCP) and its HRP-mediated stabilization in two alkali-extracted soils. Alkali extraction reduced the soil organic matter (SOM) contents of the geomaterials and enriched the residual SOM with humin C. Sorption of DCP on these sorbents was complete within 1 d. However, most of the sorbed DCP was removed from the geomaterials by water and methanol, suggesting weak solute-sorbent interactions. The addition of HRP resulted in the generation of DCP polymerization products (DPP), which partitioned between the aqueous and solid phases. The DPP phase distribution was rapid and complete within 24 h. Between 70 and 90% of the added DCP was converted to DPP and up to 43% of the initial aqueous phase contaminant was transformed into a residue that was resistant to extraction with methanol. Bound residues of DPP increased with initial aqueous phase solute concentration and remained fairly constant after 7 d of contact. Contaminant stabilization was noted to be high in the humin-mineral geomaterial. Results illustrate that HRP may be effective in stabilizing phenolic contaminants in subsoils that are likely to contain SOM enriched in humin C.

Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy

PubMed Central

Huang, Qing; Al-Azzam, Wasfi; Griebenow, Kai; Schweitzer-Stenner, Reinhard

2003-01-01

The heme structure perturbation of poly(ethylene glycol)-modified horseradish peroxidase (HRP-PEG) dissolved in benzene and toluene has been probed by resonance Raman dispersion spectroscopy. Analysis of the depolarization ratio dispersion of several Raman bands revealed an increase of rhombic B1g distortion with respect to native HRP in water. This finding strongly supports the notion that a solvent molecule has moved into the heme pocket where it stays in close proximity to one of the heme's pyrrole rings. The interactions between the solvent molecule, the heme, and the heme cavity slightly stabilize the hexacoordinate high spin state without eliminating the pentacoordinate quantum mixed spin state that is dominant in the resting enzyme. On the contrary, the model substrate benzohydroxamic acid strongly favors the hexacoordinate quantum mixed spin state and induces a B2g-type distortion owing to its position close to one of the heme methine bridges. These results strongly suggest that substrate binding must have an influence on the heme geometry of HRP and that the heme structure of the enzyme-substrate complex (as opposed to the resting state) must be the key to understanding the chemical reactivity of HRP. PMID:12719258

The Space Medicine Exploration Medical Condition List

NASA Technical Reports Server (NTRS)

Watkins, Sharmi; Barr, Yael; Kerstman, Eric

2011-01-01

Exploration Medical Capability (ExMC) is an element of NASA s Human Research Program (HRP). ExMC's goal is to address the risk of the "Inability to Adequately Recognize or Treat an Ill or Injured Crewmember." This poster highlights the approach ExMC has taken to address this risk. The Space Medicine Exploration Medical Condition List (SMEMCL) was created to define the set of medical conditions that are most likely to occur during exploration space flight missions. The list was derived from the International Space Station Medical Checklist, the Shuttle Medical Checklist, in-flight occurrence data from the Lifetime Surveillance of Astronaut Health, and NASA subject matter experts. The list of conditions was further prioritized for eight specific design reference missions with the assistance of the ExMC Advisory Group. The purpose of the SMEMCL is to serve as an evidence-based foundation for the conditions that could affect a crewmember during flight. This information is used to ensure that the appropriate medical capabilities are available for exploration missions.

Overview of an Integrated Medical System for Exploration Missions

NASA Technical Reports Server (NTRS)

Watkins, Sharmila; Rubin, David

2013-01-01

The Exploration Medical Capability (ExMC) element of the NASA Human Research Program (HRP) is charged with addressing the risk of unacceptable health and mission outcomes due to limitations of inflight medical capabilities. The Exploration Medical System Demonstration (EMSD) is a project within the ExMC element aimed at reducing this risk by improving the medical capabilities available for exploration missions. The EMSD project will demonstrate, on the ground and on ISS, the integration of several components felt to be essential to the delivery of medical care during long ]duration missions outside of low Earth orbit. The components of the EMSD include the electronic medical record, assisted medical procedure software, medical consumables tracking technology and RFID ] tagged consumables, video conferencing capability, ultrasound device and probes (ground demonstration only), peripheral biosensors, and the software to allow communication among the various components (middleware). This presentation seeks to inform our international partners of the goals and objectives of the EMSD and to foster collaboration opportunities related to this and future projects.

International Space Station Medical Projects - Full Services to Mars

NASA Technical Reports Server (NTRS)

Pietrzyk, R. A.; Primeaux, L. L.; Wood, S. J.; Vessay, W. B.; Platts, S. H.

2018-01-01

The International Space Station Medical Projects (ISSMP) Element provides planning, integration, and implementation services for HRP research studies for both spaceflight and flight analog research. Through the implementation of these two efforts, ISSMP offers an innovative way of guiding research decisions to meet the unique challenges of understanding the human risks to space exploration. Flight services provided by ISSMP include leading informed consent briefings, developing and validating in-flight crew procedures, providing ISS crew and ground-controller training, real-time experiment monitoring, on-orbit experiment and hardware operations and facilitating data transfer to investigators. For analog studies at the NASA Human Exploration Research Analog (HERA), the ISSMP team provides subject recruitment and screening, science requirements integration, data collection schedules, data sharing agreements, mission scenarios and facilities to support investigators. The ISSMP also serves as the HRP interface to external analog providers including the :envihab bed rest facility (Cologne, Germany), NEK isolation chamber (Moscow, Russia) and the Antarctica research stations. Investigators working in either spaceflight or analog environments requires a coordinated effort between NASA and the investigators. The interdisciplinary nature of both flight and analog research requires investigators to be aware of concurrent research studies and take into account potential confounding factors that may impact their research objectives. Investigators must define clear research requirements, participate in Investigator Working Group meetings, obtain human use approvals, and provide study-specific training, sample and data collection and procedures all while adhering to schedule deadlines. These science requirements define the technical, functional and performance operations to meet the research objectives. The ISSMP maintains an expert team of professionals with the knowledge and experience to guide investigators science through all aspects of mission planning, crew operations, and research integration. During this session, the ISSMP team will discuss best-practices approaches for successfully preparing and conducting studies in both the flight and analog environments. Critical tips and tricks will be shown to greatly improve your chances of successfully completing your research aboard the International Space Station and in Spaceflight Analogs.

Covalent chemical functionalization enhances the biodegradation of graphene oxide

NASA Astrophysics Data System (ADS)

Kurapati, Rajendra; Bonachera, Fanny; Russier, Julie; Rajukrishnan Sureshbabu, Adukamparai; Ménard-Moyon, Cécilia; Kostarelos, Kostas; Bianco, Alberto

2018-01-01

Biodegradation of the graphene-based materials is an emerging issue due to their estimated widespread usage in different industries. Indeed, a few concerns have been raised about their biopersistence. Here, we propose the design of surface-functionalized graphene oxide (GO) with the capacity to degrade more effectively compared to unmodified GO using horseradish peroxidase (HRP). For this purpose, we have functionalized the surface of GO with two well-known substrates of HRP namely coumarin and catechol. The biodegradation of all conjugates has been followed by Raman, dynamic light scattering and electron microscopy. Molecular docking and gel electrophoresis have been carried out to gain more insights into the interaction between GO conjugates and HRP. Our studies have revealed better binding when GO is functionalized with coumarin or catechol compared to control GOs. All results prove that GO functionalized with coumarin and catechol moieties display a faster and more efficient biodegradation over GO.

Embryonic development of pleuropodia of the cicada, Magicicada cassini

PubMed Central

Strauß, Johannes; Lakes-Harlan, Reinhard

2006-01-01

In many insects the first abdominal segment possesses embryonic appendages called pleuropodia. Here we show the embryogenesis of pleuropodial cells of the periodical cicada, Magicicada cassini (Fisher 1851) (Insecta, Homoptera, Cicadidae). An antibody, anti-horseradish perioxidase (HRP), that is usually neuron-specific strongly marked the pleuropodial anlagen and revealed their ectodermal origin shortly after limb bud formation. Thereafter the cells sank into the epidermis and their apical parts enlarged. A globular part protruded from the body wall. Filamentous structures were marked at the stem region and into the apical dilation. In later embryonic stages the pleuropodia degenerated. Despite the binding of anti-HRP the cells had no morphological neuronal characters and cannot be regarded as neurons. The binding indicates that glycosylated cell surface molecules contribute to the adhesion between the presumably glandular pleuropodial cells. In comparison, anti-HRP does not mark the pleuropodia of Orthoptera. PMID:19537987

Bilateral innervation of syringeal muscles by the hypoglossal nucleus in the jungle crow (Corvus macrorhynchos).

PubMed

Tsukahara, Naoki; Kamata, Naoki; Nagasawa, Miyuki; Sugita, Shoei

2009-08-01

Bird vocalizations are produced by contractions of syringeal muscles, which are controlled by the hypoglossal nucleus. In oscines, syringeal muscles are controlled by the hypoglossal nucleus ipsilaterally, whereas syringeal innervation is bilateral in non-oscines. We have determined the course of hypoglossal nerves in the jungle crow Corvus macrorhynchos. Our results indicate a cross-over of the hypoglossal nerve from the left side to the right side on the trachea 7 mm rostral to the Musculus sternotrachealis. We also investigated the innervation of the syringeal muscles of jungle crows from the hypoglossal nucleus using the horseradish peroxidase (HRP) method. After HRP was injected into the syringeal muscles on each side, HRP-labeled cells were found bilaterally in the hypoglossal nerve. These results suggest that the syringeal muscles of jungle crows are innervated bilaterally from the hypoglossal nucleus, although these birds are categorized as oscines.

Bilateral innervation of syringeal muscles by the hypoglossal nucleus in the jungle crow (Corvus macrorhynchos)

PubMed Central

Tsukahara, Naoki; Kamata, Naoki; Nagasawa, Miyuki; Sugita, Shoei

2009-01-01

Bird vocalizations are produced by contractions of syringeal muscles, which are controlled by the hypoglossal nucleus. In oscines, syringeal muscles are controlled by the hypoglossal nucleus ipsilaterally, whereas syringeal innervation is bilateral in non-oscines. We have determined the course of hypoglossal nerves in the jungle crow Corvus macrorhynchos. Our results indicate a cross-over of the hypoglossal nerve from the left side to the right side on the trachea 7 mm rostral to theMusculus sternotrachealis. We also investigated the innervation of the syringeal muscles of jungle crows from the hypoglossal nucleus using the horseradish peroxidase (HRP) method. After HRP was injected into the syringeal muscles on each side, HRP-labeled cells were found bilaterally in the hypoglossal nerve. These results suggest that the syringeal muscles of jungle crows are innervated bilaterally from the hypoglossal nucleus, although these birds are categorized as oscines. PMID:19490396

A simple and sensitive fluorescence based biosensor for the determination of uric acid using H2O2-sensitive quantum dots/dual enzymes.

PubMed

Azmi, Nur Ellina; Ramli, Noor Izaanin; Abdullah, Jaafar; Abdul Hamid, Mohammad Azmi; Sidek, Hamidah; Abd Rahman, Samsulida; Ariffin, Nurhayati; Yusof, Nor Azah

2015-05-15

A novel optical detection system consisting of combination of uricase/HRP-CdS quantum dots (QDs) for the determination of uric acid in urine sample is described. The QDs was used as an indicator to reveal fluorescence property of the system resulting from enzymatic reaction of uricase and HRP (horseradish peroxidase), which is involved in oxidizing uric acid to allaintoin and hydrogen peroxide. The hydrogen peroxide produced was able to quench the QDs fluorescence, which was proportional to uric acid concentration. The system demonstrated sufficient activity of uricase and HRP at a ratio of 5U:5U and pH 7.0. The linearity of the system toward uric acid was in the concentration range of 125-1000 µM with detection limit of 125 µM. Copyright © 2014 Elsevier B.V. All rights reserved.

Degradation of 2,4,6-trinitrotoluene by immobilized horseradish peroxidase and electrogenerated peroxide.

PubMed

Beom Lee, Ki; Bock Gu, Man; Moon, Seung Hyeon

2003-03-01

This paper presents horseradish peroxidase (HRP)-catalyzed removal of 2,4,6-trinitrotoluene (TNT) by an electrochemical packed-bed flow reactor operated in a circulating batch mode with the help of in situ generated hydrogen peroxide. HRP immobilized on the reticulated vitreous carbon electrode was prepared for the cyclic voltammetry of 2,4,6-TNT. Effects of pH and temperature on the TNT electroreduction in 0.2M phosphate buffer saturated with oxygen were examined. HRP immobilized carbon electrode was capable of catalyzing the oxidation and detoxification of 44 microM TNT in aqueous solution under optimized conditions. The removal rate of TNT for the electroenzymatic method was much greater than for electrochemical and biochemical methods. Stoichiometric and kinetic studies indicated that the hydrogen peroxide was utilized more effectively in the electroenzymatic method. Denitrification as intermediate reaction was also investigated.

Comparison of a PfHRP2-based rapid diagnostic test and PCR for malaria in a low prevalence setting in rural southern Zambia: implications for elimination.

PubMed

Laban, Natasha M; Kobayashi, Tamaki; Hamapumbu, Harry; Sullivan, David; Mharakurwa, Sungano; Thuma, Philip E; Shiff, Clive J; Moss, William J

2015-01-28

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum. Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR. Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 x 10(3) copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of >99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion. Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination.

Synaptic vesicle pool‐specific modification of neurotransmitter release by intravesicular free radical generation

PubMed Central

Afuwape, Olusoji A. T.; Wasser, Catherine R.; Schikorski, Thomas

2016-01-01

Key points Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane.There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct.In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli.By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa.Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Abstract Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) – a haem‐containing plant enzyme – or antibodies against synaptotagmin‐1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2O2) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization‐induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP‐tagged syt1 construct and subsequent H2O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss‐of‐function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation. PMID:27723113

Effect of Damage on Strength and Durability

DTIC Science & Technology

2010-05-01

sheets and different core materials. The HRP core has a phenolic resin matrix, the NP core has nylon modified phenolic base resin matrix and TPC core...core are 25% to 65% higher than those of NP or TPC cores. The phenolic resin of the HRP makes core stiff and brittle, resulting in cracking on impact...characteristics of graphite laminates can be improved by inserting glass or Kevlar fibers to form a hybrid laminate system. However, since glass and

Development of two highly sensitive immunoassays for detection of copper ions and a suite of relevant immunochemicals.

PubMed

Zhao, Hongwei; Nan, Tiegui; Tan, Guiyu; Gao, Wei; Cao, Zhen; Sun, Shuo; Li, Zhaohu; Li, Qing X; Wang, Baomin

2011-09-19

Availability of highly sensitive assays for metal ions can help monitor and manage the environmental and food contamination. In the present study, a monoclonal antibody against Copper(II)-ethylenediaminetetraacetic acid was used to develop two sensitive ELISAs for Cu(II) analysis. Cobalt(II)-EDTA-BSA was the coating antigen in a heterologous indirect competitive ELISA (hicELISA), whereas Co(II)-EDTA-BSA-horseradish peroxidase (HRP) was the enzyme tracer in a heterologous direct competitive ELISA (hdcELISA). Both ELISAs were validated for detecting the content of Cu(II) in environmental waters. The ELISA data agreed well with those from graphite furnace atomic absorption spectroscopy. The methods of developing the Cu(II) hicELISA and hdcELISA are potentially applicable for developing ELISAs for other metals. The chelator-protein complexes such as EDTA-BSA and EDTA-BSA-HRP can form a suite of metal complexes having the consistent hapten density, location and orientation on the conjugates except the difference of the metal core, which can be used as ideal reagents to investigate the relationship between assay sensitivity and antibody affinities for the haptens and the analytes. The strategy of conjugating a haptenated protein directly with HRP can reduce the loss of HRP activity during the conjugation reaction and thus can be applicable for the development of ELISAs for small molecules. Copyright © 2011. Published by Elsevier B.V.

Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions.

PubMed

Nguyen, Le Truc; Yang, Kun-Lin

2017-05-01

Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Malaria surveys using rapid diagnostic tests and validation of results using post hoc quantification of Plasmodium falciparum histidine-rich protein 2.

PubMed

Plucinski, Mateusz; Dimbu, Rafael; Candrinho, Baltazar; Colborn, James; Badiane, Aida; Ndiaye, Daouda; Mace, Kimberly; Chang, Michelle; Lemoine, Jean F; Halsey, Eric S; Barnwell, John W; Udhayakumar, Venkatachalam; Aidoo, Michael; Rogier, Eric

2017-11-07

Rapid diagnostic test (RDT) positivity is supplanting microscopy as the standard measure of malaria burden at the population level. However, there is currently no standard for externally validating RDT results from field surveys. Individuals' blood concentration of the Plasmodium falciparum histidine rich protein 2 (HRP2) protein were compared to results of HRP2-detecting RDTs in participants from field surveys in Angola, Mozambique, Haiti, and Senegal. A logistic regression model was used to estimate the HRP2 concentrations corresponding to the 50 and 90% level of detection (LOD) specific for each survey. There was a sigmoidal dose-response relationship between HRP2 concentration and RDT positivity for all surveys. Variation was noted in estimates for field RDT sensitivity, with the 50% LOD ranging between 0.076 and 6.1 ng/mL and the 90% LOD ranging between 1.1 and 53 ng/mL. Surveys conducted in two different provinces of Angola using the same brand of RDT and same study methodology showed a threefold difference in LOD. Measures of malaria prevalence estimated using population RDT positivity should be interpreted in the context of potentially large variation in RDT LODs between, and even within, surveys. Surveys based on RDT positivity would benefit from external validation of field RDT results by comparing RDT positivity and antigen concentration.

Catalytic resonance scattering spectral determination of ultratrace horseradish peroxidase using rhodamine S.

PubMed

Jiang, Zhiliang; Liang, Yueyuan; Huang, Guoxia; Wei, Xiaoling; Liang, Aihui; Zhong, Fuxin

2009-01-01

A highly sensitive and selective resonance scattering spectral assay was proposed for the determination of horseradish peroxidase (HRP), based on its catalytic effect on the H2O2 oxidation of KI to form I3(-). The I3(-) combined respectively with rhodamine (Rh) dye such as rhodamine S (RhS), rhodamine 6G (Rh6G), rhodamine B (RhB) and butyl-rhodamine B (b-RhB), to form association particles (Rh-I3(n). The four Rh systems all exhibit a stronger resonance scattering (RS) peak at 424 nm. For the RhS, Rh6G, RhB and b-RhB systems, HRP concentration in the range of 3.2 x 10(-12) to 4.8 x 10(-9), 2 x 10(-11) to 3.2 x 10(-9), 1.6 x 10(-11) to 3.2 x 10(-9) and 1.6 x 10(-11) to 4 x 10(-9) g/mL was linear to its RS intensity at 424 nm, with a detection limit of 2.2 x 10(-12), 2.5 x 10(-12), 4.4 x 10(-12) and 2.6 x 10(-12 )g/mL, respectively. This RhS system was most sensitive and stable, and was applied for the determination of HRP in the hepatitis B surface antibody labeling HRP and water samples, with satisfactory results.

Amperometric Choline Biosensor Fabricated through Electrostatic Assembly of Bienzyme/Polyelectrolyte Hybrid Layers on Carbon Nanotubes

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

Wang, Jun; Liu, Guodong; Lin, Yuehe

2006-03-01

We report a flow injection amperometric choline biosensors based on the electrostatic assembly of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO and horseradish peroxidase (HRP) onto multi-wall carbon nanotubes (MWCNT) modified glassy carbon (GC) electrodes. These choline biosensors were fabricated by immobilization of enzymes on the negatively charged MWCNT surface through alternatively assembling a cationic polydiallydiimethylammonium chloride (PDDA) layer and an enzyme layer. Using this layer-by-layer assembling approach, bioactive nanocomposite film of a PDDA/ChO/PDDA/HRP/PDDA/CNT (ChO/HRP/CNT) and a PDDA/ChO/PDDA/ CNT (ChO/ CNT) were fabricated on GC surface, respectively. Owning to the electrocatalytic effect of carbon nanotubes, themore » measurement of faradic responses resulting from enzymatic reactions has been realized at low potential with acceptable sensitivity. It is found the ChO/HRP/CNT biosensor is more sensitive than the ChO/CNT one. Experimental parameters affecting the sensitivity of biosensors, e.g. applied potential, flow rate, etc. were optimized and potential interference was examined. The response time for this choline biosensor is fast (less than a few seconds). The linear range of detection for the choline biosensor is from 5 x 10-5 to 5 x 10-3 M and the detection limit is determined to be about 1.0 x 10-5 M.« less

Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect.

PubMed

Fournand, David; Cathala, Bernard; Lapierre, Catherine

2003-01-01

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.

Electrochemical detection of DNA damage induced by acrylamide and its metabolite at the graphene-ionic liquid-Nafion modified pyrolytic graphite electrode.

PubMed

Qiu, Yanyan; Qu, Xiangjin; Dong, Jing; Ai, Shiyun; Han, Ruixia

2011-06-15

A new electrochemical biosensor for directly detecting DNA damage induced by acrylamide (AA) and its metabolite was presented in this work. The graphene-ionic liquid-Nafion modified pyrolytic graphite electrode (PGE) was prepared, and then horseradish peroxidase (HRP) and natural double-stranded DNA were alternately assembled on the modified electrode by the layer-by-layer method. The PGE/graphene-ionic liquid-Nafion and the construction of the (HRP/DNA)(n) film were characterized by electrochemical impedance spectroscopy. With the guanine signal in DNA as an indicator, the damage of DNA was detected by differential pulse voltammetry after PGE/graphene-ionic liquid-Nafion/(HRP/DNA)(n) was incubated in AA solution or AA+H(2)O(2) solution at 37°C. This method provides a new model to mimic and directly detect DNA damage induced by chemical pollutants and their metabolites in vitro. The results indicated that, in the presence of H(2)O(2), HRP was activated and catalyzed the transformation of AA to glycidamide, which could form DNA adducts and induce more serious damage of DNA than AA. In order to further verify these results, UV-vis spectrophotometry was also used to investigate DNA damage induced by AA and its metabolites in solution and the similar results were obtained. Copyright © 2011 Elsevier B.V. All rights reserved.

Sensitive electrochemical immunosensor for α-synuclein based on dual signal amplification using PAMAM dendrimer-encapsulated Au and enhanced gold nanoparticle labels.

PubMed

An, Yarui; Jiang, Xiaoli; Bi, Wenji; Chen, Hua; Jin, Litong; Zhang, Shengping; Wang, Chuangui; Zhang, Wen

2012-02-15

A novel electrochemical immunosensor for sensitive detection of α-synuclein (α-SYN), a very important neuronal protein, has been developed based on dual signal amplification strategy. Herein, G4-polyamidoamine dendrimer-encapsulated Au nanoparticles (PAMAM-Au nanocomposites) were covalently bound on the poly-o-aminobenzoic acid (poly-o-ABA), which was initially electropolymerized on the electrode surface to perform abundant carboxyl groups. The formed immunosensor platform, PAMAM-Au, was proved to provide numerous amino groups to allow highly dense immobilization of antigen, and facilitate the improvement of electrochemical responses as well. Subsequently, the enhanced gold nanoparticle labels ({HRP-Ab(2)-GNPs}) were fabricated by immobilizing horseradish peroxidase-secondary antibody (HRP-Ab(2)) on the surface of gold nanoparticles (GNPs). After an immunoassay process, the {HRP-Ab(2)-GNPs} labels were introduced onto the electrode surface, and produced an electrocatalytic response by reduction of hydrogen peroxide (H(2)O(2)) in the presence of enzymatically oxidized thionine. On the basis of the dual signal amplification of PAMAM-Au and {HRP-Ab(2)-GNPs} labels, the designed immunosensor displayed an excellent analytical performance with high sensitivity and stability. This developed strategy was successfully proved as a simple, cost-effective method, and could be easily extended to other protein analysis schemes. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of wondfo rapid diagnostic kit (Pf-HRP2/PAN-pLDH) for diagnosis of malaria by using nano-gold immunochromatographic assay.

PubMed

Wu, Junlin; Peng, Yunping; Liu, Xiaoyun; Li, Wenmei; Tang, Shixing

2014-06-01

Prompt and accurate diagnosis is necessary to start adequate treatment for different affecting species including P. falciparum and P. vivax. Here we described the Wondfo Rapid diagnostic Kit (Pf-HRP2/PAN-pLDH) for the detection of P. falciparum and pan-plasmodium in patient specimen by using a nano-gold immunochromatographic assay. Our rapid assay adapted nano-gold labeling techniques and the monoclonal antibodies (mAbs) against both histidine rich protein-2 (Pf HRP-2) of P. falciparum and pan plasmodium-specific pLDH (pan pLDH). The established two-antibody sandwich immunochromatographic assay could detect P. falciparum and pan-plasmodium. The sensitivity and specificity of Wondfo rapid diagnostic kit were determined by comparing with the "gold standard" of microscopic examination of blood smears. In this study1023 blood samples were collected from outpatient clinics in China and Burma, and detected by both Wondfo kit and microscopic examination. The detection sensitivity and specificity of Wondfo rapid diagnostic kit were 96.46% and 99.67% for P. falciparum (HRP2), 95.03% and 99.24% for pLDH, 96.83% and 99.74% for non-falciparum species, 96.70% and 99.74% for P. vivax, respectively. These results indicate that Wondfo rapid diagnostic assay may be useful for detecting P. falciparum and non-P. falciparum (especially P.v.) in patient specimen.

Exploration Medical Capability

NASA Technical Reports Server (NTRS)

Watkins, Sharmila; Baumann, David; Wu, Jimmy; Barsten, Kristina

2010-01-01

Exploration Medical Capability (ExMC) is an element of NASA's Human Research Program (HRP). ExMC's goal is to address the risk of the Inability to Adequately Recognize or Treat an Ill or Injured Crewmember. This poster highlights the approach ExMC has taken to address this goal and our current areas of interest. The Space Medicine Exploration Medical Condition List (SMEMCL) was created to identify medical conditions of concern during exploration missions. The list was derived from space flight medical incidents, the shuttle medical checklist, the International Space Station medical checklist, and expert opinion. The conditions on the list were prioritized according to mission type by a panel comprised of flight surgeons, physician astronauts, engineers, and scientists. From the prioritized list, the ExMC element determined the capabilities needed to address the medical conditions of concern. Where such capabilities were not currently available, a gap was identified. The element s research plan outlines these gaps and the tasks identified to achieve the desired capabilities for exploration missions. This poster is being presented to inform the audience of the gaps and tasks being investigated by ExMC and to encourage discussions of shared interests and possible future collaborations.

Normative Standards for HRpQCT Parameters in Chinese Men and Women.

PubMed

Zhu, Tracy Y; Yip, Benjamin Hk; Hung, Vivian Wy; Choy, Carol Wy; Cheng, Ka-Lo; Kwok, Timothy Cy; Cheng, Jack Cy; Qin, Ling

2018-06-12

Assessing bone architecture using high resolution peripheral quantitative computed tomography (HRpQCT) has the potential to improve fracture risk assessment. The Normal Reference Study aimed to establish sex-specific reference centile curves for HRpQCT parameters. This was an age-stratified cross-sectional study and 1,072 ambulatory Chinese men (n = 544) and women (n = 528) aged 20-79yrs, who were free from conditions and medications that could affect bone metabolism and had no history of fragility fracture, were recruited from local communities of Hong Kong. Reference centile curves for each HRpQCT parameter were constructed using Generalized Additive Models for Location, Scale and Shape with age as the only explanatory variable. Patterns of reference centile curves reflected age-related changes of bone density, microarchitecture, and estimated bone strength. In both sexes, loss of cortical bone was only evident in mid-adulthood, particularly in women with a more rapid fashion probably concurrent with the onset of menopause. In contrast, loss of trabecular bone was subtle or gradual or occurred at an earlier age. Expected values of HRpQCT parameters for a defined sex and age, and a defined percentile or z-score were obtained from these curves. T-scores were calculated using the population with the peak values as the reference and reflected age- or menopause-related bone loss in an older individual or the room to reach the peak potential in a younger individual. These reference centile curves produced a standard describing a norm or desirable target that enables value clinical judgements. Percentiles, z-scores and T-scores would be helpful in detecting abnormalities in bone density and microarchitecture arising from various conditions and establishing entry criteria for clinical trials. They also hold the potential to refine the diagnosis of osteoporosis and assessment of fracture risk. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Rapid Diagnostic Tests for Malaria Diagnosis in the Peruvian Amazon: Impact of pfhrp2 Gene Deletions and Cross-Reactions

PubMed Central

Maltha, Jessica; Gamboa, Dionicia; Bendezu, Jorge; Sanchez, Luis; Cnops, Lieselotte; Gillet, Philippe; Jacobs, Jan

2012-01-01

Background In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Methods Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Results Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)- detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%–10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. Conclusion PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern. PMID:22952633

Rapid diagnostic tests for malaria diagnosis in the Peruvian Amazon: impact of pfhrp2 gene deletions and cross-reactions.

PubMed

Maltha, Jessica; Gamboa, Dionicia; Bendezu, Jorge; Sanchez, Luis; Cnops, Lieselotte; Gillet, Philippe; Jacobs, Jan

2012-01-01

In the Peruvian Amazon, Plasmodium falciparum and Plasmodium vivax malaria are endemic in rural areas, where microscopy is not available. Malaria rapid diagnostic tests (RDTs) provide quick and accurate diagnosis. However, pfhrp2 gene deletions may limit the use of histidine-rich protein-2 (PfHRP2) detecting RDTs. Further, cross-reactions of P. falciparum with P. vivax-specific test lines and vice versa may impair diagnostic specificity. Thirteen RDT products were evaluated on 179 prospectively collected malaria positive samples. Species diagnosis was performed by microscopy and confirmed by PCR. Pfhrp2 gene deletions were assessed by PCR. Sensitivity for P. falciparum diagnosis was lower for PfHRP2 compared to P. falciparum-specific Plasmodium lactate dehydrogenase (Pf-pLDH)-detecting RDTs (71.6% vs. 98.7%, p<0.001). Most (19/21) false negative PfHRP2 results were associated with pfhrp2 gene deletions (25.7% of 74 P. falciparum samples). Diagnostic sensitivity for P. vivax (101 samples) was excellent, except for two products. In 10/12 P. vivax-detecting RDT products, cross-reactions with the PfHRP2 or Pf-pLDH line occurred at a median frequency of 2.5% (range 0%-10.9%) of P. vivax samples assessed. In two RDT products, two and one P. falciparum samples respectively cross-reacted with the Pv-pLDH line. Two Pf-pLDH/pan-pLDH-detecting RDTs showed excellent sensitivity with few (1.0%) cross-reactions but showed faint Pf-pLDH lines in 24.7% and 38.9% of P. falciparum samples. PfHRP2-detecting RDTs are not suitable in the Peruvian Amazon due to pfhrp2 gene deletions. Two Pf-pLDH-detecting RDTs performed excellently and are promising RDTs for this region although faint test lines are of concern.

Direct and mediated electrochemistry of peroxidase and its electrocatalysis on a variety of screen-printed carbon electrodes: amperometric hydrogen peroxide and phenols biosensor.

PubMed

Chekin, Fereshteh; Gorton, Lo; Tapsobea, Issa

2015-01-01

This study compares the behaviour of direct and mediated electrochemistry of horseradish peroxidase (HRP) immobilised on screen-printed carbon electrodes (SPCEs), screen-printed carbon electrodes modified with carboxyl-functionalised multi-wall carbon nanotubes (MWCNT-SPCEs) and screen-printed carbon electrodes modified with carboxyl-functionalised single-wall carbon nanotubes (SWCNT-SPCEs). The techniques of cyclic voltammetry and amperometry in the flow mode were used to characterise the properties of the HRP immobilised on screen-printed electrodes. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the HRP-modified electrodes, it was concluded that the fraction of enzyme molecules in direct electron transfer (DET) contact with the electrode varies substantially for the different electrodes. It was observed that the screen-printed carbon electrodes modified with carbon nanotubes (MWCNT-SPCEs and SWCNT-SPCEs) demonstrated a substantially higher percentage (≈100 %) of HRP molecules in DET contact than the screen-printed carbon electrodes (≈60 %). The HRP-modified electrodes were used for determination of hydrogen peroxide in mediatorless mode. The SWCNT-SPCE gave the lowest detection limit (0.40 ± 0.09 μM) followed by MWCNT-SPCE (0.48 ± 0.07 μM) and SPCE (0.98 ± 0.2 μM). These modified electrodes were additionally developed for amperometric determination of phenolic compounds. It was found that the SWCNT-SPCE gave a detection limit for catechol of 110.2 ± 3.6 nM, dopamine of 640.2 ± 9.2 nM, octopamine of 3341 ± 15 nM, pyrogallol of 50.10 ± 2.9 nM and 3,4-dihydroxy-L-phenylalanine of 980.7 ± 8.7 nM using 50 μM H2O2 in the flow carrier.

Magnetic Beads-Based Sensor with Tailored Sensitivity for Rapid and Single-Step Amperometric Determination of miRNAs.

PubMed

Vargas, Eva; Torrente-Rodríguez, Rebeca M; Ruiz-Valdepeñas Montiel, Víctor; Povedano, Eloy; Pedrero, María; Montoya, Juan J; Campuzano, Susana; Pingarrón, José M

2017-11-09

This work describes a sensitive amperometric magneto-biosensor for single-step and rapid determination of microRNAs (miRNAs). The developed strategy involves the use of direct hybridization of the target miRNA (miRNA-21) with a specific biotinylated DNA probe immobilized on streptavidin-modified magnetic beads (MBs), and labeling of the resulting heteroduplexes with a specific DNA-RNA antibody and the bacterial protein A (ProtA) conjugated with an horseradish peroxidase (HRP) homopolymer (Poly-HRP40) as an enzymatic label for signal amplification. Amperometric detection is performed upon magnetic capture of the modified MBs onto the working electrode surface of disposable screen-printed carbon electrodes (SPCEs) using the H₂O₂/hydroquinone (HQ) system. The magnitude of the cathodic signal obtained at -0.20 V (vs. the Ag pseudo-reference electrode) demonstrated linear dependence with the concentration of the synthetic target miRNA over the 1.0 to 100 pM range. The method provided a detection limit (LOD) of 10 attomoles (in a 25 μL sample) without any target miRNA amplification in just 30 min (once the DNA capture probe-MBs were prepared). This approach shows improved sensitivity compared with that of biosensors constructed with the same anti-DNA-RNA Ab as capture instead of a detector antibody and further labeling with a Strep-HRP conjugate instead of the Poly-HRP40 homopolymer. The developed strategy involves a single step working protocol, as well as the possibility to tailor the sensitivity by enlarging the length of the DNA/miRNA heteroduplexes using additional probes and/or performing the labelling with ProtA conjugated with homopolymers prepared with different numbers of HRP molecules. The practical usefulness was demonstrated by determination of the endogenous levels of the mature target miRNA in 250 ng raw total RNA (RNA t ) extracted from human mammary epithelial normal (MCF-10A) and cancer (MCF-7) cells and tumor tissues.

Nerve growth factor induced changes in the Golgi apparatus of PC-12 rat pheochromocytoma cells as studied by ligand endocytosis, cytochemical and morphometric methods.

PubMed

Hickey, W F; Stieber, A; Hogue-Angeletti, R; Gonatas, J; GOnatas, N K

1983-10-01

Cells of the PC-12 rat pheochromocytoma cell line respond to nerve growth factor (NGF) by sprouting neurites and biochemically differentiating into sympathetic ganglion-like cells. NGF-stimulated ('differentiated') and unstimulated ('undifferentiated') cells were studied by cytochemical techniques for the localization of the enzymes acid phosphatase (ACPase) and thiamine pyrophosphatase (TPPase), and by a morphometric analysis of the distribution of endocytosed wheat-germ agglutinin labelled with horseradish peroxidase (WGA-HRP). Both cytochemical stains showed the enzymes to be distributed in lysosomes and certain cisternae of the Golgi apparatus in both NGF stimulated and unstimulated cells. ACPase was not confined to GERL (Golgi-endoplasmic reticulum-lysosome) as in certain other cells. The morphometric studies demonstrated that the reaction product of the internalized WGA-HRP occupied 4.7% of the cytoplasmic area in unstimulated cells and 4.5% in NGF-stimulated ones. Despite this similarity, the distribution of the WGA-HRP among the studied intracellular compartments in these two cell groups varied. In the NGF-stimulated cells 3.3% of the WGA-HRP reaction product was found in the innermost Golgi cisterna(e) while in unstimulated cells only 0.3% was seen in this compartment. Similarly, 4.3% of the WGA-HRP stain was found in small vesicles at the 'trans' aspect of the Golgi apparatus in stimulated cells, when only 0.3% of the stain occupied this compartment in 'undifferentiated' cells. The morphometric analysis also revealed that when the PC-12 cells were stimulated with NGF, the Golgi apparatus increased in area by approximately 70%. These findings are consistent with the hypothesis that NGF induced differentiation of PC-12 cells is coupled with enhanced endocytosis of WGA and probably of its 'receptor' to the innermost Golgi cisterna(e) and the closely associated vesicles.

Design and simulation of proportional biological operational Mu-circuit.

PubMed

Xu, Dechang; Cai, Zhipeng; Liu, Ke; Zeng, Xiangmiao; Ouyang, Yujing; Dai, Cuihong; Hou, Aiju; Cheng, Dayou; Li, Jianzhong

2015-03-01

It is challenging yet desirable to quantitatively control the expression of a target gene in practice. We design a device-Proportional Biological Operational Mu-circuit (P-BOM) incorporating AND/OR gate and operational amplifier into one circuit and explore its behaviors through simulation. The results imply that will be possible to regulate input-output proportionally by manipulating the RBS of hrpR, hrpS, tetR and output gene and used in the sensing of environmental weak signals such as dioxins.

Horseradish peroxidase-labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome-specific repeat sequences.

PubMed

van Gijlswijk, R P; Wiegant, J; Vervenne, R; Lasan, R; Tanke, H J; Raap, A K

1996-01-01

We present a sensitive and rapid fluorescence in situ hybridization (FISH) strategy for detecting chromosome-specific repeat sequences. It uses horseradish peroxidase (HRP)-labeled oligonucleotide sequences in combination with fluorescent tyramide-based detection. After in situ hybridization, the HRP conjugated to the oligonucleotide probe is used to deposit fluorescently labeled tyramide molecules at the site of hybridization. The method features full chemical synthesis of probes, strong FISH signals, and short processing periods, as well as multicolor capabilities.

Glucose biosensor based on GOx/HRP bienzyme at liquid-crystal/aqueous interface.

PubMed

Khan, Mashooq; Park, Soo-Young

2015-11-01

Glucose oxidase (GOx) and horseradish peroxidase (HRP) were co-immobilized to the polyacrylicacid block of a poly(acrylicacid-b-4-cyanobiphenyl-4'-undecylacrylate) (PAA-b-LCP) copolymer in water. PAA-b-LCP was strongly anchored by the LCP block in 4-cyano-4'-pentylbiphenyl (5CB) which was contained in a transmission electron microscope (TEM) grid for glucose detection. The optimal conditions for the performance of the TEM grid glucose biosensor were studied in terms of the activity and stability of the immobilized enzymes. Glucose in water was detected by the 5CB changing from a planar to a homeotropic orientation, as observed through a polarized optical microscope. The TEM biosensor detected glucose concentrations at ⩾0.02 mM, with an optimal GOx/HRP molar ratio of 3/1. This glucose biosensor has characteristics of enzyme sensitivity and stability, reusability, the ease and selective glucose detection which may provide a new way of detecting glucose. Copyright © 2015 Elsevier Inc. All rights reserved.

Immobilization effects on the photocatalytic activity of CdS quantum Dots-Horseradish peroxidase hybrid nanomaterials.

PubMed

Iñarritu, Iker; Torres, Eduardo; Topete, Antonio; Campos-Terán, José

2017-11-15

The potential use of hybrid nanomaterials based on inorganic optically active nanoparticles known as quantum dots (QDs) and horseradish peroxidase (HRP) has been proposed by several authors as light-controllable nanocatalyzers, moreover, the immobilization within or over silica based supports represents an advantage over bulk-dispersed systems. However, the implications of the immobilization of such hybrid photoactivatable catalyzing systems have not been clarified with detail. Here, we present a thorough study of the functional photoactive efficiency and recycling of immobilized CdS QDs and HRP systems with different configurations, immobilized over silanized silica quartz crystal microbalance (QCM) sensors, allowing an accurate measure of the immobilized mass of each component and its correlation with the initial reaction rate of conversion of Amplex Red (AR) to resorufin. As well, the conversion efficiency is compared between the different systems and also to non-immobilized QD-HRP complexed systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin

NASA Astrophysics Data System (ADS)

Ni, Yuyang; Li, Jun; Huang, Zhenzhen; He, Ke; Zhuang, Jiaqi; Yang, Wensheng

2013-11-01

The using of macromolecular additives is known to be a simple and effective way to improve the activity of immobilized enzymes on solid support, yet the mechanism has not been well understood. Taking horseradish peroxidase (HRP) as an example, only 30 % of its catalytic activity was kept after being immobilized on the surface of 25-nm Au nanoparticles, mainly attributed to the conformational change of the heme-containing active site. The catalytic activity of HRP was significantly improved to 80 % when a certain amount of bovine serum albumin (BSA) was added at the initial stage of the immobilization. Systematic spectral investigation indicated that the addition of BSA inhibited the tertiary structure change around the active site, which was a prerequisite for improved activity of the immobilized HRP. Steady-state kinetic analyses revealed that the introduction of BSA could effectively improve the turnover rate of substrate to product in spite of slight reduced affinity to substrates, which also contributed to the improved catalytic activity.

An electrochemical ELISA-like immunosensor for miRNAs detection based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes.

PubMed

Tran, H V; Piro, B; Reisberg, S; Huy Nguyen, L; Dung Nguyen, T; Duc, H T; Pham, M C

2014-12-15

We design an electrochemical immunosensor for miRNA detection, based on screen-printed gold electrodes modified with reduced graphene oxide and carbon nanotubes. An original immunological approach is followed, using antibodies directed to DNA.RNA hybrids. An electrochemical ELISA-like amplification strategy was set up using a secondary antibody conjugated to horseradish peroxidase (HRP). Hydroquinone is oxidized into benzoquinone by the HRP/H2O2 catalytic system. In turn, benzoquinone is electroreduced into hydroquinone at the electrode. The catalytic reduction current is related to HRP amount immobilized on the surface, which itself is related to miRNA.DNA surface density on the electrode. This architecture, compared to classical optical detection, lowers the detection limit down to 10 fM. Two miRNAs were studied: miR-141 (a prostate biomarker) and miR-29b-1 (a lung cancer biomarker). Copyright © 2014 Elsevier B.V. All rights reserved.

A rapid and visual aptasensor for Lipopolysaccharides detection based on the bulb-like triplex turn-on switch coupled with HCR-HRP nanostructures.

PubMed

Xu, Wentao; Tian, Jingjing; Shao, Xiangli; Zhu, Longjiao; Huang, Kunlun; Luo, Yunbo

2017-03-15

For previously reported aptasensor, the sensitivity and selectivity of aptamers to targets were often suppressed due to the reporter label of single-stranded molecular beacon or hindrance of the duplex DNA strand displacement. To solve the affinity declining of aptamers showed in traditional way and realize on-site rapid detection of Lipopolysaccharides (LPS), we developed an ingenious structure-switching aptasensor based on the bulb-like triplex turn-on switch (BTTS) as the effective molecular recognition and signal transduction element and streptavidin-horseradish peroxidase modified hybridization chain reaction (HCR-HRP) nanocomposites as the signal amplifier and signal report element. In the presence of LPS, the bulb-like LPS-aptamer (BLA) and LPS formed the LPS/aptamer complex, while the BTTS disassembled and liberated the dissociative bridge probes (BP) to achieve molecular recognition and signal transduction. Immobilized BP, captured by immobilized capture probes (CP), triggered hybridization chain reactions (HCR) to amplify the switching signal, and the HCR products were then modified with streptavidin-horseradish peroxidase (SA-HRP) to form HCR-HRP nanostructures to output colorimetric signals. In less than four hours, the proposed biosensor showed a detection limit of 50pg/mL of LPS quantitatively with the portable spectrophotometer and the observation limit of 20ng/mL semi-quantitatively with the naked eye, opening up new opportunities for LPS detection in future clinical diagnosis, food security and environment monitoring. Copyright © 2016 Elsevier B.V. All rights reserved.

Monkey extensor digitorum communis motoneuron pool: Proximal dendritic trees and small motoneurons.

PubMed

Jenny, Arthur B; Cheney, Paul D; Jenny, Andrew K

2018-05-14

Transverse sections of the monkey cervical spinal cord from a previous study (Jenny and Inukai, 1983) were reanalyzed using Neurolucida to create a three-dimensional display of extensor digitorum communis (EDC) motoneurons and proximal dendrites that had been labeled with horse radish peroxidase (HRP). The EDC motoneuron pool was located primarily in the C8 and T1 segments of the spinal cord. Small motoneurons (cell body areas less than 500 μm 2 and presumed to be gamma motoneurons) comprised about ten percent of the motoneurons and were located throughout the length of the motoneuron pool. Most small motoneurons were oblong in shape and had one or two major dendrites originating from the cell body in the transverse plane of section. The majority of the HRP labeled dendritic trees were directed either superiorly, dorsal-medially to the mid zone area between the base of the dorsal horn and the upper portion of the ventral horn, or medially to the ventromedial gray matter. The longer HRP labeled dendrites usually continued in the same radial direction as when originating from the cell body. As such we considered the radial direction of the longer proximal HRP labeled dendrites to be a reasonable estimate of the radial direction of the more distal dendritic tree. Our data suggest that the motoneuron dendritic tree as seen in transverse section has direction-oriented dendrites that extend toward functional terminal regions. Copyright © 2018 Elsevier B.V. All rights reserved.

A hot-spot-active magnetic graphene oxide substrate for microRNA detection based on cascaded chemiluminescence resonance energy transfer.

PubMed

Bi, Sai; Chen, Min; Jia, Xiaoqiang; Dong, Ying

2015-02-28

Herein, a cascaded chemiluminescence resonance energy transfer (C-CRET) process was demonstrated from horseradish peroxidase (HRP)-mimicking DNAzyme-catalyzed luminol-H2O2 to fluorescein and further to graphene oxide (GO) when HRP-mimicking DNAzyme/fluorescein was in close proximity to the GO surface. The proposed C-CRET system was successfully implemented to construct three modes of C-CRET hot-spot-active substrates (modes I, II and III) by covalently immobilizing HRP-mimicking DNAzyme/fluorescein-labeled hairpin DNAs (hot-spot-generation probes) on magnetic GO (MGO), resulting in a signal "off" state due to the quenching of the luminol/H2O2/HRP-mimicking DNAzyme/fluorescein CRET system by GO. Upon the introduction of microRNA-122 (miRNA-122), the targets (mode I) or the new triggers that were generated through a strand displacement reaction (SDR) initiated by miRNA-122 (modes II and III) hybridized with the loop domains of hairpin probes on MGO to form double-stranded (modes I and II) or triplex-stem structures (mode III), causing an "open" configuration of the hairpin probe and a CRET signal "on" state, thus achieving sensitive and selective detection of miRNA-122. More importantly, the substrate exhibited excellent controllability, reversibility and reproducibility through SDR and magnetic separation (modes II and III), especially sequence-independence for hairpin probes in mode III, holding great potential for the development of a versatile platform for optical biosensing.

Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1.

PubMed

Sun, Linlin; Zhao, Qiang

2018-03-01

Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination. Copyright © 2017 Elsevier B.V. All rights reserved.

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

PubMed

Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

2015-12-15

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

Two overlapping two-component systems in Xanthomonas oryzae pv. oryzae contribute to full fitness in rice by regulating virulence factors expression

PubMed Central

Zheng, Dehong; Yao, Xiaoyan; Duan, Meng; Luo, Yufeng; Liu, Biao; Qi, Pengyuan; Sun, Ming; Ruan, Lifang

2016-01-01

Two-component signal transduction systems (TCSs) are widely used by bacteria to adapt to the environment. In the present study, StoS (stress tolerance-related oxygen sensor) and SreKRS (salt response kinase, regulator, and sensor) were found to positively regulate extracellular polysaccharide (EPS) production and swarming in the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). Surprisingly, the absence of stoS or sreKRS did not attenuate virulence. To better understand the intrinsic functions of StoS and SreKRS, quantitative proteomics isobaric tags for relative and absolute quantitation (iTRAQ) was employed. Consistent with stoS and sreK mutants exhibiting a similar phenotype, the signalling circuits of StoS and SreKRS overlapped. Carbohydrate metabolism proteins and chemotaxis proteins, which could be responsible for EPS and swarming regulation, respectively, were reprogrammed in stoS and sreK mutants. Moreover, StoS and SreKRS demonstrated moderate expression of the major virulence factor, hypersensitive response and pathogenicity (Hrp) proteins through the HrpG-HrpX circuit. Most importantly, Xoo equipped with StoS and SreKRS outcompetes strains without StoS or SreKRS in co-infected rice and grows outside the host. Therefore, we propose that StoS and SreKRS adopt a novel strategy involving the moderation of Hrp protein expression and the promotion of EPS and motility to adapt to the environment. PMID:26957113

Microfluidic immunosensor for rapid and highly-sensitive salivary cortisol quantification.

PubMed

Pinto, V; Sousa, P; Catarino, S O; Correia-Neves, M; Minas, G

2017-04-15

This paper presents a novel poly(dimethylsiloxane) (PDMS) microfluidic immunosensor that integrates a complementary metal-oxide-semiconductor (CMOS) optical detection system for a rapid and highly-sensitive quantification of salivary cortisol. The simple and non-invasive method of saliva sampling provides an interesting alternative to the blood, allowing a fast sampling at short intervals, relevant for many clinical diagnostic applications. The developed approach is based on the covalent immobilization of a coating antibody (Ab), a polyclonal anti-IgG, onto a treated PDMS surface. The coating Ab binds the capture Ab, an IgG specific for cortisol, allowing its correct orientation. Horseradish peroxidase (HRP)-labelled cortisol is added to compete with the cortisol in the sample, for the capture Ab binding sites. The HRP-labelled cortisol, bonded to the capture Ab, is measured through the HRP enzyme and the tetramethylbenzidine (TMB) substrate reaction. The cortisol quantification is performed by colorimetric detection of HRP-labelled cortisol, through optical absorption at 450nm, using a CMOS silicon photodiode as the photodetector. Under the developed optimized conditions presented here, e.g., microfluidic channels geometry, immobilization method and immunoassay conditions, the immunosensor shows a linear range of detection between 0.01-20ng/mL, a limit of detection (LOD) of 18pg/mL and an analysis time of 35min, featuring a great potential for point-of-care applications requiring continuous monitoring of the salivary cortisol levels during a circadian cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

Multilayers enzyme-coated carbon nanotubes as biolabel for ultrasensitive chemiluminescence immunoassay of cancer biomarker.

PubMed

Bi, Sai; Zhou, Hong; Zhang, Shusheng

2009-06-15

A novel and ultrasensitive chemiluminescence immunoassay (CLIA) method based on multiple enzyme layers assembled multiwall carbon nanotubes (MWCNTs) as signal amplification labels was developed by employing luminol-H(2)O(2)-HRP-bromophenol blue (BPB) enhanced chemiluminescence (CL) system for the detection of a cancer biomarker in human serum samples, as exemplified by the measurement of alpha-fetoprotein (AFP) as a model protein. In this study, horseradish peroxidase (HRP) was assembled onto MWCNTs templates layer-by-layer (LBL) through electrostatic interactions with polyion PDDA, and further conjugated with AFP secondary antibodies (Ab(2)) as the enzyme label. The resulting LBL assembly could maximize the ratio of HRP/Ab(2) which could amplify the sensitivity greatly. To the best of our knowledge, it was the first time for this strategy applied in CLIA to date. Under the optimum conditions of luminol-H(2)O(2)-HRP-BPB CL system and the sandwich immunoreactions, a linear range from 0.02 to 2.0 ng/mL (R=0.9980) was obtained with the detection limit of 8.0 pg/mL (3sigma) which was two orders of magnitude lower than standard ELISA method. Furthermore, accurate detection of AFP in human serum samples was also demonstrated by comparison to ELISA assays. From the above results, such signal amplification strategy proposed by the novel CNT-LBL enzyme label showed an excellent promise for ultrasensitive detection of cancer biomarkers in clinical laboratory.

Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv. aptata.

PubMed

Minardi, P

1995-01-01

A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.

Effect of body weight loss on cardiopulmonary function assessed by 6-minute walk test and arterial blood gas analysis in obese dogs.

PubMed

Manens, J; Ricci, R; Damoiseaux, C; Gault, S; Contiero, B; Diez, M; Clercx, C

2014-01-01

Few studies show the detrimental effect of canine obesity on cardiopulmonary function (CPF). The 6-Minute Walk Test (6MWT) is a noninvasive exercise test easy to perform in clinical settings. The aim of this study was to investigate the effect of obesity and body weight loss (BWL) on CPF assessed by the 6MWT and arterial blood gas analysis. Six experimental Beagles and 9 privately owned obese dogs were enrolled in a diet-induced BWL program. Arterial blood gas analysis and 6MWT were repeated in obese subjects (BCS 8-9/9), in the middle of BWL (overweight, BCS 6-7/9), and in lean dogs (BCS 5/9). Heart rate (HRp) and oxygen saturation (SpO2 ) were measured by pulse oximetry before the 6MWT, at midtest, and during a 5-minute recovery period. Twelve dogs completed the BWL program (initial BW, 27.3 ± 2.9 kg; final BW, 20.85 ± 2.9, lsmeans ± SE, P ≤ .001). BWL caused a significant increase in 6MWT walked distance (WD; obese: 509 ± 35 m; overweight: 575 ± 36 m; lean: 589 ± 36 m; P ≤ .05). Resting arterial blood gas results were not influenced by BWL. Including all time points, obese dogs showed higher HRp and lower SpO2 compared to overweight and lean dogs. SpO2 at the end of the walk was significantly lower in obese dogs. Obesity negatively affects 6MWT performances in dogs. The 6MWT may be used to demonstrate the efficacy of BWL to improve CPF and quality of life in obese dogs. Although BWL induced significant improvement of cardiopulmonary parameters before ideal BW, WD improved until the end of the BWL program. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Electrical Microengineering of Redox Enzymes

DTIC Science & Technology

1994-03-31

coating applied to the electrode 41 to prevent lose of mediator graphite foil potassium -~0.02a 0.03  electrolyte wus dioxane with 15% 42...hexacyanoferrats(ll)d aqueous buffer carbon fiber’ none h 40-5000 biotin/avidin complex used to obtain a 43 surface layer of HRP Pt organic metal, potassium ...OFFICE OF NAVAL RESEARCH AD-A279 015 FINAL REPORT FOR CONTRACT NOOO 14-91 -J- 1656 R&T CODE 413h007---05 ELECTRICAL MICROENGINEERING OF REDOX ENZYMES

Spectrofluorimetric determination of hydrogen peroxide with 2-hydroxy-1-naphthaldehyde salicyloylhydrazone (HNSH) as the substrate for horseradish peroxidase (HRP).

PubMed

Tang, Bo; Wang, Yan; Sun, Ying; Shen, Han Xi

2002-01-01

The oxidation reaction of HNSH with H2O2 under the catalysis of HRP was studied in detail. The possible reaction mechanism was discussed. Under optimum experimental conditions, the oxidized product of HNSH had excitation and emission maxima at 296 and 414 nm, respectively. A study to prove the existence of -O-O-H in polyethylene glycols was carried out. The proposed method was successfully applied to the determination of -O-O-H in polyethylene glycols.

Embryonic Lethality Due to Arrested Cardiac Development in Psip1/Hdgfrp2 Double-Deficient Mice.

PubMed

Wang, Hao; Shun, Ming-Chieh; Dickson, Amy K; Engelman, Alan N

2015-01-01

Hepatoma-derived growth factor (HDGF) related protein 2 (HRP2) and lens epithelium-derived growth factor (LEDGF)/p75 are closely related members of the HRP2 protein family. LEDGF/p75 has been implicated in numerous human pathologies including cancer, autoimmunity, and infectious disease. Knockout of the Psip1 gene, which encodes for LEDGF/p75 and the shorter LEDGF/p52 isoform, was previously shown to cause perinatal lethality in mice. The function of HRP2 was by contrast largely unknown. To learn about the role of HRP2 in development, we knocked out the Hdgfrp2 gene, which encodes for HRP2, in both normal and Psip1 knockout mice. Hdgfrp2 knockout mice developed normally and were fertile. By contrast, the double deficient mice died at approximate embryonic day (E) 13.5. Histological examination revealed ventricular septal defect (VSD) associated with E14.5 double knockout embryos. To investigate the underlying molecular mechanism(s), RNA recovered from ventricular tissue was subjected to RNA-sequencing on the Illumina platform. Bioinformatic analysis revealed several genes and biological pathways that were significantly deregulated by the Psip1 knockout and/or Psip1/Hdgfrp2 double knockout. Among the dozen genes known to encode for LEDGF/p75 binding factors, only the expression of Nova1, which encodes an RNA splicing factor, was significantly deregulated by the knockouts. However the expression of other RNA splicing factors, including the LEDGF/p52-interacting protein ASF/SF2, was not significantly altered, indicating that deregulation of global RNA splicing was not a driving factor in the pathology of the VSD. Tumor growth factor (Tgf) β-signaling, which plays a key role in cardiac morphogenesis during development, was the only pathway significantly deregulated by the double knockout as compared to control and Psip1 knockout samples. We accordingly speculate that deregulated Tgf-β signaling was a contributing factor to the VSD and prenatal lethality of Psip1/Hdgfrp2 double-deficient mice.

The accuracy of the first response histidine-rich protein2 rapid diagnostic test compared with malaria microscopy for guiding field treatment in an outbreak of falciparum malaria.

PubMed

Ghouth, Abdulla Salim Bin; Nasseb, Faraj Mubarak; Al-Kaldy, Khaled Hussin

2012-01-01

Recent WHO guidelines recommended a universal "test and treat" strategy for malaria mainly by use of the rapid diagnostic test (RDT) in all areas. There are concerns about RDT that use the antigen histidine-rich protein2 (HRP2) to detect Plasmodium falciparum, because infection can persist after effective treatment. The aim of this paper is to describe the accuracy of the first response (HRP2)-RDT compared with malaria microscopy used for guiding the field treatment of patients in an outbreak situation in the Al-Rahabah area in Al-Rydah district in Hadramout/Yemen. An ad hoc cross sectional survey of all febrile patients in the affected area was conducted in May 2011. The field team was developed including the case management group and the entomology group. The group of case management prepared their plan based on "test and treat" strategy by using First Response Malaria Antigen HRP2 rapid diagnostic test for falciparum malaria, artemsinin-based combination therapy (ACT) according to the national policy of anti-malaria drugs in Yemen were supplied to treat those who were found to be RDT positive in the field; also blood smear films were taken from every patient with fever in order to validate the use of the RDT in the field. Blood film slides prepared and read by skilled lab technicians, the fourth reading was done by one lab expert in the malaria referral lab. The accuracy parameters of HRP2 compared with microscopy are: Sensitivity (74%), specificity (94%). The positive predictive value is 68% and the negative predictive value is 96%. Total agreement is 148/162 (93%) and the overall prevalence is 14%. All the positive malaria cases were of P. falciparum either coming from RDT or microscopy. HRP2-rapid test is an acceptable test as a guide for field treatment in an outbreak situation where prompt response is indicated. Good prepared blood film slides should be used as it is feasible to evaluate the accuracy of RDTs as a quality control tool.

Nanoparticle-based sandwich electrochemical immunoassay for carbohydrate antigen 125 with signal enhancement using enzyme-coated nanometer-sized enzyme-doped silica beads.

PubMed

Tang, Dianping; Su, Biling; Tang, Juan; Ren, Jingjing; Chen, Guonan

2010-02-15

A novel nanoparticle-based electrochemical immunoassay of carbohydrate antigen 125 (CA125) as a model was designed to couple with a microfluidic strategy using anti-CA125-functionalized magnetic beads as immunosensing probes. To construct the immunoassay, thionine-horseradish peroxidase conjugation (TH-HRP) was initially doped into nanosilica particles using the reverse micelle method, and then HRP-labeled anti-CA125 antibodies (HRP-anti-CA125) were bound onto the surface of the synthesized nanoparticles, which were used as recognition elements. Different from conventional nanoparticle-based electrochemical immunoassays, the recognition elements of the immunoassay simultaneously contained electron mediator and enzyme labels and simplified the electrochemical measurement process. The sandwich-type immunoassay format was used for the online formation of the immunocomplex in an incubation cell and captured in the detection cell with an external magnet. The electrochemical signals derived from the carried HRP toward the reduction of H(2)O(2) using the doped thionine as electron mediator. Under optimal conditions, the electrochemical immunoassay exhibited a wide working range from 0.1 to 450 U/mL with a detection limit of 0.1 U/mL CA125. The precision, reproducibility, and stability of the immunoassay were acceptable. The assay was evaluated for clinical serum samples, receiving in excellent accordance with results obtained from the standard enzyme-linked immunosorbent assay (ELISA) method. Concluding, the nanoparticle-based assay format provides a promising approach in clinical application and thus represents a versatile detection method.

Chemiluminescence generation and detection in a capillary-driven microfluidic chip

NASA Astrophysics Data System (ADS)

Ramon, Charlotte; Temiz, Yuksel; Delamarche, Emmanuel

2017-02-01

The use of microfluidic technology represents a strong opportunity for providing sensitive, low-cost and rapid diagnosis at the point-of-care and such a technology might therefore support better, faster and more efficient diagnosis and treatment of patients at home and in healthcare settings both in developed and developing countries. In this work, we consider luminescence-based assays as an alternative to well-established fluorescence-based systems because luminescence does not require a light source or expensive optical components and is therefore a promising detection method for point-of-care applications. Here, we show a proof-of-concept of chemiluminescence (CL) generation and detection in a capillary-driven microfluidic chip for potential immunoassay applications. We employed a commercial acridan-based reaction, which is catalyzed by horseradish peroxidase (HRP). We investigated CL generation under flow conditions using a simplified immunoassay model where HRP is used instead of the complete sandwich immunocomplex. First, CL signals were generated in a capillary microfluidic chip by immobilizing HRP on a polydimethylsiloxane (PDMS) sealing layer using stencil deposition and flowing CL substrate through the hydrophilic channels. CL signals were detected using a compact (only 5×5×2.5 cm3) and custom-designed scanner, which was assembled for less than $30 and comprised a 128×1 photodiode array, a mini stepper motor, an Arduino microcontroller, and a 3D-printed housing. In addition, microfluidic chips having specific 30-μm-deep structures were fabricated and used to immobilize ensembles of 4.50 μm beads functionalized with HRP so as to generate high CL signals from capillary-driven chips.

Emotional Dissonance and Sickness Absence Among Employees Working With Customers and Clients: A Moderated Mediation Model via Exhaustion and Human Resource Primacy.

PubMed

Indregard, Anne-Marthe R; Ulleberg, Pål; Knardahl, Stein; Nielsen, Morten B

2018-01-01

Emotional dissonance, i.e., a discrepancy between required and felt emotions, has been established as a predictor of sickness absence in studies, but little is known about mechanisms that can explain this association. In order to prevent and reduce the impact of emotional dissonance on sickness absence, there is a need for greater attention to variables explaining when and how emotional dissonance is related to sickness absence. The overarching aim of this study was to examine whether emotional dissonance has an indirect association with sickness absence through exhaustion. In addition, we examined whether human resource primacy (HRP), which is the employer's degree of concern for human resources, moderates this indirect effect. A sample of 7758 employees, all working with customers and clients, were recruited from 96 Norwegian organizations. Emotional dissonance, exhaustion, and HRP were measured through surveys and then linked to registry data on medically certified sickness absence for the year following the survey assessment. Results showed that exhaustion is a mediator for the relationship between emotional dissonance and sickness absence. Furthermore, higher levels of HRP were found to reduce the positive association between emotional dissonance and exhaustion, and the indirect effect of emotional dissonance on sickness absence through exhaustion is found to be weaker when HRP is high. By testing this moderated mediation model, the current study contributes to the literature on emotion work by clarifying mechanisms that are crucial for the development of targeted interventions that aim to reduce and prevent sickness absence in client-driven work environments.

Investigating the effect of gallium curcumin and gallium diacetylcurcumin complexes on the structure, function and oxidative stability of the peroxidase enzyme and their anticancer and antibacterial activities.

PubMed

Jahangoshaei, Parisa; Hassani, Leila; Mohammadi, Fakhrossadat; Hamidi, Akram; Mohammadi, Khosro

2015-10-01

Curcumin has a wide spectrum of biological and pharmacological activities including anti-inflammatory, antioxidant, antiproliferative, antimicrobial and anticancer activities. Complexation of curcumin with metals has gained attention in recent years for improvement of its stability. In this study, the effect of gallium curcumin and gallium diacetylcurcumin on the structure, function and oxidative stability of horseradish peroxidase (HRP) enzyme were evaluated by spectroscopic techniques. In addition to the enzymatic investigation, the cytotoxic effect of the complexes was assessed on bladder, MCF-7 breast cancer and LNCaP prostate carcinoma cell lines by MTT assay. Furthermore, antibacterial activity of the complexes against S. aureus and E. coli was explored by dilution test method. The results showed that the complexes improve activity of HRP and also increase its tolerance against the oxidative condition. After addition of the complexes, affinity of HRP for hydrogen peroxide substrate decreases, while the affinity increases for phenol substrate. Circular dichroism, intrinsic and synchronous fluorescence spectra showed that the enzyme structure around the catalytic heme group becomes less compact and also the distance between the heme group and tryptophan residues increases due to binding of the complexes to HRP. On the whole, it can be concluded that the change in the enzyme structure upon binding to the gallium curcumin and gallium diacetylcurcumin complexes results in an increase in the antioxidant efficiency and activity of the peroxidise enzyme. The result of anticancer and antibacterial activities suggested that the complexes exhibit the potential for cancer treatment, but they have no significant antibacterial activity.

Projections of Somatosensory Cortex and Frontal Eye Fields onto Incertotectal Neurons in the Cat

PubMed Central

Perkins, Eddie; Warren, Susan; Lin, Rick C.-S.; May, Paul J.

2014-01-01

The goal of this study was to determine whether the input-output characteristics of the zona incerta (ZI) are appropriate for it to serve as a conduit for cortical control over saccade-related activity in the superior colliculus. The study utilized the neuronal tracers wheat germ agglutinin-horseradish peroxidase (WGA-HRP) and biotinylated dextran amine (BDA) in the cat. Injections of WGA-HRP into primary somatosensory cortex (SI) revealed sparse, widespread nontopographic projections throughout ZI. In addition, region-specific areas of more intense termination were present in ventral ZI, although strict topography was not observed. In comparison, the frontal eye fields (FEF) also projected sparsely throughout ZI, but terminated more heavily, medially, along the border between the two sublaminae. Furthermore, retrogradely labeled incertocortical neurons were observed in both experiments. The relationship of these two cortical projections to incertotectal cells was also directly examined by retrogradely labeling incertotectal cells with WGA-HRP in animals that had also received cortical BDA injections. Labeled axonal arbors from both SI and FEF had thin, sparsely branched axons with numerous en passant boutons. They formed numerous close associations with the somata and dendrites of WGA-HRP-labeled incertotectal cells. In summary, these results indicate that both sensory and motor cortical inputs to ZI display similar morphologies and distributions. In addition, both display close associations with incertotectal cells, suggesting direct synaptic contact. From these data, we conclude that inputs from somatosensory and FEF cortex both play a role in controlling gaze-related activity in the superior colliculus by way of the inhibitory incertotectal projection. PMID:17083121

Neurochemical diversity of afferent neurons that transduce sensory signals from dog ventricular myocardium

PubMed Central

Hoover, Donald B.; Shepherd, Angela V.; Southerland, E. Marie; Armour, J. Andrew; Ardell, Jeffrey L.

2008-01-01

While much is known about the influence of ventricular afferent neurons on cardiovascular function in the dog, identification of the neurochemicals transmitting cardiac afferent signals to central neurons is lacking. Accordingly, we identified ventricular afferent neurons in canine dorsal root ganglia (DRG) and nodose ganglia by retrograde labeling after injecting horseradish peroxidase (HRP) into the anterior right and left ventricles. Primary antibodies from three host species were used in immunohistochemical experiments to simultaneously evaluate afferent somata for the presence of HRP and markers for two neurotransmitters. Only a small percentage (2%) of afferent somata were labeled with HRP. About half of the HRP-identified ventricular afferent neurons in T3 DRG also stained for substance P (SP), calcitonin gene-related peptide (CGRP), or neuronal nitric oxide synthase (nNOS), either alone or with two markers colocalized. Ventricular afferent neurons and the general population of T3 DRG neurons showed the same labeling profiles; CGRP (alone or colocalized with SP) being the most common (30–40% of ventricular afferent somata in T3 DRG). About 30% of the ventricular afferent neurons in T2 DRG displayed CGRP immunoreactivity and binding of the putative nociceptive marker IB4. Ventricular afferent neurons of the nodose ganglia were distinct from those in the DRG by having smaller size and lacking immunoreactivity for SP, CGRP, and nNOS. These findings suggest that ventricular sensory information is transferred to the central nervous system by relatively small populations of vagal and spinal afferent neurons and that spinal afferents use a variety of neurotransmitters. PMID:18558516

Magnetic bead and gold nanoparticle probes based immunoassay for β-casein detection in bovine milk samples.

PubMed

Li, Y S; Meng, X Y; Zhou, Y; Zhang, Y Y; Meng, X M; Yang, L; Hu, P; Lu, S Y; Ren, H L; Liu, Z S; Wang, X R

2015-04-15

In this work, a double-probe based immunoassay was developed for rapid and sensitive determination of β-casein in bovine milk samples. In the method, magnetic beads (MBs), employed as supports for the immobilization of anti-β-casein polyclonal antibody (PAb), were used as the capture probe. Colloidal gold nanoparticles (AuNPs), employed as a bridge for loading anti-β-casein monoclonal antibody (McAb) and horseradish peroxidase (HRP), were used as the amplification probe. The presence of β-casein causes the sandwich structures of MBs-PAb-β-casein-McAb-AuNPs through the interaction between β-casein and the anti-β-casein antibodies. The HRP, used as an enzymatic-amplified tracer, can catalytically oxidize the substrate 3,3',5,5'-tetramethylbenzidine (TMB), generating optical signals that are proportional to the quantity of β-casein. The linear range of the immunoassay was from 6.5 to 1520ngmL(-1). The limit of detection (LOD) was 4.8ngmL(-1) which was 700 times lower than that of MBs-antibody-HRP based immunoassay and 6-7 times lower than that from the microplate-antibody-HRP based assay. The recoveries of β-casein from bovine milk samples were from 95.0% to 104.3% that had a good correlation coefficient (R(2)=0.9956) with those obtained by an official standard Kjeldahl method. For higher sensitivity, simple sample pretreatment and shorter time requirement of the antigen-antibody reaction, the developed immunoassay demonstrated the viability for detection of β-casein in bovine milk samples. Copyright © 2014. Published by Elsevier B.V.

Ultrastructural study of the GABAergic and cerebellar input to the nucleus reticularis tegmenti pontis.

PubMed

Verveer, C; Hawkins, R K; Ruigrok, T J; De Zeeuw, C I

1997-08-22

The nucleus reticularis tegmenti pontis is an intermediate of the cerebrocerebellar pathway and serves as a relay centre for sensorimotor and visual information. The central nuclei of the cerebellum provide a dense projection to the nucleus reticularis tegmenti pontis, but it is not known to what extent this projection is excitatory or inhibitory, and whether the terminals of this projection contact the neurons in the nucleus reticularis tegmenti pontis that give rise to the mossy fibre collaterals innervating the cerebellar nuclei. In the present study the nucleus reticularis tegmenti pontis of the cat was investigated at the ultrastructural level following anterograde and retrograde transport of wheat germ agglutinin coupled to horseradish peroxidase (WGA-HRP) from the cerebellar nuclei combined with postembedding GABA immunocytochemistry. The neuropil of this nucleus was found to contain many WGA-HRP labeled terminals, cell bodies and dendrites, but none of these pre- or postsynaptic structures was double labeled with GABA. The vast majority of the WGA-HRP labeled terminals contained clear spherical vesicles, showed asymmetric synapses, and contacted intermediate or distal dendrites. Many of the postsynaptic elements of the cerebellar afferents in the nucleus reticularis tegmenti pontis were retrogradely labeled with WGA-HRP, while relatively few were GABAergic. We conclude that all cerebellar terminals in the nucleus reticularis tegmenti pontis of the cat are nonGABAergic and excitatory, and that they contact predominantly neurons that project back to the cerebellum. Thus, the reciprocal circuit between the cerebellar nuclei and the nucleus reticularis tegmenti pontis appears to be well designed to function as an excitatory reverberating loop.

Emotional Dissonance and Sickness Absence Among Employees Working With Customers and Clients: A Moderated Mediation Model via Exhaustion and Human Resource Primacy

PubMed Central

Indregard, Anne-Marthe R.; Ulleberg, Pål; Knardahl, Stein; Nielsen, Morten B.

2018-01-01

Emotional dissonance, i.e., a discrepancy between required and felt emotions, has been established as a predictor of sickness absence in studies, but little is known about mechanisms that can explain this association. In order to prevent and reduce the impact of emotional dissonance on sickness absence, there is a need for greater attention to variables explaining when and how emotional dissonance is related to sickness absence. The overarching aim of this study was to examine whether emotional dissonance has an indirect association with sickness absence through exhaustion. In addition, we examined whether human resource primacy (HRP), which is the employer’s degree of concern for human resources, moderates this indirect effect. A sample of 7758 employees, all working with customers and clients, were recruited from 96 Norwegian organizations. Emotional dissonance, exhaustion, and HRP were measured through surveys and then linked to registry data on medically certified sickness absence for the year following the survey assessment. Results showed that exhaustion is a mediator for the relationship between emotional dissonance and sickness absence. Furthermore, higher levels of HRP were found to reduce the positive association between emotional dissonance and exhaustion, and the indirect effect of emotional dissonance on sickness absence through exhaustion is found to be weaker when HRP is high. By testing this moderated mediation model, the current study contributes to the literature on emotion work by clarifying mechanisms that are crucial for the development of targeted interventions that aim to reduce and prevent sickness absence in client-driven work environments. PMID:29670556

Neurochemical diversity of afferent neurons that transduce sensory signals from dog ventricular myocardium.

PubMed

Hoover, Donald B; Shepherd, Angela V; Southerland, E Marie; Armour, J Andrew; Ardell, Jeffrey L

2008-08-18

While much is known about the influence of ventricular afferent neurons on cardiovascular function in the dog, identification of the neurochemicals transmitting cardiac afferent signals to central neurons is lacking. Accordingly, we identified ventricular afferent neurons in canine dorsal root ganglia (DRG) and nodose ganglia by retrograde labeling after injecting horseradish peroxidase (HRP) into the anterior right and left ventricles. Primary antibodies from three host species were used in immunohistochemical experiments to simultaneously evaluate afferent somata for the presence of HRP and markers for two neurotransmitters. Only a small percentage (2%) of afferent somata were labeled with HRP. About half of the HRP-identified ventricular afferent neurons in T(3) DRG also stained for substance P (SP), calcitonin gene-related peptide (CGRP), or neuronal nitric oxide synthase (nNOS), either alone or with two markers colocalized. Ventricular afferent neurons and the general population of T(3) DRG neurons showed the same labeling profiles; CGRP (alone or colocalized with SP) being the most common (30-40% of ventricular afferent somata in T(3) DRG). About 30% of the ventricular afferent neurons in T(2) DRG displayed CGRP immunoreactivity and binding of the putative nociceptive marker IB(4). Ventricular afferent neurons of the nodose ganglia were distinct from those in the DRG by having smaller size and lacking immunoreactivity for SP, CGRP, and nNOS. These findings suggest that ventricular sensory information is transferred to the central nervous system by relatively small populations of vagal and spinal afferent neurons and that spinal afferents use a variety of neurotransmitters.

Exploration Medical System Demonstration (EMSD) Project

NASA Technical Reports Server (NTRS)

Chin, Duane

2012-01-01

The Exploration Medical System Demonstration (EMSD) is a project under the Exploration Medical Capability (ExMC) element managed by the Human Research Program (HRP). The vision for the EMSD is to utilize ISS as a test bed to show that several medical technologies needed for an exploration mission and medical informatics tools for managing evidence and decision making can be integrated into a single system and used by the on-orbit crew in an efficient and meaningful manner. Objectives: a) Reduce and even possibly eliminate the time required for on-orbit crew and ground personnel (which include Surgeon, Biomedical Engineer (BME) Flight Controller, and Medical Operations Data Specialist) to access and move medical data from one application to another. b) Demonstrate that the on-orbit crew has the ability to access medical data/information using an intuitive and crew-friendly software solution to assist/aid in the treatment of a medical condition. c) Develop a common data management framework and architecture that can be ubiquitously used to automate repetitive data collection, management, and communications tasks for all crew health and life sciences activities.

Rapid and Sensitive Quantification of Vibrio cholerae and Vibrio mimicus Cells in Water Samples by Use of Catalyzed Reporter Deposition Fluorescence In Situ Hybridization Combined with Solid-Phase Cytometry

PubMed Central

Schauer, Sonja; Sommer, Regina; Farnleitner, Andreas H.

2012-01-01

A new protocol for rapid, specific, and sensitive cell-based quantification of Vibrio cholerae/Vibrio mimicus in water samples was developed. The protocol is based on catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) in combination with solid-phase cytometry. For pure cultures, we were able to quantify down to 6 V. cholerae cells on one membrane with a relative precision of 39% and down to 12 cells with a relative precision of 17% after hybridization with the horseradish peroxidase (HRP)-labeled probe Vchomim1276 (specific for V. cholerae and V. mimicus) and signal amplification. The corresponding position of the probe on the 16S rRNA is highly accessible even when labeled with HRP. For the first time, we were also able to successfully quantify V. cholerae/V. mimicus via solid-phase cytometry in extremely turbid environmental water samples collected in Austria. Cell numbers ranged from 4.5 × 101 cells ml−1 in the large saline lake Neusiedler See to 5.6 × 104 cells ml−1 in an extremely turbid shallow soda lake situated nearby. We therefore suggest CARD-FISH in combination with solid-phase cytometry as a powerful tool to quantify V. cholerae/V. mimicus in ecological studies as well as for risk assessment and monitoring programs. PMID:22885749

Virulence Factors of Erwinia amylovora: A Review.

PubMed

Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M

2015-06-05

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3'-5')-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

Spatial and temporal control of microwave triggered chemiluminescence: a protein detection platform.

PubMed

Previte, Michael J R; Aslan, Kadir; Geddes, Chris D

2007-09-15

We have combined the principles of microwave circuitry and antenna design and our recent work in microwave-triggered metal-enhanced chemiluminescence to now "trigger" chemically and enzyme-catalyzed chemiluminescent reactions with spatial and temporal control. With this technology platform, we achieve spatial and temporal control of enzyme and chemically catalyzed chemiluminescence reactions to achieve more than 500-fold increases in "on-demand" photon flux from chemically catalyzed chemiluminescent reactions. We also report a 6-fold increase in photon flux from HRP-catalyzed assays on disposable coverslips functionalized with HRP and placed proximal to the substrates modified with thin-film aluminum triangle disjointed "bow-tie" structures. In addition, we demonstrate the applicability of this technology to develop multiplexed or high-throughput chemiluminescent assays. We also demonstrate the clinical and biological relevance of this technology platform by affixing aluminum structures in proximity to HRP protein immobilized on nitrocellulose to improve the sensitivity for this model Western blot scheme by 50-fold. We believe analytical applications that rely on enzyme-catalyzed chemiluminescence, such as immunoassays, may greatly benefit from this new platform technology.

Evidence of a gustatory-vestibular pathway for protein transport.

PubMed

Gacek, Richard; Lyon, Michael J

2010-02-01

To demonstrate anatomically a pathway for protein transport from the palate to the vestibular system. The vestibulofacial anastomosis and associated ganglion cells were identified in a collection of 160 horizontally sectioned human temporal bones that had been stained with hematoxylin and eosin. Wheat germ agglutinin-horseradish peroxidase (HRP) was applied to the greater superficial petrosal nerve in 4 Sprague-Dawley rats. After 30 hours, the rats were killed by intracardiac perfusion, and the seventh and eighth nerves with adjacent brainstem removed. Frozen sections cut at 30 mum through this block were then reacted for HRP, counterstained with neutral red, and mounted on slides for examination in the light microscope. Thirty-two of the 160 human temporal bones contained sections through the vestibulofacial anastomosis and its ganglion. In all cases, the ganglion was incorporated into the vestibular ganglion (VG) adjacent to the nervus intermedius. In all 4 experimental rats, HRP reaction product labeled a small number of ganglion cells in the VG adjacent to the nervus intermedius and facial nerve. These observations support the presence of a pathway from receptors in the palate to the VG.

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

PubMed

Gill, Pooria; Forouzandeh, Mehdi; Rahbarizadeh, Fatemeh; Ramezani, Reihaneh; Rasaee, Mohammad Javad

2006-01-01

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Stable and general-purpose chemiluminescent detection system for horseradish peroxidase employing a thiazole compound enhancer and some additives.

PubMed

Iwata, R; Ito, H; Hayashi, T; Sekine, Y; Koyama, N; Yamaki, M

1995-10-10

A stable and highly sensitive chemiluminescent detection system for horseradish peroxidase (HRP)/luminol/hydrogen peroxide using a newly designed thiazole compound enhancer has been established. Some additives for the chemiluminescent reaction were explored to overcome some defects of the reaction such as rapid decay and high background of light emission. Recrystallization of luminol and the addition of several detergents into the reacting solution were effective to increase specific light emissions. The addition of skim milk into the reacting solution reduced the background. Consequently, skim milk combined with a detergent increased the signal to noise ratio about 20 times compared with the reactions in the absence of both additives. The optimal concentration of enhancer and the addition of egg albumin stabilized the emission. In the new method, 6x 10(-18) mol of HRP was detectable. This would be the most sensitive enhanced chemiluminescent detection system for HRP. Furthermore, we could detect picogram per milliliter (10(-17) mol) concentrations of a trace component in biological materials such as endothelin-1 by employing this reaction.

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections.

PubMed

Park, J S; Kurman, R J; Kessis, T D; Shah, K V

1991-01-01

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and 35S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe but not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.

Gold nanoparticles bound on microgel particles and their application as an enzyme support

NASA Astrophysics Data System (ADS)

Xu, Jing; Zeng, Fang; Wu, Shuizhu; Liu, Xinxing; Hou, Chao; Tong, Zhen

2007-07-01

Submicron-sized poly(N-isopropyl acrylamide)/polyethyleneimine core-shell microgels were prepared in aqueous media by using tert-butyl hydroperoxide (TBHP) as an initiator, and then the gold nanoparticles (~8 nm) were formed on the surface of the microgels. The amino groups on the polyethyleneimine (PEI) chains act as the binder for the assembly of the gold nanoparticles/microgel complex. In aqueous media the microgels are highly stable with the gold nanoparticles on their extended PEI chains, and this multi-scale nanoparticle complex can be recovered from water and redispersed in water. The nanogold/microgel particles were conjugated with the enzymes horseradish peroxidase (HRP) and urease. It is found that under identical assay conditions the enzyme/nanogold/microgel systems exhibit enhanced biocatalytic activity over free enzymes in solution, especially at lower enzyme concentrations. In addition, compared to free HRP, the HRP/nanogold/microgel systems show higher activity at varied pHs and temperatures, as well as higher storage stability. Thus the novel nanogold/microgel particles can serve as an excellent support for enzymes.

Polydimethylsiloxane microfluidic chemiluminescence immunodevice with the signal amplification strategy for sensitive detection of human immunoglobin G.

PubMed

Li, Huifang; Zhao, Mei; Liu, Wei; Chu, Weiru; Guo, Yumei

2016-01-15

A polydimethylsiloxane (PDMS) microfluidic chemiluminescence (CL) immunodevice for sensitive detection of human immunoglobin G (IgG) with the signal amplification strategy was developed in this work. The immunodevice was prepared by covalently immobilizing capture antibodies (Abs) on the silanized microchannel of microfluidic chip. Gold nanoparticles (AuNPs) functionalized with a high molar ratio of horseradish peroxidase (HRP) were used as an Ab label for signal amplification. Using a sandwich immunoassay, the multi-HRP conjugated AuNPs can catalyze the luminol-H2O2 CL system to achieve the high sensitivity. In addition, the double spiral flow-channel was adopted here, which can still contribute to the high sensitivity. Based on signal amplification strategy, the performance of human IgG tests revealed a lower detection limit (DL) of 0.03ng/mL and showed an increase of 7.4-fold in detection sensitivity compared to a commercial Ab-HRP conjugation. This microfluidic immunodevice can provide an alternative approach for sensitive detection of human IgG in the field of clinic diagnostic and therapeutic. Copyright © 2015 Elsevier B.V. All rights reserved.

Type III secretion system effector proteins: double agents in bacterial disease and plant defense.

PubMed

Alfano, James R; Collmer, Alan

2004-01-01

Many phytopathogenic bacteria inject virulence effector proteins into plant cells via a Hrp type III secretion system (TTSS). Without the TTSS, these pathogens cannot defeat basal defenses, grow in plants, produce disease lesions in hosts, or elicit the hypersensitive response (HR) in nonhosts. Pathogen genome projects employing bioinformatic methods to identify TTSS Hrp regulon promoters and TTSS pathway targeting signals suggest that phytopathogenic Pseudomonas, Xanthomonas, and Ralstonia spp. harbor large arsenals of effectors. The Hrp TTSS employs customized cytoplasmic chaperones, conserved export components in the bacterial envelope (also used by the TTSS of animal pathogens), and a more specialized set of TTSS-secreted proteins to deliver effectors across the plant cell wall and plasma membrane. Many effectors can act as molecular double agents that betray the pathogen to plant defenses in some interactions and suppress host defenses in others. Investigations of the functions of effectors within plant cells have demonstrated the plasma membrane and nucleus as subcellular sites for several effectors, revealed some effectors to possess cysteine protease or protein tyrosine phosphatase activity, and provided new clues to the coevolution of bacterium-plant interactions.

Nanogold-enwrapped graphene nanocomposites as trace labels for sensitivity enhancement of electrochemical immunosensors in clinical immunoassays: Carcinoembryonic antigen as a model.

PubMed

Zhong, Zhaoyang; Wu, Wei; Wang, Dong; Wang, Dan; Shan, Jinlu; Qing, Yi; Zhang, Zhimin

2010-06-15

A new, highly sensitive electrochemical immunosensor with a sandwich-type immunoassay format was designed to quantify carcinoembryonic antigen (CEA), as a model tumor marker, using nanogold-enwrapped graphene nanocomposites (NGGNs) as trace labels in clinical immunoassays. The device consisted of a glassy carbon electrode coated with Prussian Blue (PB) on whose surface gold nanoparticles were electrochemically deposited to the further modified with the specific analyte-capturing molecule, anti-CEA antibodies. The immunoassay was performed using horseradish peroxidase (HRP)-conjugated anti-CEA as secondary antibodies attached on the NGGN surface (HRP-anti-CEA-NGGN). The method using HRP-anti-CEA-NGGNs as detection antibodies shows high signal amplification, and exhibits a dynamic working range of 0.05-350 ng/mL with a low detection limit of 0.01 ng/mL CEA (at 3s). The assayed results of serum samples with the sensor received an acceptable agreement with the reference values. Importantly, the methodology provides a promising ultrasensitive assay strategy for clinical applications. Copyright 2010 Elsevier B.V. All rights reserved.

Can We Trust Computational Modeling for Medical Applications?

NASA Technical Reports Server (NTRS)

Mulugeta, Lealem; Walton, Marlei; Nelson, Emily; Myers, Jerry

2015-01-01

Operations in extreme environments such as spaceflight pose human health risks that are currently not well understood and potentially unanticipated. In addition, there are limited clinical and research data to inform development and implementation of therapeutics for these unique health risks. In this light, NASA's Human Research Program (HRP) is leveraging biomedical computational models and simulations (M&S) to help inform, predict, assess and mitigate spaceflight health and performance risks, and enhance countermeasure development. To ensure that these M&S can be applied with confidence to the space environment, it is imperative to incorporate a rigorous verification, validation and credibility assessment (VV&C) processes to ensure that the computational tools are sufficiently reliable to answer questions within their intended use domain. In this presentation, we will discuss how NASA's Integrated Medical Model (IMM) and Digital Astronaut Project (DAP) have successfully adapted NASA's Standard for Models and Simulations, NASA-STD-7009 (7009) to achieve this goal. These VV&C methods are also being leveraged by organization such as the Food and Drug Administration (FDA), National Institute of Health (NIH) and the American Society of Mechanical Engineers (ASME) to establish new M&S VV&C standards and guidelines for healthcare applications. Similarly, we hope to provide some insight to the greater aerospace medicine community on how to develop and implement M&S with sufficient confidence to augment medical research and operations.

Genetic deletion of HRP2 and HRP3 in Indian Plasmodium falciparum population and false negative malaria rapid diagnostic test.

PubMed

Kumar, Navin; Pande, Veena; Bhatt, R M; Shah, Naman K; Mishra, Neelima; Srivastava, Bina; Valecha, Neena; Anvikar, Anupkumar R

2013-01-01

Genetic polymorphisms in diagnostic antigens are important factors responsible for variable performance of rapid diagnostic tests. Additionally, the failure of antigen expression due to gene deletion may also contribute to variable performance. We report Indian Plasmodium falciparum field isolates lacking both Pfhrp2 and Pfhrp3 genes leading to false negative results of rapid diagnostic tests. The study highlights need to determine the prevalence of P. falciparum isolates lacking these genes in larger field populations in India. Copyright © 2012 Elsevier B.V. All rights reserved.

A novel and efficient oxidative functionalization of lignin by layer-by-layer immobilised Horseradish peroxidase.

PubMed

Perazzini, Raffaella; Saladino, Raffaele; Guazzaroni, Melissa; Crestini, Claudia

2011-01-01

Horseradish peroxidase (HRP) was chemically immobilised onto alumina particles and coated by polyelectrolytes layers, using the layer-by-layer technique. The reactivity of the immobilised enzyme was studied in the oxidative functionalisation of softwood milled wood and residual kraft lignins and found higher than the free enzyme. In order to investigate the chemical modifications in the lignin structure, quantitative (31)P NMR was used. The immobilised HRP showed a higher reactivity with respect to the native enzyme yielding extensive depolymerisation of lignin. Copyright © 2010 Elsevier Ltd. All rights reserved.

Meta-analysis: accuracy of rapid tests for malaria in travelers returning from endemic areas.

PubMed

Marx, Arthur; Pewsner, Daniel; Egger, Matthias; Nüesch, Reto; Bucher, Heiner C; Genton, Blaise; Hatz, Christoph; Jüni, Peter

2005-05-17

Microscopic diagnosis of malaria is unreliable outside specialized centers. Rapid tests have become available in recent years, but their accuracy has not been assessed systematically. To determine the accuracy of rapid diagnostic tests for ruling out malaria in nonimmune travelers returning from malaria-endemic areas. The authors searched MEDLINE, EMBASE, CAB Health, and CINAHL (1988 to September 2004); hand-searched conference proceedings; checked reference lists; and contacted experts and manufacturers. Diagnostic accuracy studies in nonimmune individuals with suspected malaria were included if they compared rapid tests with expert microscopic examination or polymerase chain reaction tests. Data on study and patient characteristics and results were extracted in duplicate. The main outcome was the likelihood ratio for a negative test result (negative likelihood ratio) for Plasmodium falciparum malaria. Likelihood ratios were combined by using random-effects meta-analysis, stratified by the antigen targeted (histidine-rich protein-2 [HRP-2] or parasite lactate dehydrogenase [LDH]) and by test generation. Nomograms of post-test probabilities were constructed. The authors included 21 studies and 5747 individuals. For P. falciparum, HRP-2-based tests were more accurate than parasite LDH-based tests: Negative likelihood ratios were 0.08 and 0.13, respectively (P = 0.019 for difference). Three-band HRP-2 tests had similar negative likelihood ratios but higher positive likelihood ratios compared with 2-band tests (34.7 vs. 98.5; P = 0.003). For P. vivax, negative likelihood ratios tended to be closer to 1.0 for HRP-2-based tests than for parasite LDH-based tests (0.24 vs. 0.13; P = 0.22), but analyses were based on a few heterogeneous studies. Negative likelihood ratios for the diagnosis of P. malariae or P. ovale were close to 1.0 for both types of tests. In febrile travelers returning from sub-Saharan Africa, the typical probability of P. falciparum malaria is estimated at 1.1% (95% CI, 0.6% to 1.9%) after a negative 3-band HRP-2 test result and 97% (CI, 92% to 99%) after a positive test result. Few studies evaluated 3-band HRP-2 tests. The evidence is also limited for species other than P. falciparum because of the few available studies and their more heterogeneous results. Further studies are needed to determine whether the use of rapid diagnostic tests improves outcomes in returning travelers with suspected malaria. Rapid malaria tests may be a useful diagnostic adjunct to microscopy in centers without major expertise in tropical medicine. Initial decisions on treatment initiation and choice of antimalarial drugs can be based on travel history and post-test probabilities after rapid testing. Expert microscopy is still required for species identification and confirmation.

The Research Plan: Closing the ExMC Med02 "Pharmacy" Gap

NASA Technical Reports Server (NTRS)

Daniels, Vernie; Bayuse, Tina; Mulcahy, Robert; Shah, Ronak; Antonsen, Erik

2017-01-01

HRP Human Research Roadmap: Risk and Gap Risk of Adverse Health Outcomes and Decrements in Performance due to Inflight Medical Conditions. Med02 "Pharmacy" Gap: We do not have the capability to provide a safe and effective medication formulary for exploration missions delivering a recommendation for a chemically stable, safe, and effective medication formulary that will support the operational needs of exploration space missions research strategy evidence-based formulary and models innovative analytical tools and methodologies novel treatments and preventive measures Planned review by a panel of experts from the pharmaceutical industry, regulatory, and academic scientific communities Formulary Selection Formulary Potency and Shelf life Formulary Safety and Toxicity Novel Technology Proof-of-Concept Portable real-time chemical analysis Innovative drug development / design

Peroxidase gene discovery from the horseradish transcriptome.

PubMed

Näätsaari, Laura; Krainer, Florian W; Schubert, Michael; Glieder, Anton; Thallinger, Gerhard G

2014-03-24

Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Peroxidase gene discovery from the horseradish transcriptome

PubMed Central

2014-01-01

Background Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. Results In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. Conclusions This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes. PMID:24666710

Antigen persistence of rapid diagnostic tests in pregnant women in Nanoro, Burkina Faso, and the implications for the diagnosis of malaria in pregnancy.

PubMed

Kattenberg, Johanna H; Tahita, Christian M; Versteeg, Inge A J; Tinto, Halidou; Traoré-Coulibaly, Maminata; Schallig, Henk D F H; Mens, Petra F

2012-05-01

To evaluate persistence of several Plasmodium antigens in pregnant women after treatment and compare diagnostics during treatment follow-up. Thirty-two pregnant women (N = 32) with confirmed malaria infection by a histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) and microscopy were followed for 28 days after artemisinin-based combination therapy (ACT). A Plasmodium lactate dehydrogenase (pLDH)-based RDT and two ELISAs based on the detection of dihydrofolate reductase-thymidylate synthase (DHFR-TS) and haeme detoxification protein (HDP) were compared with each other and to RT-PCR at each visit. The mean visit number (95% confidence interval) on which the HRP2-based RDT was still positive after treatment was 3.4 (2.7-4.1) visits with some patients still positive at day 28. This is significantly later than the pLDH-based RDT [0.84 (0.55-1.1)], microscopy (median 1, range 1-3), DHFR-TS-ELISA [1.7 (1.1-2.3)] and RT-PCR (median 2, range 1-5) (P < 0.05), but not significantly later than HDP-ELISA [2.1 (1.6-2.7)]. Lower gravidity and higher parasite density at day 0 resulted in significantly longer positive results with most tests (P < 0.05). HRP2 can persist up to 28 days after ACT treatment; therefore, this test is not suitable for treatment follow-up in pregnant women and can generate problems when using this test during intermittent preventive treatment (IPTp). DHFR-TS is less persistent than HRP2, making it a potentially interesting target for diagnosis. © 2012 Blackwell Publishing Ltd.

Plasmodium falciparum diagnostic tools in HIV-positive under-5-year-olds in two ART clinics in Ghana: are there missed infections?

PubMed

Owusu, Ewurama D A; Djonor, Samson K; Brown, Charles A; Grobusch, Martin P; Mens, Petra F

2018-02-23

Plasmodium falciparum, the most dominant species in sub-Saharan Africa, causes the most severe clinical malaria manifestations. In resource-limited Ghana, where malaria and HIV geographically overlap, histidine-rich protein 2 (HRP2)-based rapid diagnostic test (RDT) is a faster, easier and cheaper alternative to clinical gold standard light microscopy. However, mutations in parasite hrp2 gene may result in missed infections, which have severe implications for malaria control. The performance of a common HRP2-based RDT and expert light microscopy in HIV-positive and HIV-negative children under 5 years old was compared with PCR as laboratory gold standard. Finger-prick capillary blood was tested with First Response ® Malaria Ag P. falciparum (HRP2). Giemsa-stained thick and thin blood films were examined with ≥ 200 high power fields and parasites counted per 200 white blood cells. Nested PCR species identification of P. falciparum was performed and resolved on agarose gel. False negatives from RDT were further tested for deleted pfhrp2/3 and flanking genes, using PCR. The study was performed in two anti-retroviral therapy clinics in Accra and Atibie. Out of 401 participants enrolled, 150 were HIV positive and 251 HIV negative. Malaria was more prevalent in children without HIV. Microscopy had a higher sensitivity [100% (99-100)] than RDT [83% (53.5-100)]. Parasites with pfhrp2/3 deletions contributed to missed infections from RDT false negatives. Circulation of malaria parasites with pfrhp2/3 deletions in this population played a role in missed infections with RDT. This ought to be addressed if further strides in malaria control are to be made.

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes.

PubMed

Smith, Shavannor M; Steinau, Martin; Trick, Harold N; Hulbert, Scot H

2010-06-01

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.

A repeatable assembling and disassembling electrochemical aptamer cytosensor for ultrasensitive and highly selective detection of human liver cancer cells.

PubMed

Sun, Duanping; Lu, Jing; Chen, Zuanguang; Yu, Yanyan; Mo, Manni

2015-07-23

In this work, a repeatable assembling and disassembling electrochemical aptamer cytosensor was proposed for the sensitive detection of human liver hepatocellular carcinoma cells (HepG2) based on a dual recognition and signal amplification strategy. A high-affinity thiolated TLS11a aptamer, covalently attached to a gold electrode through Au-thiol interactions, was adopted to recognize and capture the target HepG2 cells. Meanwhile, the G-quadruplex/hemin/aptamer and horseradish peroxidase (HRP) modified gold nanoparticles (G-quadruplex/hemin/aptamer-AuNPs-HRP) nanoprobe was designed. It could be used for electrochemical cytosensing with specific recognition and enzymatic signal amplification of HRP and G-quadruplex/hemin HRP-mimicking DNAzyme. With the nanoprobes as recognizing probes, the HepG2 cancer cells were captured to fabricate an aptamer-cell-nanoprobes sandwich-like superstructure on a gold electrode surface. The proposed electrochemical cytosensor delivered a wide detection range from 1×10(2) to 1×10(7) cells mL(-1) and high sensitivity with a low detection limit of 30 cells mL(-1). Furthermore, after the electrochemical detection, the activation potential of -0.9 to -1.7V was performed to break Au-thiol bond and regenerate a bare gold electrode surface, while maintaining the good characteristic of being used repeatedly. The changes of gold electrode behavior after assembling and desorption processes were investigated by electrochemical impedance spectroscopy and cyclic voltammetry techniques. These results indicate that the cytosensor has great potential in disease diagnostic of cancers and opens new insight into the reusable gold electrode with repeatable assembling and disassembling in the electrochemical sensing. Copyright © 2015 Elsevier B.V. All rights reserved.

Microgel coating of magnetic nanoparticles via bienzyme-mediated free-radical polymerization for colorimetric detection of glucose

NASA Astrophysics Data System (ADS)

Wu, Qing; Wang, Xia; Liao, Chuanan; Wei, Qingcong; Wang, Qigang

2015-10-01

This study describes a new strategy for the fabrication of magnetic core-shell microgels by free-radical polymerization triggered by the cascade reaction of glucose oxidase (GOx) and horseradish peroxidase (HRP). The mild polymerization around the interface of the magnetic nanoparticles permits the mild coating of the microgel layer with excellent characteristics for various applications in biocatalysis and medical diagnostics, as well as in clinical fields. The immobilized bienzyme within the microgel has a largely retained activity relative to the non-immobilized one. The confining effect of the microgel and the well designed distance between the two enzymes can benefit the diffusion of intermediates to the HRP active site. The final microgels can be incontestably employed as sensitive biosensors for colorimetric glucose detection.This study describes a new strategy for the fabrication of magnetic core-shell microgels by free-radical polymerization triggered by the cascade reaction of glucose oxidase (GOx) and horseradish peroxidase (HRP). The mild polymerization around the interface of the magnetic nanoparticles permits the mild coating of the microgel layer with excellent characteristics for various applications in biocatalysis and medical diagnostics, as well as in clinical fields. The immobilized bienzyme within the microgel has a largely retained activity relative to the non-immobilized one. The confining effect of the microgel and the well designed distance between the two enzymes can benefit the diffusion of intermediates to the HRP active site. The final microgels can be incontestably employed as sensitive biosensors for colorimetric glucose detection. Electronic supplementary information (ESI) available: Experimental details and ESI figures. See DOI: 10.1039/c5nr05716g

[Effects of electromagnetic pulse exposure on the permeability of inner blood-retinal barrier model in vitro].

PubMed

Li, Hai-juan; Yang, Long-long; Tian, Wei; Liu, Jun-ju; Xie, Xue-jun; Guo, Guo-zhen

2012-03-01

To establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro. RF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro. TEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure. The inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

Comparative proteomic analysis of Xanthomonas citri ssp. citri periplasmic proteins reveals changes in cellular envelope metabolism during in vitro pathogenicity induction.

PubMed

Artier, Juliana; da Silva Zandonadi, Flávia; de Souza Carvalho, Flávia Maria; Pauletti, Bianca Alves; Leme, Adriana Franco Paes; Carnielli, Carolina Moretto; Selistre-de-Araujo, Heloisa Sobreiro; Bertolini, Maria Célia; Ferro, Jesus Aparecido; Belasque Júnior, José; de Oliveira, Julio Cezar Franco; Novo-Mansur, Maria Teresa Marques

2018-01-01

Citrus canker is a plant disease caused by Gram-negative bacteria from the genus Xanthomonas. The most virulent species is Xanthomonas citri ssp. citri (XAC), which attacks a wide range of citrus hosts. Differential proteomic analysis of the periplasm-enriched fraction was performed for XAC cells grown in pathogenicity-inducing (XAM-M) and pathogenicity-non-inducing (nutrient broth) media using two-dimensional electrophoresis combined with liquid chromatography-tandem mass spectrometry. Amongst the 40 proteins identified, transglycosylase was detected in a highly abundant spot in XAC cells grown under inducing condition. Additional up-regulated proteins related to cellular envelope metabolism included glucose-1-phosphate thymidylyltransferase, dTDP-4-dehydrorhamnose-3,5-epimerase and peptidyl-prolyl cis-trans-isomerase. Phosphoglucomutase and superoxide dismutase proteins, known to be involved in pathogenicity in other Xanthomonas species or organisms, were also detected. Western blot and quantitative real-time polymerase chain reaction analyses for transglycosylase and superoxide dismutase confirmed that these proteins were up-regulated under inducing condition, consistent with the proteomic results. Multiple spots for the 60-kDa chaperonin and glyceraldehyde-3-phosphate dehydrogenase were identified, suggesting the presence of post-translational modifications. We propose that substantial alterations in cellular envelope metabolism occur during the XAC infectious process, which are related to several aspects, from defence against reactive oxygen species to exopolysaccharide synthesis. Our results provide new candidates for virulence-related proteins, whose abundance correlates with the induction of pathogenicity and virulence genes, such as hrpD6, hrpG, hrpB7, hpa1 and hrpX. The results present new potential targets against XAC to be investigated in further functional studies. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

PubMed Central

2010-01-01

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation. PMID:20470441

Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and Drosophila TAR DNA-binding protein 43 (TDP-43).

PubMed

Romano, Maurizio; Buratti, Emanuele; Romano, Giulia; Klima, Raffaella; Del Bel Belluz, Lisa; Stuani, Cristiana; Baralle, Francisco; Feiguin, Fabian

2014-03-07

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.

The Xanthomonas oryzae pv. oryzae PhoPQ Two-Component System Is Required for AvrXA21 Activity, hrpG Expression, and Virulence▿ †

PubMed Central

Lee, Sang-Won; Jeong, Kyu-Sik; Han, Sang-Wook; Lee, Seung-Eun; Phee, Bong-Kwan; Hahn, Tae-Ryong; Ronald, Pamela

2008-01-01

The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH. PMID:18203830

Development and performance evaluation of a novel immunofluorescence chromatographic assay for histidine-rich protein 2 of Plasmodium falciparum.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Huang, Yongping; Xie, Mingquan; Luo, Xiaochun; Li, Wenmei; Wang, Jihua

2015-05-30

The low sensitivity and specificity of Plasmodium falciparum diagnostic tests pose a serious health threat to people living in endemic areas. The objective of the study was to develop a rapid assay for the detection of histidine-rich protein 2 (HRP2) of P. falciparum in whole blood by immunofluorescence chromatographic technology. A total of 1163 positive and negative blood samples were screened. The double-antibody sandwich assay was used to establish the kit and its performance was evaluated for sensitivity, specificity, accuracy, precision, stability, and clinical effectiveness. The cut-off level of detection of the kit was 25 parasites/μl. Common interfering substances in human blood specimens, such as bilirubin, triglyceride and cholesterol had no significant effect on HRP2 antigen detection. The precision of the kit was run with different concentration of standard calibrators and the values were less than 10 %. The performance of this diagnostic kit in the detection of the calibrators has shown that a shelf life of about 12 months gives a more reliable result. Among clinical samples tested, the HRP2 test kit and the reference products had good coincidence rate in a parallel experiment and this test kit had a more sensitive detecting level to the target protein than the reference kits used in this study. The specificity and sensitivity for this test were 99.6 % (800/803) and 99.7 % (1160/1163), respectively. A novel HRP2 immunofluorescence detection method was developed in this study. Overall performance evaluation indicated that the kit has a rapid, high sensitivity and on-spot method for detecting P. falciparum.

The nucleus of the optic tract (NOT) and the dorsal terminal nucleus (DTN) of opossums (Didelphis marsupialis aurita).

PubMed

Vargas, C D; Volchan, E; Nasi, J P; Bernardes, R F; Rocha-Miranda, C E

1996-01-01

Wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected unilaterally into the pretectocollicular region of opossums (Didelphis marsupialis aurita), primarily to investigate the existence of a commissural subcortical pathway but also to reveal afferents and efferents of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in this species. Labelled cells and terminals were observed in the contralateral NOT-DTN. Furthermore, HRP was injected bilaterally in the region of the inferior olive (IO) to verify if the distribution of labelled cells in the NOT-DTN overlapped the region of commissural labelled cells. The two subpopulations of retrogradely labelled cells coincided, being distributed within the retinal terminal field attributed to the NOT-DTN, as revealed by contralateral eye injections of HRP. The commissural cells were located slightly more ventral than the olivary cells in the optic tract. The pretectocollicular WGA-HRP injections also labelled cells and terminals bilaterally in the lateral terminal nucleus (LTN), interstitial nucleus of the superior fasciculus, posterior fibers (INSFp), ventral lateral geniculate nucleus (vLGN), and superior colliculus (SC) and ipsilaterally in the medial terminal nucleus (MTN). In addition, further caudally, labelled cells and terminals were observed bilaterally in the nuclei prepositus hypoglossi (PH) and in the medial (MVN) and lateral (LVN) vestibular nuclei. Labelled terminals were found in the ipsilateral nucleus reticularis tegmenti pontis (NRTP) and in the IO with ipsilateral predominance. This study allowed an anatomical delimitation of the NOT-DTN in this opossum species, as defined by the olivary and commissural subpopulations, as well as a hodological evaluation of this region. The existence of some common anatomical aspects with other mammalian species is discussed.

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests.

PubMed

Jimenez, Alfons; Rees-Channer, Roxanne R; Perera, Rushini; Gamboa, Dionicia; Chiodini, Peter L; González, Iveth J; Mayor, Alfredo; Ding, Xavier C

2017-03-24

Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity. Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins. The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH. The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.

Glial-derived neurotrophic factor is essential for blood-nerve barrier functional recovery in an experimental murine model of traumatic peripheral neuropathy.

PubMed

Dong, Chaoling; Helton, E Scott; Zhou, Ping; Ouyang, Xuan; d'Anglemont de Tassigny, Xavier; Pascual, Alberto; López-Barneo, José; Ubogu, Eroboghene E

2018-06-18

There is emerging evidence that glial-derived neurotrophic factor (GDNF) is a potent inducer of restrictive barrier function in tight junction-forming microvascular endothelium and epithelium, including the human blood-nerve barrier (BNB) in vitro. We sought to determine the role of GDNF in restoring BNB function in vivo by evaluating sciatic nerve horseradish peroxidase (HRP) permeability in tamoxifen-inducible GDNF conditional knockout (CKO) adult mice following non-transecting crush injury via electron microscopy, with appropriate wildtype (WT) and heterozygous (HET) littermate controls. A total of 24 age-, genotype- and sex-matched mice >12 weeks of age were injected with 30 mg/kg HRP via tail vein injection 7 or 14 days following unilateral sciatic nerve crush, and both sciatic nerves were harvested 30 minutes later for morphometric assessment by light and electron microscopy. The number and percentage of HRP-permeable endoneurial microvessels were ascertained to determine the effect of GDNF in restoring barrier function in vivo. Following sciatic nerve crush, there was significant upregulation in GDNF protein expression in WT and HET mice that was abrogated in CKO mice. GDNF significantly restored sciatic nerve BNB HRP impermeability to near normal levels by day 7, with complete restoration seen by day 14 in WT and HET mice. A significant recovery lag was observed in CKO mice. This effect was independent on VE-Cadherin or claudin-5 expression on endoneurial microvessels. These results imply an important role of GDNF in restoring restrictive BNB function in vivo, suggesting a potential strategy to re-establish the restrictive endoneurial microenvironment following traumatic peripheral neuropathies.

Prevalence of pfhrp2 and/or pfhrp3 Gene Deletion in Plasmodium falciparum Population in Eight Highly Endemic States in India

PubMed Central

Bharti, Praveen Kumar; Chandel, Himanshu Singh; Ahmad, Amreen; Krishna, Sri; Udhayakumar, Venkatachalam; Singh, Neeru

2016-01-01

Background Plasmodium falciparum encoded histidine rich protein (HRP2) based malaria rapid diagnostic tests (RDTs) are used in India. Deletion of pfhrp2 and pfhrp3 genes contributes to false negative test results, and large numbers of such deletions have been reported from South America, highlighting the importance of surveillance to detect such deletions. Methods This is the first prospective field study carried out at 16 sites located in eight endemic states of India to assess the performance of PfHRP2 based RDT kits used in the national malaria control programme. In this study, microscopically confirmed P. falciparum but RDT negative samples were assessed for presence of pfhrp2, pfhrp3, and their flanking genes using PCR. Results Among 1521 microscopically positive P. falciparum samples screened, 50 were negative by HRP2 based RDT test. Molecular testing was carried out using these 50 RDT negative samples by assuming that 1471 RDT positive samples carried pfhrp2 gene. It was found that 2.4% (36/1521) and 1.8% (27/1521) of samples were negative for pfhrp2 and pfhrp3 genes, respectively. However, the frequency of pfhrp2 deletions varied between the sites ranging from 0–25% (2.4, 95% CI; 1.6–3.3). The frequency of both pfhrp2 and pfhrp3 gene deletion varied from 0–8% (1.6, 95% CI; 1.0–2.4). Conclusion This study provides evidence for low level presence of pfhrp2 and pfhrp3 deleted P. falciparum parasites in different endemic regions of India, and periodic surveillance is warranted for reliable use of PfHRP2 based RDTs. PMID:27518538

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity.

PubMed

Samuni, Amram; Maimon, Eric; Goldstein, Sara

2017-08-01

Horseradish peroxidase (HRP) catalyzes H 2 O 2 dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H 2 O 2 -induced inactivation, have been investigated. HRP reaction with H 2 O 2 was studied by following H 2 O 2 depletion, O 2 evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. Nitroxide protects HRP against H 2 O 2 -induced inactivation. The rate of H 2 O 2 dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H 2 O 2 . The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO)>4-OH-TPO>3-carbamoyl proxyl>4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III. Nitroxide catalytically protects HRP against inactivation induced by H 2 O 2 while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex. Nitroxides catalytically protect heme proteins against inactivation induced by H 2 O 2 revealing an additional role played by nitroxide antioxidants in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Visual function in anterior ischemic optic neuropathy: effect of Vision Restoration Therapy--a pilot study.

PubMed

Jung, Cecilia S; Bruce, Beau; Newman, Nancy J; Biousse, Valérie

2008-05-15

To evaluate the effects of Vision Restoration Therapy (VRT) on the visual function of patients with anterior ischemic optic neuropathy. Randomized controlled double-blind pilot trial. 10 patients with stable anterior ischemic optic neuropathy (AION). All patients were evaluated before VRT and after 3 and 6 months of treatment by Early Treatment Diabetic Retinopathy Study (ETDRS) visual acuity, contrast sensitivity, reading speed, 24-2 SITA-standard Humphrey visual field (HVF), High Resolution Perimetry (HRP) (perimetry obtained during VRT), and vision-based quality of life questionnaire. Patients were randomized between two VRT strategies (5 in each group): I) VRT in which stimulation was performed in the seeing VF of the affected eye ("seeing field-VRT"); II) VRT in which stimulation was performed along the area of central fixation and in the ARV (areas of residual vision) of the affected eye ("ARV-VRT"). The results of the HRP, HVF, and clinical assessment of visual function were compared for each patient and between the two groups at each evaluation. Visual acuity qualitatively improved in the ARV-VRT group, however the change was not statistically significant (p=0.28). Binocular reading speed significantly improved in the ARV-VRT group (p=0.03). HVF foveal sensitivity increased mildly in both groups (p=0.059). HRP analysis showed a similar increase in stimulus accuracy in both groups (mean improvement of about 15%). All patients reported functional improvement after VRT. Despite a small sample, the study showed a trend toward improvement of visual function in the ARV-VRT group. Improvement of HRP in both groups may reflect diffusely increased visual attention (neuronal activation), or improvement of an underlying sub-clinical abnormality in the "seeing" visual field of patients with optic neuropathies.

Triply responsive films in bioelectrocatalysis with a binary architecture: combined layer-by-layer assembly and hydrogel polymerization.

PubMed

Yao, Huiqin; Hu, Naifei

2011-05-26

In this work, triply responsive films with a specific binary architecture combining layer-by-layer assembly (LbL) and hydrogel polymerization were successfully prepared. First, concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}(5) LbL layers on electrode surface by the lectin-sugar biospecific interaction between them. The poly(N,N-diethylacrylamide) (PDEA) hydrogels with entrapped horseradish peroxidase (HRP) were then synthesized by polymerization on the surface of LbL inner layers, forming {Con A/Dex}(5)-(PDEA-HRP) films. The films demonstrated reversible pH-, thermo-, and salt-responsive on-off behavior toward electroactive probe Fe(CN)(6)(3-) in its cyclic voltammetric responses. This multiple stimuli-responsive films could be further used to realize triply switchable electrochemical reduction of H(2)O(2) catalyzed by HRP immobilized in the films and mediated by Fe(CN)(6)(3-) in solution. The responsive mechanism of the films was explored and discussed. The pH-sensitive property of the system was attributed to the electrostatic interaction between the {Con A/Dex}(5) inner layers and the probe at different pH, and the thermo- and salt-responsive behaviors should be ascribed to the structure change of PDEA hydrogels for the PDEA-HRP outermost layers under different conditions. The concept of binary architecture was also used to fabricate {Con A/Dex}(5)-(PDEA-GOD) films on electrodes, where GOD = glucose oxidase, which was applied to realize the triply switchable bioelectrocatalysis of glucose by GOD in the films with ferrocenedicarboxylic acid as the mediator in solution. This film system with the unique binary architecture may establish a foundation for fabricating a novel type of multicontrollable biosensors based on bioelectrocatalysis with immobilized enzymes.

Space Mission Human Reliability Analysis (HRA) Project

NASA Technical Reports Server (NTRS)

Boyer, Roger

2014-01-01

The purpose of the Space Mission Human Reliability Analysis (HRA) Project is to extend current ground-based HRA risk prediction techniques to a long-duration, space-based tool. Ground-based HRA methodology has been shown to be a reasonable tool for short-duration space missions, such as Space Shuttle and lunar fly-bys. However, longer-duration deep-space missions, such as asteroid and Mars missions, will require the crew to be in space for as long as 400 to 900 day missions with periods of extended autonomy and self-sufficiency. Current indications show higher risk due to fatigue, physiological effects due to extended low gravity environments, and others, may impact HRA predictions. For this project, Safety & Mission Assurance (S&MA) will work with Human Health & Performance (HH&P) to establish what is currently used to assess human reliabiilty for human space programs, identify human performance factors that may be sensitive to long duration space flight, collect available historical data, and update current tools to account for performance shaping factors believed to be important to such missions. This effort will also contribute data to the Human Performance Data Repository and influence the Space Human Factors Engineering research risks and gaps (part of the HRP Program). An accurate risk predictor mitigates Loss of Crew (LOC) and Loss of Mission (LOM).The end result will be an updated HRA model that can effectively predict risk on long-duration missions.

New Pharmacology Studies on the ISS

NASA Technical Reports Server (NTRS)

Wotring, Virginia E.; Pour, S.

2015-01-01

It is known that medications degrade over time and that extreme storage conditions will hasten their degradation. This is the basis of the HRP Risk of Ineffective or Toxic Medications Due to Long Term Storage. Gaps include questions about the effects of the spaceflight environment and about the potential for safe use of medications beyond their expiration dates. There are also open questions regarding effects of the spaceflight environment on human physiology and subsequent changes in how medications act on the body; these unanswered questions gave rise to the HRP Concern of Clinically Relevant Unpredicted Effects of Medication. Studies designed to address this Risk and Concern are described below.

Parkia pendula lectin as histochemistry marker for meningothelial tumour.

PubMed

Beltrão, E I C; Medeiros, P L; Rodrigues, O G; Figueredo-Silva, J; Valença, M M; Coelho, L C B B; Carvalho, L B

2003-01-01

Lectins have been intensively used in histochemical techniques for cell surface characterization. These proteins are involved in several biological processes and their use as histochemical markers have been evaluated since they can indicate differences in cell surfaces. Parkia pendula lectin (PpeL) was evaluated as histochemical marker for meningothelial meningioma biopsies. Tissue slices were incubated with PpeL conjugated to horseradish peroxidase (PpeL-HRP) and Concanavalin A-HRP (ConA-HPR) and the binding visualized with diaminobenzidine and hydrogen peroxide. The lectin-tissue binding was inhibited with D-glucose. PpeL showed to be a useful tool for the characterization of meningothelial tumour and clinico-pathological diagnosis.

Preparation of a bio-composite of sericin-g-PMMA via HRP-mediated graft copolymerization.

PubMed

He, Min; Hu, Haoran; Wang, Ping; Fu, Haitian; Yuan, Jiugang; Wang, Qiang; Fan, Xuerong

2018-05-26

Silk sericin (SS) derived from silkworms has the characteristics of anti-oxidation, antibacterial, and biocompatibility, however, high solubility in water restricted its applications in biomedical fields. In the present work, SS was enzymatically graft-copolymerized with a hydrophobic vinyl monomer of methyl methacrylate (MMA), through a free radical reaction, through the combination use of hydrogen peroxide and horseradish peroxidase (HRP). Efficacy of the HRP-mediated reaction was examined by means of FTIR, SDS-PAGE, and SEC chromatogram. A bio-composite of SS-graft-polymethyl methacrylate (SS-g-PMMA) was constructed subsequently, the corresponding wettability, thermal behavior, and biocompatibility of the obtained composite were examined, respectively. The data reveal that MMA was successfully copolymerized with the reactive sites in sericin chains, resulting in a noticeable increase in the molecular weight. For the membrane of SS-g-PMMA, the surface hydrophobicity was evidently improved compared to that of the untreated, according to the determined data of water contact angle and dissolution ratio. The current work develops an eco-friendly technique for reuse of the industrial waste like sericin, and provides a novel method for preparation of the sericin-based biomaterials as well. Copyright © 2018 Elsevier B.V. All rights reserved.

Step-Up Synthesis of Periodic Mesoporous Organosilicas with a Tyrosine Framework and Performance in Horseradish Peroxidase Immobilization.

PubMed

Wang, Jianqiang; Zhang, Wenqi; Gu, Changqing; Zhang, Wenpei; Zhou, Man; Wang, Zhiwei; Guo, Cheng; Sun, Linbing

2017-12-14

New amino-acid-bridged periodic mesoporous organosilicas (PMOs) were constructed by hydrolysis and condensation reactions under acid conditions in the presence of a template. The tyrosine bissilylated organic precursor (TBOS) was first prepared through a multistep reaction by using tyrosine (a natural amino acid) as the starting material. PMOs with the tyrosine framework (Tyr-PMOs) were constructed by simultaneously using TBOS and tetraethoxysilane as complex silicon sources in the condensation process. All the Tyr-PMOs materials were characterized by XRD, FTIR spectroscopy, N 2 adsorption-desorption, TEM, SEM, and solid-state 29 Si NMR spectroscopy to confirm the structure. The horseradish peroxidase (HRP) enzyme was first immobilized on these new Tyr-PMOs materials. Optimal conditions for enzyme adsorption included a temperature of 40 °C, a time of 8 h, and a pH value of 7. Furthermore, the novel Tyr-PMOs materials could store HRP for approximately 40 days and maintained the enzymatic activity, and the Tyr-PMOs-10 % HRP with the best immobilization effect could be reused at least eight times. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly sensitive microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer for the detection of neuron-specific enolase.

PubMed

Yang, Tingzhen; Vdovenko, Marina; Jin, Xue; Sakharov, Ivan Yu; Zhao, Shulin

2014-07-01

A microfluidic competitive enzyme immunoassay based on chemiluminescence resonance energy transfer (CRET) was developed for highly sensitive detection of neuron-specific enolase (NSE). The CRET system consisted of horseradish peroxidase (HRP)/luminol as a light donor and fluorescein isothiocyanate as an acceptor. When fluorescein isothiocyanate-labeled antibody binds with HRP-labeled antigen to form immunocomplex, the donor and acceptor are brought close each other and CRET occurs in the immunocomplex. In the MCE, the immunocomplex and excess HRP-NSE were separated, and the chemiluminescense intensity of immunocomplex was used to estimate NSE concentration. The calibration curve showed a linearity in the range of NSE concentrations from 9.0 to 950 pM with a correlation coefficient of 0.9964. Based on a S/N of 3, the detection limit for NSE determination was estimated to be 4.5 pM, which is two-order magnitude lower than that of without CRET detection. This assay was applied for NSE quantification in human serum. The obtained results demonstrated that the proposed immunoassay may serve as an alternative tool for clinical analysis of NSE. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of self-assembled redox polymer and antibody molecules on thiolated gold electrodes.

PubMed

Calvo, E J; Danilowicz, C; Lagier, C M; Manrique, J; Otero, M

2004-05-15

Multilayer immobilization of antibody and redox polymer molecules on a gold electrode was achieved, as a strategy for the potential development of an amperometric immunosensor. The step-by-step assembly of antibiotin IgG on Os(bpy)(2)ClPyCH(2)NH poly(allylamine) redox polymer (PAH-Os) adsorbed on thiolated gold electrodes was proved by quartz crystal microbalance (QCM) and atomic force microscopy (AFM) experiments, confirming the electrochemical evidence. The increase of redox charge during the layer-by-layer deposition demonstrated that charge propagation within the layers is feasible. The multilayer structure proved to be effective for the molecular recognition of horseradish peroxidase-biotin conjugate (HRP-biotin), as confirmed by the QCM measurements and the electrocatalytic reduction current obtained upon H(2)O(2) addition. The catalytic current resulting from PAH-Os mediation was shown to increase with the number of assembled layers. Furthermore, the inventory of IgG molecules on the supramolecular self-assembled structure and the specific and non-specific binding of HRP-biotin conjugate were confirmed by the QCM transient studies, giving information on the kinetics of IgG deposition and HRP-biotin conjugate binding to the IgG.

A sensitive, colorimetric immunosensor based on Cu-MOFs and HRP for detection of dibutyl phthalate in environmental and food samples.

PubMed

Zhu, Nuanfei; Zou, Yanmin; Huang, Menglu; Dong, Shuaibing; Wu, Xiangyang; Liang, Guoxi; Han, Zhixiang; Zhang, Zhen

2018-08-15

A sensitive and artful colorimetric immunosensor based on horseradish peroxidase (HRP) was designed by labelling metal-organic frameworks (Cu-MOFs) on the second antibody (Cu-MOFs@Ab 2 ) as signal amplification for the detection of trace dibutyl phthalate (DBP). In this system, when Cu-MOFs@Ab 2 was captured by antigen- primary antibody (Ab 1 ) complex, tremendous Cu(II) will be released from Cu-MOFs in the presence of nitric acid (HNO 3 ), and Cu(II) will be further reduced to Cu(I) after the addition of sodium ascorbate (SA), consequently, inhibiting the HRP to catalyse the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (ox TMB). Under the optimized conditions, the limit of detection (LOD) was 1 μg L -1 , which was almost 60 times lower than that using a conventional ELISA with the same antibody. In addition, our method showed good accuracy and reproducibility (recoveries of 87.73-103.4%; CV values of 1.46-5.95%) through a spike-recovery analysis. The proposed immunosensor indicated great potential for trace DBP determination from environmental and food samples. Copyright © 2018. Published by Elsevier B.V.

Electron Tomography Reveals Posttranscriptional Binding of Pre-Mrnps to Specific Fibers in the Nucleoplasm

PubMed Central

Miralles, Francesc; Öfverstedt, Lars-Göran; Sabri, Nafiseh; Aissouni, Youssef; Hellman, Ulf; Skoglund, Ulf; Visa, Neus

2000-01-01

Using electron tomography, we have analyzed whether the Balbiani ring (BR) pre-mRNP particles in transit from the gene to the nuclear pore complex (NPC) are bound to any structure that could impair free diffusion through the nucleoplasm. We show that one-third of the BR particles are in contact with thin connecting fibers (CFs), which in some cases merge into large fibrogranular clusters. The CFs have a specific protein composition different from that of BR particles, as shown by immuno-EM. Moreover, we have identified hrp65 as one of the protein components of the CFs. The sequencing of hrp65 cDNA reveals similarities with hnRNP proteins and splicing factors. However, hrp65 is likely to have a different function because it does not bind to nascent pre-mRNA and is not part of the pre-mRNP itself. Taken together, our observations indicate that pre-mRNPs are not always freely diffusible in the nucleoplasm but interact with fibers of specific structure and composition, which implies that some of the posttranscriptional events that the pre-mRNPs undergo before reaching the NPC occur in a bound state. PMID:10648560

The Exosome Associates Cotranscriptionally with the Nascent Pre-mRNP through Interactions with Heterogeneous Nuclear Ribonucleoproteins

PubMed Central

Hessle, Viktoria; Björk, Petra; Sokolowski, Marcus; de Valdivia, Ernesto González; Silverstein, Rebecca; Artemenko, Konstantin; Tyagi, Anu; Maddalo, Gianluca; Ilag, Leopold; Helbig, Roger; Zubarev, Roman A.

2009-01-01

Eukaryotic cells have evolved quality control mechanisms to degrade aberrant mRNA molecules and prevent the synthesis of defective proteins that could be deleterious for the cell. The exosome, a protein complex with ribonuclease activity, is a key player in quality control. An early quality checkpoint takes place cotranscriptionally but little is known about the molecular mechanisms by which the exosome is recruited to the transcribed genes. Here we study the core exosome subunit Rrp4 in two insect model systems, Chironomus and Drosophila. We show that a significant fraction of Rrp4 is associated with the nascent pre-mRNPs and that a specific mRNA-binding protein, Hrp59/hnRNP M, interacts in vivo with multiple exosome subunits. Depletion of Hrp59 by RNA interference reduces the levels of Rrp4 at transcription sites, which suggests that Hrp59 is needed for the exosome to stably interact with nascent pre-mRNPs. Our results lead to a revised mechanistic model for cotranscriptional quality control in which the exosome is constantly recruited to newly synthesized RNAs through direct interactions with specific hnRNP proteins. PMID:19494042

Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag.

PubMed

Song, Xuezheng; Lasanajak, Yi; Rivera-Marrero, Carlos; Luyai, Anthony; Willard, Margaret; Smith, David F; Cummings, Richard D

2009-12-15

Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by Pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core alpha1,3-fucose and core alpha1,2-xylose. After simple Fmoc deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.

Comparison of peroxidase-labeled DNA probes with radioactive RNA probes for detection of human papillomaviruses by in situ hybridization in paraffin sections

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

Park, J.S.; Kurman, R.J.; Kessis, T.D.

1991-01-01

A study comparing in situ hybridization using nonradioactive DNA probes directly conjugated with horseradish peroxidase (HRP), and {sup 35}S-labeled antisense RNA probes for human papillomavirus (HPV) types 6/11, 16, and 18 was performed on formalin-fixed, paraffin-embedded tissue from 34 lesions of the cervix and vulva. These lesions included exophytic condylomas and intraepithelial and invasive neoplasms. HPV 6/11 was detected in two of four condylomata acuminata by both in situ techniques. HPV 16 was detected in 13 of 30 cases of intraepithelial and invasive neoplasms by both methods. Discordance between the two methods occurred in two instances. The radiolabeled probe butmore » not the HRP probe detected HPV 16 in one case of cervical intraepithelial neoplasia (CIN 3), whereas the converse occurred in one case of vulvar intraepithelial neoplasia (VIN 3). HPV 18 was not detected in any of the specimens by either method. This study demonstrates that nonradioactive HRP-labeled probes for the detection of specific HPV types are as sensitive as the more laborious and potentially hazardous radioactive probes.« less

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples.

PubMed

Prieto-Simón, Beatriz; Campàs, Mònica; Marty, Jean-Louis; Noguer, Thierry

2008-02-28

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.

The bacterial alarmone (p)ppGpp activates the type III secretion system in Erwinia amylovora.

PubMed

Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In; Zhao, Youfu

2015-04-01

The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp(0)) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp(0) and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp(0) and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

PubMed Central

Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In

2015-01-01

ABSTRACT The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for the regulation of the T3SS and virulence in plant-pathogenic bacteria. Furthermore, the recovery of an spoT null mutant, which displayed very unique phenotypes, suggested that small proteins containing a single ppGpp hydrolase domain are functional. PMID:25666138

Serum from Chronic Hepatitis B Patients Promotes Growth and Proliferation via the IGF-II/IGF-IR/MEK/ERK Signaling Pathway in Hepatocellular Carcinoma Cells.

PubMed

Ji, Yuanyuan; Wang, Zhidong; Chen, Haiyan; Zhang, Lei; Zhuo, Fei; Yang, Qingqing

2018-05-09

Chronic hepatitis B virus (HBV) infection (CHB) plays a central role in the etiology of hepatocellular carcinoma (HCC). Emerging evidence implicates insulin-like growth factor (IGF)-II as a major risk factor for the growth and development of HCC. However, the relationship between HBV infection and IGF-II functions remains to be elucidated. Levels of circulating IGF-II and IGF-I receptor (IGF-IR) in healthy donors (HDs) and CHB patients were tested by ELISA. Human HCC cell lines (HepG-2, SMMC-7721, MHCC97-H) were incubated with serum from HDs and CHB patients at various concentrations for 24, 48, and 72 h. MTT and plate colony formation assays, BrdU ELISA, ELISA, small-interfering RNA (siRNA) transfection, quantitative real-time PCR, and western blot were applied to assess the functional and molecular mechanisms in HCC cell lines. Serum levels of IGF-II and IGF-IR were significantly higher in CHB patients than in HDs. Additionally, serum from CHB patients directly induced cell growth, proliferation, IGF-II secretion, and HDGF-related protein-2 (HRP-2) and nuclear protein 1 (NUPR1) mRNA and protein expression in HCC cells. Moreover, serum from CHB patients increased IGF-II-induced cell growth, proliferation, and HRP-2 and NUPR1 mRNA and protein expression in HCC cells. Blockade of IGF-IR clearly inhibited the above effects. Most importantly, interference with IGF-II function markedly repressed the cell proliferation and HRP-2 and NUPR1 mRNA and protein expression induced by serum from CHB patients. Furthermore, serum from CHB patients induced ERK phosphorylation via IGF-IR, with the MEK inhibitor PD98059 significantly decreasing CHB patient serum-induced IGF-II secretion, cell proliferation, and HRP-2 and NUPR1 mRNA and protein expression. Serum from CHB patients increases cell growth and proliferation and enhances HRP-2 and NUPR1 expression in HCC cells via the IGF-II/IGF-IR/MEK/ERK signaling pathway. These findings help to explain the molecular mechanisms underlying HBV-related HCC and may lead to the development of effective therapies. © 2018 The Author(s). Published by S. Karger AG, Basel.

Virulence Factors of Erwinia amylovora: A Review

PubMed Central

Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M.

2015-01-01

Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them. PMID:26057748

Identification and Evaluation of Integration and Cross Cutting Issues Across HRP Risks

NASA Technical Reports Server (NTRS)

Steinberg, S. L.; Shelhamer, Mark

2015-01-01

The HRP Integrated Research Plan contains the research plans for the 32 risks requiring research to characterize and mitigate. These risks to human health and performance in spaceflight are identified by evidence and each one focuses on a single aspect of human physiology or performance. They are further categorized by aspects of the spaceflight environment, such as altered gravity or space radiation, that that play a major role in their likelihood and consequence. From its inception the "integrate" in the Research Plan has denoted the integrated nature of risks to human health and performance, the connectedness of physiological systems within the human body regardless of the spaceflight environment, and the integrated response of the human body to the spaceflight environment. Common characteristics of the spaceflight environment include altered gravity, atmospheres and light/dark cycles, space radiation, isolation, noise, and periods of high or low workload. Long term exposure to this unique environment produces a suite of physiological effects such as stress; vision, neurocognitive and anthropometric changes; circadian misalignment; fluid shifts, deconditioning; immune dysregulation; and altered nutritional requirements. Matrix diagraming was used to systematically identify, analyze and rate the many-to-many relationships between environmental characteristics and the suite of physiological effects. It was also to identify patterns in the relationships of common physiological effects to each other. Analyses of patterns or relationships in these diagrams help to identify issues that cut across multiple risks. Cross cutting issues benefit from a multidisciplinary approach that synthesizes concepts or data from two or more disciplines to identify and characterize risk factors or develop countermeasures relevant to multiple risks. They also help to illuminate possible problem areas that may arise when a countermeasure impacts risks other than those which it was developed to mitigate, or identify groupings of physiological changes that are likely to occur that may impact the overall risk posture.